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1

Monteiro, Emiliano Carneiro. "O sistema digestivo do \"bicho-pau\" Phibalosoma phyllinum (Phasmida, Phasmatidae): uma análise morfológica, fisiológica e bioquímica." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/41/41131/tde-24082012-092811/.

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Este trabalho consiste em um estudo detalhado do sistema digestivo de Phibalosoma phyllinum (Phasmida, Phasmatidae) num enfoque morfofuncional. Do ponto de vista anatômico, o seu sistema digestivo é constituído por um intestino anterior, um intestino médio e um intestino posterior. O intestino anterior é composto por uma cavidade bucal (onde se abre o ducto da glândula salivar bilobada), uma faringe, que se continua para o esôfago, terminando em um proventrículo, que forma uma válvula em seu interior. O intestino médio é formado por um ventrículo tubular, dividido em três regiões: ventrículo anterior (VA), ventrículo médio (VM) e ventrículo posterior (VP). Este último foi, ainda, subdividido em proximal (VPI) e distal (VPII). Na região do VPI, foi constatada a presença de estruturas denominadas de apêndices ventriculares, formadas por protuberâncias, que se inserem no tubo digestivo, e canalículos, que se projetam da região apical de cada uma delas e terminam em fundo cego. O intestino posterior é dividido em um íleo e um reto que termina no ânus. A análise histológica mostrou que o intestino anterior é formado por um epitélio simples, composto por células pavimentosas, revestido por uma cutícula, que se modifica na forma de pequenas espículas ao longo desta região. O proventriculo é composto por uma válvula muscular simples, que separa o intestino anterior e o intestino médio. Este último é formado por um epitélio simples, constituído por células do tipo colunar, chamadas de enterócitos, principais responsáveis pela secreção de enzimas digestivas e pela absorção dos nutrientes, por células regenerativas, que se apresentam reunidas em ninhos na base do epitélio, e por células endócrinas. Os apêndices ventriculares, por sua vez, possuem um epitélio simples e contínuo com o epitélio ventricular. As células das protuberâncias apresentam-se grandes e arredondadas, enquanto que os canalículos são compostos por um epitélio de células achatadas, semelhante ao dos túbulos de Malpighi. Na região de transição entre o epitélio ventricular e o intestino posterior se inserem os túbulos de Malpighi. O epitélio do intestino posterior é do tipo cúbico, simples, revestido por cutícula. A luz do intestino médio (ou ventrículo) é revestida por uma estrutura tubular, denominada de membrana peritrófica, cuja existência foi comprovada por microscopia de fluorescência com a utilização da técnica WGA-FITC (aglutinina do gérmen do trigo conjugada à fluoresceína). Com a finalidade de identificar regiões específicas do ventrículo onde ocorre a absorção ou secreção de água através do epitélio, foram realizados experimentos fisiológicos de ingestão e injeção do corante amaranto em solução. Verificou-se que, quando o corante é ingerido pelos insetos, o VA apresenta-se corado, tanto em animais em jejum quanto alimentados, indicando ser este o possível sítio de absorção de água no ventrículo. Por sua vez, a partir de experimentos com a injeção do mesmo corante na hemolinfa, foi constatada a sua tomada pelos túbulos de Malpighi, indicando serem estes os principais sítios de tomada de água da hemolinfa e de sua secreção para a luz do tubo digestivo. Tanto em animais em jejum, quanto alimentados, o corante injetado é encontrado na luz do intestino posterior. Entretanto, nos animais em jejum, o corante é capaz de se difundir, também, pelo ventrículo até a sua região anterior. A análise ultraestrutural do intestino médio revelou que a superfície apical dos enterócitos constituintes apresenta-se modificada na forma de microvilosidades. Nas membranas laterais, observam-se especializações juncionais na forma de desmossomos apicais, seguidos por junções septadas lisas. A membrana plasmática basal, por sua vez, exibe diversas invaginações, formando uma rede complexa de canais com mitocôndrias associadas. Nas células do VA e VM, essa rede exibe um número limitado de aberturas para a lâmina basal, o que indica um maior potencial de absorção de água pelo epitélio ventricular, a partir de sua luz. Nas células da região do VP, o número de aberturas é bem maior, o que sugere que pode ocorrer secreção de água e íons através desta região, não detectável nos experimentos com corantes. Por outro lado, as células regenerativas exibem características típicas de células indiferenciadas, com núcleo grande e poucas organelas. Células endócrinas, por sua vez, são, eventualmente, detectadas na base do epitélio, sem se prolongarem até a superfície apical do epitélio. Foi observado, ainda, que tanto os canalículos ventriculares, quanto os túbulos de Malpighi são formados por células achatadas, com microvilosidades apicais modificadas portando mitocôndrias em seu interior e membrana plasmática basal formando um labirinto complexo, com muitas aberturas para a lâmina basal e com muitas mitocôndrias associadas. Com relação a atividade secretora do epitélio ventricular, verifica-se a existência de grandes quantidades de retículo endoplasmático rugoso e diversas áreas de Golgi concentradas, principalmente, no citoplasma perinuclear. Nas regiões VA e VM, é detectado um grande número de vesículas secretoras, cujo mecanismo de secreção parece ser o merócrino. Ao longo de todo VP, por outro lado, vesículas secretoras estão, aparentemente, ausentes no citoplasma apical, mas pode ser constatada a presença de dilatações nas microvilosidades, algumas das quais apresentem pequenas vesículas em seu interior. Esta observação é sugestiva da ocorrência de secreção do tipo microapócrina nesta região. Devem ocorrer, portanto, dois mecanismos de secreção diferentes ao longo do ventrículo de P. phyllinum: o merócrino (no VA e no VM) e o microapócrino (no VP) O local de produção e secreção das enzimas amilase e tripsina pôde ser localizado através de experimentos de imunomarcação ultraestrutural com a utilização de anticorpos heterólogos. Ambas as enzimas foram detectadas na região do VA e do VM, podendo ser traçadas nas áreas de Golgi, nas vesículas secretoras e na luz do epitélio, junto às microvilosidades, seguindo, pois, a rota secretora. Ambas as enzimas foram identificadas no interior do mesmo tipo de vesículas de secreção, indicando que devem ser eliminadas conjuntamente através da mesma via secretora. Medidas de pH luminal revelaram grandes diferenças de pH ao longo do tubo digestivo nesta espécie. Enquanto que na região anterior o pH é mais ácido (5,3 no intestino anterior e 5,6 no VA), ele vai se tornando mais alcalino nas regiões mais posteriores do ventrículo (6,3 no VM anterior, 8,0 no VM posterior, 9,1 no VPI e 8,5 no VPII) e é neutro no intestino posterior (7,3). Foi determinado, ainda, o efeito do pH sobre a atividade das enzimas tripsina e amilase. A maior atividade para tripsina foi observada no pH 9,0. Já para amilase, a maior atividade foi observada em pH 5,0. No que diz respeito a atividade de enzimas digestivas, ficou evidente que a atividade de amilase e tripsina se concentra no conteúdo do intestino anterior e VA.
This work presents a detailed morphofunctional study of the digestive system of Phibalosoma phyllinum (Phasmida, Phasmatidae). From an anatomical point of view, the digestive system of this insect is formed by a foregut, a midgut and a hindgut. The foregut is composed by the buccal cavity (where the salivary gland\'s duct open), a pharynx and an aesophagus that ends in the proventriculus. The midgut consists of a tubular ventriculus and it can be divided in three regions: the anterior ventriculus (AV), the middle ventriculus (MV), and the posterior ventriculus (PV) which can be further subdivided in its proximal (PVI), and distal (PVII) sub-regions. The presence of a complex system of ventricular appendices was observed in the PVI sub-region, which are formed by protuberances, in the external ventricular surface, and by blind-ended canaliculi, which are connected to the protuberances. The hindgut is divided into an ileum and a rectum that ends in the anus. Seen by light microscopy, the foregut is made up by a simple epithelium, which is composed of squamous cells and covered by a cuticle layer. Through the whole foregut the cuticle forms spines. The proventriculus is formed by a simple muscular valve that separates the foregut from the midgut. The midgut itself is formed by a simple epithelium, which is made up of three different cell types: columnar cells (enterocytes), which are the main site of enzyme production and secretion, and nutrient absorption, the regenerative cells, which are clustered in nidi at the basal portion of the epithelium, and the endocrine cells. The ventricular appendices, in turn, are formed by a simple epithelium that is continuous with the ventricular epithelium. Cells from the protuberances are large showing a round shape, while canalicular cells are short, and very similar to the ones presented in the Malpighian tubules. In the transition between the midgut and the hindgut, several Malpighian tubes branch off. The hindgut is formed by a simple epithelium, lined by cuticle. The ventricular lumen is covered by a tubular structure called peritrophic membrane whose presence was confirmed by fluorescence microscopy, using chitin-binding lectin WGA (wheat germ agglutinin) coupled with FITC (fluorescein isothiocyanate). In order to identify specific ventricular regions where water absorption and secretion through the epithelium take place, physiological experiments, using amaranth dye solution were performed. This solution was either orally administered, or injected in the hemolinph of both fed and starved insects. These experiments revealed that, both in fed and starved animals, the AV is the main site of water absorption, whereas the Malpighian tubules are the main sites of water secretion into the ventricular lumen. Ultrastructural analysis showed that enterocytes present an apical surface modified into well-developed microvilli. In their lateral surface, the adjacent plasma membranes are linked by desmosomes and smooth septate junctions. The basal plasma membrane shows several infoldings, forming a labyrinth of channels with associated mitochondria. In the AV and MV, these infoldings present a limited number of openings to the basal lamina, which indicates a greater water absorption potential of these regions from the gut lumen. In the PV cells, the number of openings is greater, indicating that these regions may be involved in water secretion (although occurring in levels that are undetectable through dye experiments). Regenerative cells nidi, in turn, can be observed in the basal portion of the epithelium throughout the midgut. This cell type shows characteristics of undifferentiated cells, such as large nuclei and few organelles. Endocrine cells are confined to the basal portion of the epithelium. Both the ventricular canaliculi and the Malpighian tubules are made up by cuboidal cells with modified apical microvilli bearing mitochondria in their interior, and basal plasma membrane forming a labirinth with many well developed infolds and openings to the basal lamina, as well as many associated mitochondria. An intense secretory activity was observed along the entire midgut. Large amounts of rough endoplasmic reticulum, well developed Golgi areas were observed in the enterocytes. In the AV and MV a large number of secretory vesicles could be observed concentrated in the apical portion of the cells, and their contents are probably eliminated by a merocrine mechanism. Along the PV, secretory vesicles are apparently absent but microvilli display dilated tips frequently showing small vesicles in their interior. This observation may indicate the occurrence of a microapocrine secretory mechanism in this ventricular region. The enzymes amylase and trypsin were immunolocalized in the cells from the AV and MV. In these regions, the two enzymes were detected in Golgi areas, secretory vesicles and in the the ventricular lumen, between the microvilli. Thus, both enzymes seem to be produced and eliminated through the same secretory pathway. Luminal pH measurements revealed a great variation of the pH along the intestine of this species. While in its anterior region the pH is acid (5,3 in the foregut and 5,6 in the AV), it becomes gradually alkaline towards the posterior regions (6,3 in the anterior MV, 8,0 in the posterior MV, 9,1 in the PVI and 8,5 in the PVII); in the hindgut the pH is neutral (7,3). The effect of the pH on amylase and trypsin activities was also measured. It was determined that trypsin\'s optimal pH was 9,0, and amylase\'s optimal pH was 5,0. Biochemical assays of digestive enzymes revealed the presence of amylase and trypsin in the luminal contents, mainly in the foregut and AV. In spite of that, it is possible that trypsin\'s activity is greater in the MV and PV, where the alkaline luminal pH matches the optimal pH for this enzyme. Chymotrypsin is also present in the lumen of the foregut and AV, but, unlike amylase and trypsin, its major activity is found in the AV. Aminopeptidase is found in the ventricular epithelium, mainly in the MV and PV, and maltase is detected associated with the microvillar glicocalix in the AV and PV regions. The hindgut showed low levels of digestive enzyme excretion, both in fed and starved animals, suggesting that these enzymes are recovered during the digestive process. Thus, these results point to the occurrence of an endo-ectoperitrophic circulation of digestive enzymes in both fed and starved animals, in which the AV is the main absorption site of water and the Malpighian tubules, the main secretion site. Initial carbohydrates digestion should occur in the foregut and in the AV, in the endoperitrophic space, whereas its final digestion should take place in the epithelial surface. Protein digestion should take place in the MV and in the PV; initial digestion should occur in the luminal endoperitrophic space, whereas final digestion takes place in the epithelial surface of these regions. Carbonic anhydrase assays revealed a high activity of this enzyme in the ventricular appendices. This fact, along with the morphological similarity between ventricular canaliculi and Malpighian tubules suggest that the canaliculi and the Malpighian tubules are homologous structures. Along the evolutionary process, the canaliculi probably acquired the capability to promote the alkalization of the PV lumen (mainly in the PVI region) thus affecting the digestive process
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2

Hames, C. A. C. "The digestive system and metals in terrestrial isopods." Thesis, University of Reading, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.234697.

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3

Man, Siu-yin. "Adrenomedullin in the rat digestive system response to starvation /." Click to view the E-thesis via HKUTO, 2005. http://sunzi.lib.hku.hk/hkuto/record/B32032298.

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4

Man, Siu-yin, and 文小燕. "Adrenomedullin in the rat digestive system: response to starvation." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2005. http://hub.hku.hk/bib/B32032298.

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5

Filietáz, Carlos Felipe Tasso. "Padrões de especificidade e expressão das lipases digestivas durante o desenvolvimento e o processo infeccioso no mosquito Aedes aegypti." Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-23022017-120752/.

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O mosquito Aedes aegypti é vetor de doenças como a febre amarela, dengue, chinkungunya e zika. O sistema digestório é responsável pela digestão e absorção de nutrientes, é também uma interface com o ambiente externo sendo a porta de entrada de organismos infecciosos. A presença de duas lipases digestivas foi confirmada por qPCR, uma na fase larval (L-Aa7051) e outra na fase adulta (L-Aa7055). Estas enzimas foram agrupadas na família das lipases neutras e apresentam alterações em resíduos envolvidos na especificidade, domínio tampa e alça β9. A L-Aa7055 recombinante foi expressa heterologamente em Escherichia coli na porção insolúvel, com atividade após a renaturação. Observamos que a expressão da lipase L-Aa7055 sofre uma redução de 30% na infecção Plasmodium gallinaceum, não sendo afetada pelo vírus dengue sorotipo 2 (DENV2). A digestão de lipídeos é importante na fase larval, com altos níveis de transcrito. Um estudo mais aprofundado ainda será necessário para compreender completamente o papel das lipases no processo infeccioso.
The Aedes aegypti borne diseases yellow fever, dengue fever, chinkungunya and zica are important public healthy problems. The digestion and absorption of nutrients are performed in the digestive system, which is also an external environment interface that allows the infection by pathogenic microorganisms. The presence of two digestive lipases were identified by qPCR, L-Aa7051 in the larval phase and L-Aa7055 in the adult female. The lipase sequences were grouped in the neutral family, and exhibit alterations in residues involved in specificity, lid domain and β9 loop. The recombinant L-Aa7055 was expressed in the insoluble fraction, and show activity after a renaturation process. We notice that the expression levels of L-Aa7055 are reduced by 30% in the Plasmodium gallinaceum infection and were not affected by serotype 2 dengue virus (DENV2). The lipid digestion is important in the larval phase, with higher transcript levels. New studies will be necessary to the complete understanding of lipase contribution in the infectious process.
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Akhlaq, Maham. "Investigation of Cten signalling and regulation in colorectal cancer." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/32802/.

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Cten (also known as Tensin4) is the fourth member of the Tensin gene family. It lacks the N terminal actin binding domain while retaining the C terminal SH2 and PTB domains. This helps to bind Cten to the intracytoplasmic tail of β1 integrin and puts it at the heart of focal adhesions. It is reported to be a tumour suppressor in kidney and prostate cancer where normal tissues show high expression. However in a number of tumours, including colorectal cancer, Cten has been labelled as an oncogene. Cten which normally is a cytoplasmic protein gives nuclear staining in colorectal metastatic deposits. It increases motility, invasion and colony formation in colorectal cancer cells. In this study we have tried toexplore the mechanism of functional activity and regulation of Cten. We looked at Cten in the nucleus in vitro and identified new downstream binding targets. In addition we investigated the role of the SH2 domain of Cten concentrating on its downstream signalling molecules and binding partners. Furthermore, we explored regulators of Cten. In this study we have forced nuclear localisation of Cten by tagging it with a nuclear localisation sequence (NLS) and found a significant increase in cell motility. In order to investigate the SH2 domain we used site directed mutagenesis to change potentially important amino acids namely Arginine at 474 to Alanine (R474A), which is important for binding tyrosine phosphorylated proteins. Moreover, we displayed that Cten underwent tyrosine phosphorylation and additionally changed three tyrosine residues i.e. Y449F, Y479F and Y530F via site directed mutagenesis. We found R474 and Y479 to be important in regulating cell motility and that known downstream targets such as ILK and FAK are dependent on an intact SH2 domain. Furthermore we have identified Cten to be physically bound to FAK in the cytoplasm and nucleus and new downstream targets identified such as Src and Paxillin. Regarding possible regulators of Cten, we found that Cten might be a possible substrate for calpain. Another regulator considered was CD24 due to its role in movement of integrins into lipid rafts and we found it was a positive regulator of Cten. In conclusion localisation of Cten into the nucleus causes an augmentation of its motility enhancing functions. Cten regulates cell motility via its SH2 domain. Arginine 474 and Tyrosine 479 are important for its function. Cten regulates levels of ILK, FAK, Src and Paxillin through its SH2 domain and binds to FAK in both cytoplasm and nucleus. Calpain and CD24 were found to possible regulators of Cten in colorectal cancer. Future studies are needed to define its role in signalling at focal adhesions and these studies should be validated in other cancer cell models as well to establish Cten as regulator of cell motility in cancer.
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Jain, Kamini. "Prevention of treatment related adverse effects in cystic fibrosis." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/55105/.

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Cystic fibrosis (CF) is one of the commonest life-limiting genetic disorders in the Caucasian population. Management involves frequent administration of antibiotics including aminoglycosides. With improving survival, it is time to focus on various age-related and treatment-associated adverse influences. The objective of this research was to evaluate renal function in CF, determine the effects of cumulative antibiotic exposure and to identify ways to reduce associated comorbidity. A cross-sectional study showed that a small number of adults and children with CF had low glomerular filtration rate (GFR), and there was no association between GFR and cumulative antibiotic exposure. An above normal GFR was identified in one in four children with CF. Estimated GFR calculated by creatinine-based equations did not accurately predict the GFR measured by the gold standard 51Cr-EDTA (51chromium-ethylenediamine tetraacetic Acid). Pure tone audiograms identified a raised hearing threshold in one in four people with CF, which did not correlate with increasing aminoglycoside exposure. A randomised controlled study established that there is no difference in the pharmacokinetics of tobramycin when administered intravenously in the morning or evening. A Cochrane systematic review concluded that there was insufficient evidence to support a routine use of bronchoalveolar lavage in the management of pulmonary infections with Pseudomonas aeruginosa in children with CF below 5 years old. CF gene (Cystic Fibrosis Transmembrane Conductance Regulator, CFTR) is expressed in pig kidneys. Histological and molecular experiments established that there is no difference between the newborn pigs with genotypes CFTR -/- (knockout) and CFTR +/- (heterozygous) or CFTR +/+ (wild-type) pig kidneys in the renal morphology and in the expression of various renal endocytic receptor proteins. The vascular haemodynamic parameter, augmentation index ascertained in a small group of children with CF suggests a possibility that the vascular age may be advanced in people with CF right from their childhood. In summary, these studies have established a low prevalence of renal disease in CF and a lack of association between cumulative antibiotic exposure and GFR. Further research is needed to evaluate the natural history of high GFR in paediatric CF population. Kidneys from pig model of CF may provide an alternative model to investigate the renal disease in CF.
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Alsulaiman, Abdullah. "Investigation of the role of CD24 in metastatic colorectal cancer." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/55401/.

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CD24 is a small (81 amino acids) GPI anchored protein which is involved in promoting cell motility and stemness and may be a part of the metastatic process. It is a heavily glycosylated molecule and contains numerous O-glycosylation sites together with two N-glycosylation sites. N-glycosylation is thought to be important in protein function, and therefore, the aim of this study is to (a) investigate the importance of N-glycosylation in the function of CD24, (b) identify other potentially functional sites in CD24 by deletion mapping, (c) define downstream targets of CD24, and (d) identify the extrinsic signals of which activate CD24. (a) Through site-directed mutagenesis, we changed the glycosylated residues N32 (ACC to CAA) and Q52 (AAT to CAG) in CD24. Mutating each of these sites individually, when compared to pCCD24WT (wild-type CD24), caused a partial reduction in ability to induce cell motility and cell invasion (cell motility p=0.0001 cell invasion p=0.0001) and, unexpectedly, resulted in significantly enhanced cell proliferation (p=0.0001). Mutation of both sites resulted in a near loss of motility induction and retained cell proliferation. (b) We mapped the functional sites of CD24 by deleting seven amino acid segments of the whole of the mature peptide. Apart from the N-glycosylation sites, no other functional domains were identified which altered cell motility or proliferation. (c) Previously, in our lab it has been shown that Cten is downstream motility-inducing target of CD24. We hypothesised that CD24 may signal through the Notch pathway since Notch1 has an important role in maintaining CSCs. Results showed that forced expression of CD24 upregulates Notch1 and Cten whilst knockdown of CD24 causes loss of Notch1 and Cten expression. However, forced expression of CD24 with simultaneous knockdown of Notch1 resulted in failure to induce Cten. (d) CD24 is reported to act as a ligand of P-selectin. We found that stimulating CD24 expressing cell lines induced with P-selectin induced cell motility (p=0.0011) and caused an increased in the protein expression of downstream targets of CD24. Stimulating cell lines expressing CD24 with mutant glycosylation sites resulted in a failure to induce motility or CD24 targets. We conclude, the removal of the N-glycosylation sites in CD24 resulted in a loss of cell migration and invasion, thereby suggesting the importance of these sites in mediating the migration and invasion functions of CD24. Unexpectedly, these mutations also appeared to stimulate cell proliferation, suggesting that wild type CD24 can functionally inhibit cell proliferation. Deletion mapping did not reveal any other functional sites on the mature CD24 suggesting that O-glycosylation is relatively affecting the glycosylation in the biology of CD24. Notch1 was to be an important downstream target of CD24 and a regulator of Cten. The binding of P-selectin with CD24 resulted in increased motility of CD24 which is also dependent on N-glycosylation.
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Couch, Daniel. "The actions of cannabidiol and palmitoylethanolamide on inflammation and permeability of the gut." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/51804/.

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In health, the gut provides a barrier between the external and internal environment. This selectively permeable barrier allows absorption of nutrients and water from the gastrointestinal contents, whilst preventing the transfer of noxious material such as bacteria. During episodes of inflammation, this barrier becomes compromised, allowing transfer of noxious material into the systemic circulation, leading to disease states such as inflammatory bowel disease and septic shock. There are no clinically available compounds to combat this increase in permeability directly. The endocannabinoid system is a group of endogenous lipid signalling molecules which activate membrane-bound receptors. Plant-derived and synthetic compounds also act at these receptors, generating a wide variety of secondary effects. The aims of this study were to identify compounds with action on the endocannabinoid system which could be used clinically to treat inflammation and hyperpermeability of the gastrointestinal tract, exploring mechanisms of action. Systematic review and meta-analysis of existing literature revealed 51 preclinical studies, and 2 clinical studies examining the effect of cannabinoid compounds. In preclinical studies, cannabinoid drugs reduced myeloperoxidase activity in the gastrointestinal mucosa within mouse and rodent models of colitis (standard mean difference -1.26, 95% confidence interval (CI)-1.54 to -0.97, I2=48.1%) and macroscopic disease activity scores (standard mean difference -1.36, 95% CI -1.62 to -1.09, I2=61%). Clinical trials found no overall benefit of cannabinoid drugs in Crohn’s disease (mean difference -74.97, 95% CI –229 to 0.79, I2=75%). Two compounds, cannabidiol and palmitoylethanolamide, possessing positive outcomes and preferable side effect profiles, were put forward for further study to examine potential clinical benefit. The mechanism of action of palmitoylethanolamide and cannabidiol were explored further by examination of their effects on the immune response, permeability of cultured cell monolayers, intracellular signalling pathways, expression of membrane-bound proteins governing permeability and receptors of the cannabinoid system. We found that these agents were anti-inflammatory in both cultured Caco-2 cells and explant human colonic tissue, prevented increases in permeability secondary to inflammation, and were likely to act through adenylyl cyclase, protein kinase A and extracellular signal-regulated kinases. The downstream effects of these compounds prevented down-regulation of the TRPV1 receptor, upregulation of aquaporin 3 expression, and prevention of downregulation of claudin-3. The effects of palmitoylethanolamide and cannabidiol were then examined on permeability in the human colon in vivo by means of a double blinded, randomised controlled trial. This study demonstrated that aspirin increased the permeability of the human gut, determined by increases in urinary concentrations of lactulose and D-mannitol, quantified by mass spectrometry. Groups receiving oral cannabidiol or palmitoylethanolamide demonstrated lower urinary concentrations of lactulose and D-mannitol, suggesting that these two drugs could be used clinically to prevent disease-induced hyperpermeability. In conclusion, cannabidiol and palmitoylethanolamide have shown consistent anti-inflammatory actions in colonic ex vivo and in vitro models, and also prevented increases in intestinal permeability in vitro and also in vivo in a randomised, double blind, placebo-controlled trial. Their clinical use in IBD should now be assessed in phase II clinical trials.
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Asiri, Abdulaziz. "Investigations into the role of Cten signalling in colorectal cancer." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/52233/.

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C-terminal tensin-like (Cten, also known as Tensin4) is the member of the tensin gene family. Cten functions as an oncogene in a variety of cancer types and its expression is commonly associated with poor prognosis and metastasis in colorectal cancer (CRC). Although several studies have shown that Cten has a critical role in the regulation of cell motility and invasion in different tumour tissues, the underlying signalling mechanisms have not been fully elucidated. This thesis investigated the biological activity of Cten in four different ways in order to further elucidate the mechanisms of Cten signalling in CRC cells. Potential downstream targets of Cten signalling involved in the regulation of epithelial-to-mesenchymal transition (EMT) induced cell motility i.e. Rho-associated protein kinase1 (ROCK1), Src and Snail were investigated. Cten expression was manipulated in different cell lines using multiple approaches including forced expression, gene knockdown and constitutive depletion (through Crispr/Cas9 gene deletion) to eliminate artefacts of methodology and cell line specific effects. Snail, Src and ROCK1 were identified as novel downstream targets of Cten signalling and additionally, Cten was shown to increase the stabilisation of both Src and Snail proteins. The functional relevance of Cten-Snail, Cten-Src and Cten-ROCK1 signalling was assessed, and the overall findings demonstrated that Cten could promote cell motility and colony formation directly through the positive regulation of the Src/ROCK1/Snail dependent axis. To gain a deeper insight into the mechanisms of Cten’s biological function, mutations, at two important residues (i.e. arginine 474 and tyrosine 479) in the Src homology 2 (SH2) domain of Cten were introduced into one construct (GFP-CtenR474A+Y479F) using site directed mutagenesis. These two residues in the SH2 domain of Cten were found to not only be important for interacting with Src, ROCK1, or Snail signalling, but also for regulating cell motility and colony formation efficiency. Numerous Cten regulatory factors have been identified, however, little is known about how Cten is activated and regulated in cancer cells. The relationship between transforming growth factor beta 1 (TGFβ1) and Cten was investigated and stimulation of cells with TGFβ1 or knockdown of TGFβ1 resulted in changes in Cten expression as well as its downstream targets of ROCK1, Src, Snail, and N-cadherin. Furthermore, this positive interaction between TGFβ1 and Cten was functionally relevant and caused changes in cell motility. and the nuclear translocation of ROCK1, Src, and Snail protein increased by TGFβ1 is probably mediated via upregulation of the Cten signalling pathway The biological function of Cten in the nucleus was further investigated and shown to increase nuclear localisation of Src, ROCK1, and Snail, further promoting the migratory capability and colony formation efficiency in CRC cells. Finally, Cten expression was shown to positively correlate with both ROCK1 and Src expression in a series of primary CRCs. This correlation was consistent with that observed following manipulation of Cten expression in CRC cell lines. In conclusion, this study has revealed a number of novel findings regarding the biological function of Cten signalling in CRC. However, further validation of the findings may enhance the understanding of the role of Cten in the invasion-metastasis cascade in the future.
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11

De, Rosa Antonella. "Oestrogen receptors in oesophageal cancer." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/52399/.

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Introduction: Oesophageal cancer is more common in men than women. Oestrogen, which mediates its effects via oestrogen receptors (ERs), may be responsible for the gender disparity. This thesis investigates the role of ERs in oesophageal cancer development and explores potential therapeutic possibilities. Methods: ERα and ERβ expression in oesophageal AC cell lines (OE19 and OE33) was knocked down using siRNA, and the effect of knockdown on the expression of proliferation-associated proteins (Ki67, PCNA, E-cadherin, Cyclin D) was assessed. The effect of the SERM, tamoxifen, on oesophageal cancer cell proliferation was investigated using proliferation assays and by evaluating the effect of tamoxifen on proliferation-associated proteins. Finally, a pilot study of tamoxifen in patients with oesophageal cancer was undertaken to assess feasibility of a clinical trial and to determine the short-term biological effect of tamoxifen on proliferation, assessed by a change in the immunohistochemical expression of Ki67 between paired biopsies. Results: ERα and ERβ are expressed at the mRNA (RT-PCR) and protein level (Western Blotting) in the OE19 and OE33 cell lines. ERβ mRNA knock down was achieved in the OE33 cell line (p = < 0.0001). However, reproducible significant ERβ protein knockdown was not demonstrated, and there was no change in the expression of proliferation-associated proteins. Treatment with tamoxifen significantly inhibited OE33 cell proliferation in a dose-dependent manner (p = < 0.0001). Interestingly, treatment with tamoxifen decreased the expression of E-cadherin, but failed to change the expression of the remaining proliferation-associated proteins. Eight patients (6 male with AC and 2 female with SCC) included in the pilot study completed a median on 30 days (range: 28 – 45 days) tamoxifen treatment; the mean Ki67 Labelling Index between paired biopsies increased by 0.625% (ns). Of the two women included, Ki67 expression decreased with tamoxifen treatment. A correlation was demonstrated between a reduction in Ki67 and mean ERβ expression (r= -0.2272, ns). Discussion: ERβ is the dominant ER subtype expressed in oesophageal cancer cell lines and human cancer tissue. Tamoxifen inhibits the proliferation of oesophageal cancer cell lines in-vitro. Further studies to define the role of the ERβ subtype in oesophageal cancer and a clinical trial of tamoxifen in patients with oesophageal cancer is needed.
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Maitra, Rudra Krishna. "Evolving new drug treatments for faecal incontinence." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/44517/.

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Faecal incontinence is an embarrassing and socially debilitating condition which is primarily acquired and increases in prevalence with age. Current conservative measures are aimed at dietary modification and changing the consistency of stool with no targeted treatments available to address the underlying cause of incontinence. Surgical treatments are either unsatisfactory or carry significant morbidity. There is currently increasing interest in the use of α adrenoceptor agonists to increase the tone of the anus and thereby improve continence. One of the potential drugs, L-erythro-methoxamine, has been shown to increase mean anal resting tone in healthy volunteers and is well tolerated as suppositories. Extensive data exists on the neuromyogenic properties of the human internal anal sphincter (IAS) and its response to various drugs, particularly α adrenoceptor agonists. Little data exists on the response of the rectum to α agonists. The ideal drug treatment for incontinence would cause a contraction in the IAS and a relaxation in the rectum – increasing the reservoir of stool while augmenting the sphincter to aid continence. We performed in vitro experiments on sheep internal anal sphincter (IAS) using an organ bath method and subjected the tissues to electrical field stimulation to mimic nerve stimulation. Our results were comparable to results of previous authors who also examined the sheep IAS. Using this validated protocol, we investigated the sheep rectum to identify the neuronal mediators of the EFS response and to investigate the effect of α1 adrenoceptor agonists. Our results showed that sheep rectum relaxes in response to nerve stimulation and this relaxation is the result of the release of nitric oxide. Contraction in response to nerve stimulation is primarily mediated by acetylcholine acting on muscarinic receptors. Methoxamine caused a contraction in the sheep rectum. We also examined the pig IAS and rectum in vitro. An identical organ bath technique was used with Electrical Field Stimulation to mimic nerve stimulation. For both IAS and rectum in the pig, nerve stimulation caused a relaxation via nitric oxide and a contraction mediated primarily by noradrenaline acting on α1 adrenoceptors with a small component mediated by acetylcholine acting via muscarinic receptors. Methoxamine caused a contraction in both IAS and rectal tissue with similar potency in each. We were part of an industry-sponsored multi-centre randomised placebo-controlled clinical trial investigating the safety and efficacy of L-erythro-methoxamine on patients with faecal incontinence. The nine patients recruited from our centre showed no significant improvement in the number of episodes of incontinence or in the questionnaire scores measuring the impact of incontinence on quality of life after eight weeks of daily suppositories. The drug was well tolerated with few adverse events. There were no significant safety concerns although there was a prolongation of the PR interval in post-treatment ECGs and a positive correlation between the QT intervals on ECGs with serum concentrations of the drug. Our results were typical of those obtained in the other centres. The overall result of the trial was that there was no improvement in episodes of faecal incontinence following treatment with L-erythro-methoxamine at the chosen doses. The results from our in vitro experiments suggest that α adrenoceptor agonists may not be the best methods of treating faecal incontinence. The results from the clinical study support this finding. We believe that more in vitro studies need to be performed on human rectal tissue to confirm our findings that α adrenoceptor agonists cause a contraction in the rectum.
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13

Saunders, John. "Ex vivo modelling of oesophago-gastric cancer." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/47574/.

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Introduction The response to neoadjuvant chemotherapy in oesophago-gastric (OG) cancer is only 40%, so over half of the patient’s disease will progress, whilst they also suffer the toxic chemotherapy side-effects. A model to predict chemotherapy response would provide a marked clinical benefit, by enabling personalised treatment of OG cancer. Methods Live chemo-naïve tumour biopsies were obtained following informed consent at staging endoscopy, before patients underwent their routine neoadjuvant chemotherapy. Tumour cells from the endoscopic biopsies were expanded, using an in vitro feeder layer system and supplemented medium. With ethics committee approval and under Home Office guidance, these individual patient cancer cells were engrafted into immuno-compromised mice, where they formed representative tumour xenografts. Primary patient tissue, the corresponding individual patient cancer cells and their matching xenografts were analysed using immunohistochemistry, demonstrating that the in vitro and in vivo cells had retained the characteristics of the original patient’s oesophageal adenocarcinoma. To model the human tumour micro-environment (TME), a three dimensional tumour growth assay (3D-TGA) was developed, whereby the individual patient’s primary tumour cells were grown as 3D cancer cell clusters. This was performed by seeding individual patient’s primary tumour cells within a biological basement membrane extract, rich in extracellular matrix (ECM) components, with and without human mesenchymal stem cells to provide stromal support. The individual patient cancer clusters in the 3D-TGA were subjected to detailed chemotherapeutic assessment, to quantify their chemo-sensitivity to the standard chemotherapy which was administered to the patient in the clinic. This 3D-TGA predicted chemo-sensitivity was then compared with the patient’s actual clinical chemotherapy response, as measured by the histological tumour regression grade, which directly relates to prognosis. In combination with standard platinum-based chemotherapy, the 3D-TGA was assessed as a platform for evaluating new chemotherapeutics: the novel emerging HDAC inhibitor Panobinostat, and the phosphodiesterase type 5 inhibitor Vardenafil, which has recently been shown to be active against cancer stroma, were evaluated. Results Individual patient tumours were grown from primary endoscopic biopsy tissue in over half of samples obtained within a clinically applicable timescale of 2-4 weeks. Incorporating human mesenchymal cells into the 3D-TGA significantly changed the growth and drug resistance profiles (p < 0.005). This 3D-TGA chemo-response in the presence of stroma reflected the clinical chemo-sensitivity, with an accurate correlation between the 3D-TGA predicted chemo-resistance and actual clinical response for the patients evaluated. As well as predicting potential chemo-sensitivity for individual patients, the method allows individual drugs and combinations to be evaluated, trends in chemo-sensitivity between patients to be appraised, and analysis of the effect of the TME on tumour growth and chemotherapy resistance. Combination with Panobinostat enhanced response and proved efficacious in otherwise chemo-resistant tumours. Addition of PDE5i demonstrated an overall significantly enhanced chemotherapeutic response (p=0.003), and consequently provided efficacy in 60% of the otherwise chemo-resistant tumours. Discussion The novel method of growing individual patient OG cancers, using a 3D model with specific components of the tumour micro-environment in particular ECM and mesenchymal cells, provides a clinically-relevant oesophageal cancer model with application for chemo-sensitivity testing. Mesenchymal cells have a significant effect enhancing chemotherapy drug resistance in OG cancer, and this 3D model allowed identification of patients in which stromal targeting using PDE5i provided a significant reduction in chemotherapy drug resistance. In these patients, addition of PDE5i to routine chemotherapy could result in a marked change in the clinical efficacy of their chemotherapy regimen. The 3D model’s chemo-response accurately reflects individual patients’ clinical chemo-sensitivity and so this research has direct clinical application: if this assay proves to be predictive across a wider patient population, then following clinical trials, it could potentially be used to routinely guide individual patient therapy in the clinic, with administration of tailored chemotherapy for individual patient benefit.
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Boereboom, Catherine L. "Determining the feasibility and effectiveness of high intensity interval training in preoperative colorectal cancer patients." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/47342/.

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Colorectal cancer (CRCa) is the 4th most common cancer in the United Kingdom with 41,265 new cases diagnosed in 2014 (Cancer Research UK, 2017). Advancing age is an established risk factor for the development of CRCa (Figure 1.1); between 2012 and 2014 44% of new cancers were diagnosed in patients aged 75 years and over (Cancer Research UK, 2017). Due to our ageing population, national screening programmes and improved diagnostic techniques, a greater number of older people are now being diagnosed with CRCa. However older people are not surviving the disease as well as their younger counterparts. The 5 year survival rate from diagnosis of bowel cancer between 2009 and 2013 was 67.5% for 60-69 year old men and 45.5% for 80-99 year old men. This illustrates an increased burden of this disease in an ageing population (Cancer Research UK, 2017). In general terms, due to improvements in health screening and perioperative care, mortality from CRCa in all populations is decreasing over time. Indeed, the England and Wales age standardised 5 year survival rates for men with CRCa have increased from 24% in the early 1970s to 59% in 2010-11 (Cancer Research UK, 2017). Overall CRCa incidence is increasing over time but this is mirrored by improved survival rates. Age is a significant factor in reduced survival from colorectal cancer and efforts to improve outcomes in elderly CRCa patients should be investigated.
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15

Palaniyappan, Naaventhan. "Quantitative magnetic resonance imaging in evaluating haemodynamic changes in portal hypertension." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/45274/.

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The majority of complications in patients with cirrhosis result from the development and progression of portal hypertension characterised by increased intrahepatic resistance and progressive splanchnic vasodilation. Hepatic venous pressure gradient (HVPG) measurement is the only validated technique to accurately evaluate changes in portal pressure. However, HVPG measurements are invasive and available only in specialised hepatology units, precluding its use in routine clinical practice. In the first study, we evaluated the use of non-contrast quantitative magnetic resonance imaging (MRI) as a surrogate measure of portal pressure. 30 patients undergoing HVPG measurement were prospectively recruited. MR parameters of longitudinal relaxation time (T1), perfusion of the liver and spleen (by arterial spin labelling), and blood flow in the portal, splanchnic and collateral circulation (by phase-contrast MRI) were assessed. We estimated the liver stiffness measurement (LSM) and Enhanced Liver Fibrosis (ELF) score. The correlation of all non-invasive parameters with HVPG was evaluated. The mean (range) HVPG of the patients was 9.8 (1-22) mmHg, and 14 patients (48%) had clinically significant portal hypertension (CSPH, HVPG ≥10 mmHg). Liver T1 relaxation time, splenic artery and superior mesenteric artery velocity correlated significantly with HVPG. Using multiple linear regression, liver T1 and splenic artery velocity remained as the two parameters in the multivariate model significantly associated with HVPG (R=0.90, p < 0.001). This correlation was maintained in patients with CSPH (R=0.85, p < 0.001). A validation cohort (n=10) showed this linear model provided a good prediction of HVPG. LSM and ELF score correlated significantly with HVPG in the whole population but the correlation was absent in CSPH. In conclusion, MR parameters related to both hepatic architecture and splanchnic haemodynamics correlate significantly with HVPG. This proposed model, confirmed in a validation cohort, could replace the invasive HVPG measurement. In the second part, we studied the use non-invasive MRI to dynamically assess changes in hepatic and collateral blood flow, liver perfusion and oxygenation in response to ingestion of a test meal (meal challenge), and hyperoxia and hypercapnia (gas challenge). These changes were compared between healthy subjects and patients with compensated cirrhosis (CC). In the meal challenge study, we evaluated the blood flow in the portal vein, hepatic artery and azygos vein, liver perfusion and blood oxygenation (using transverse relaxation time (T2*) mapping). Measures were collected at baseline and at 6-7 minute intervals from 20 to 65 minutes following a test meal (440 ml; 660 kcal) in 10 healthy participants and 10 CC patients. In healthy participants, we observed a postprandial increase in portal vein flow from baseline coupled with a reduction in hepatic artery flow from baseline, reflecting the hepatic artery buffer response (HABR). In CC patients, changes in portal vein and hepatic artery flow were smaller, with no clear HABR. In healthy participants, postprandial liver perfusion increased, but not in CC patients. There was no change in liver T2* for either group. In the gas challenge study, we evaluated the blood flow in portal vein and hepatic artery, liver perfusion and liver T2* during hyperoxia and hypercapnia in 10 healthy subjects and 4 patients with compensated cirrhosis. A sequential gas delivery breathing circuit and a prospective, feed-forward gas delivery system (RespiractTM, Thornhill Research Inc., Toronto, Canada) was used to control and monitor end-tidal O2 (PETO2) and CO2 (PETCO2) partial pressures. Hyperoxia was targeted at PETO2 ~500mmHg and hypercapnia was aimed at PETCO2 ~6mmHg above resting value. The study design consisted of 5 blocks. Blocks 1, 3 and 5 were 5 min periods at resting PETCO2 and PETO2. Blocks 2 and 4 were, in a random order, 5 mins of hyperoxia (with a 2 min transition up and down) or 5 mins of hypercapnia. We observed an increase in portal vein velocity during hypercapnia among the healthy subjects and patients with cirrhosis. There was no significant changes in liver T2* but the full-width-half-maximum (FWHM) of the distribution of the liver T2* increased in response to hyperoxia and hypercapnia in both groups. Subjects with low T2* at baseline exhibited a smaller change in FWHM following the gas challenge. The within session and inter-session coefficient of variation (CoV) the blood flow measurement using phase-contrast MRI in healthy subjects was less than 15%. If our findings are confirmed in external validation studies, non-invasive MRI can be used as a surrogate measure of HVPG. Assessment of postprandial dynamic changes in hepatic, splanchnic and collateral circulation using MRI could potentially be used to stratify patients with portal hypertension and study the effects of potential novel treatments for portal hypertension.
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16

Vitish-Sharma, Parveen. "The effect of Trendelenburg positioning in laparoscopic colorectal surgery on intraocular pressure and cognitive function." Thesis, University of Nottingham, 2018. http://eprints.nottingham.ac.uk/48966/.

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Trendelenburg positioning is frequently used during laparoscopic surgery particularly when access to the pelvis is required. With improvements in laparoscopic skills, high risk patients and more complex procedures are now frequently being performed laparoscopically. (Improvement, 2016) The aim of this thesis is to investigate the effect of Trendelenburg positioning on intraocular pressure (IOP) and cognitive function. Chapters 2 and 4 look at the effect of Trendelenburg positioning on IOP. Perioperative vision loss occurs rarely but it is a life changing complication. A rise in IOP is a recognised risk factor for POVL. The incidence of POVL following laparoscopic colorectal surgery has been quoted as 1.24 in 10,000 cases. (Pinkney et al., 2012) Chapter 2 is an observational study during which IOP was monitored during laparoscopic colorectal surgery. This was correlated with the degree of Trendelenburg tilt used during surgery. This study revealed an increase in IOP occurred which was dependent on the degree of Trendelenburg tilt as well as the time spent in this position (Pearson’s correlation coefficient was 0.78). Patients undergoing left-sided colonic resections had a mean maximum IOP rise of 15.2mmHg. Chapter 4 is a follow-on study which looked at acetazolamide as a method of reducing the IOP rise that occurred whilst in the Trendelenburg position. This was a randomised placebo controlled cross-over healthy volunteer study. After 4 hours of Trendelenburg, the mean IOP increase was 3.17mmHg in the placebo group compared to 0.02mmHg in the acetazolamide group (P < 0.05). This suggests acetazolamide has a role in reducing the IOP rise that occurs from Trendelenburg positioning. The second half of this thesis focuses on the effect of Trendelenburg positioning on cognitive function. Post-operative cognitive decline (POCD) is defined as cognitive impairment following surgical intervention. It is associated with increased hospital stay, longer return to work/normal functioning, and in patients with existing cognitive impairment a further decline can result in loss of ability to carry out activities of daily living. (Moller et al., 1998) Chapter 5 is an observational study that explores the incidence of short- or long-term POCD following laparoscopic colorectal surgery. Post-resectional surgery, 55.4% of patients had evidence of POCD on Day 1 and 31.6% at long-term follow-up. On Day 2, 11.6% had POCD following right-sided resection compared to 16.3% in the left-sided resection group. Chapter 6 and 7 look at the effect of Trendelenburg positioning on cognitive function in healthy volunteers. Chapter 6 assessed changes in brain function using magnetoencephalography and n-back testing as well as looking at MRI structural changes after 2 hours in Trendelenburg position. Although the difference was not statistically significant, there was an increase in brain volume after 2 hours in Trendelenburg compared to pre-Trendelenburg MRI scan suggesting an element of cerebral oedema. Chapter 7 was a volunteer study designed to assess the effect of time spent in Trendelenburg position on cognitive function using cognitive tests (n back, stroop and lexical decision making tasks). This was carried out at regular intervals whilst in the Trendelenburg position and again once the volunteer was placed supine. After 3 hours in the Trendelenburg position, 40% had cognitive decline compared to 26.7% after 30 minutes.
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17

Prokopchuk, O. V. "Criteria for digestive system dysfunction in newborns with perinatal pathology." Thesis, БДМУ, 2022. http://dspace.bsmu.edu.ua:8080/xmlui/handle/123456789/19808.

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Lam, Ching Yin. "Biomarkers to assess an anti-inflammatory treatment for irritable bowel syndrome : mast cell assays and magnetic resonance imaging." Thesis, University of Nottingham, 2015. http://eprints.nottingham.ac.uk/28803/.

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Irritable bowel syndrome (IBS) remains a heterogeneous condition and is a common condition. The causes of IBS remain poorly understood and there is a lack in biomarkers to distinguish this condition. Recently, there have been reports on the release of immune mediators leading to symptoms of irritable bowel syndrome. Mast cells, which can be activated by allergy or stress, are thought to be important cause of symptoms in some IBS patients because they can release chemicals, which cause pain and diarrhoea. Currently, there are few effective treatments available to alleviate these symptoms. Recent small studies have shown that Mesalazine, an ‘anti-inflammatory’ drug, may be able to modify and reverse the symptoms of IBS with diarrhoea. One small study suggested Mesalazine reduced mast cell numbers. This current study is one of the largest studies looking at the use of Mesalazine as a form of treatment for IBS with diarrhoea. Unfortunately, this study did not show any beneficial effect of Mesalazine treatment in unselected patients with IBS and diarrhoea. Potentially, there is a subgroup of IBS patients who developed their symptoms following a bout of gastroenteritis who appeared to benefit from Mesalazine treatment but a larger study is needed to confirm this. In this study, the mast cell mediators released from mucosal biopsies was not a useful marker of disease since it failed to correlate with any symptoms. Magnetic resonance imaging (MRI) is a potentially useful tool to assess the physiology of the gastrointestinal tract in patients with functional gut disorders as it does not involve radiation and is not invasive. So far, there is a lack of biomarkers to assist in diagnosis and treatment of irritable bowel syndrome. The MRI marker pill used in the multiple studies in Chapter 3 to assess whole gut transit time is very promising as it is now applied, in the research setting, to patients with chronic constipation such as slow transit constipation and irritable bowel syndrome with constipation. Further use of the MRI and adding a stimulus such as laxative in patients with chronic constipation is helpful to distinguish between functional constipation and irritable bowel syndrome with constipation; thus helping with its medical management. The use of MRI as a biomarker for diagnosis of irritable bowel syndrome remains promising although it was not demonstrated in this thesis.
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19

Tobias, Amanda Jane. "Gastrin interactions which impact upon gastric, colonic, pancreatic and oesophageal carcinogenesis." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11348/.

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20

Albasri, Abdulkader. "An investigation of the role of C-terminal tensin-like (Cten) gene in colorectal cancer." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12055/.

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The C-terminal tensin-like (Cten) gene is a member of the tensin family and is localised at the cytoplasmic tail of β-integrin. It is involved in various biological events although the role of in the development of colorectal cancer (CRC) is uncertain. In order to study this, the expression of Cten during the development of CRC was initially evaluated using immunohistochemistry (IHC) on colorectal adenomas and carcinomas. Positive immunoreactivity for Cten was observed in 317/342 (92.6%) of CRC and 19/20 (90%) of colorectal adenoma. High Cten expression was significantly associated with advance Dukes stage (p=0.001), lymph node metastasis (p<0.001), extra-mural vascular invasion (p=0.001) and distant metastases (p=0.008). Survival analysis demonstrated that patients with high Cten expression had significantly shorter disease free survival (DFS) on univariate analysis (p<0.001) a trend towards Cten expression as an independent predictor of DFS on multivariate analysis (p=0.071). To further test the association with metastasis, the role of Cten in metastasis was tested by (a) intrasplenic injection of CRC cells stably transfected with Green Fluorescent Protein (GFP) tagged Cten into nude mice and (b) testing a series of primary CRCs and their metastases by IHC. Compared with control mice (injected with cells transfected with GFP empty vector), mice injected with cells expressing Cten developed larger tumours in the spleen (p=0.03) and liver (p=0.05). Compared with primary tumours, the metastatic deposits had a significantly higher frequency of nuclear localisation of Cten (p=0.002). To further investigate the potential role of Cten in metastasis, in-vitro models were used to investigate Cten function. Ectopic expression of Cten in the HCT116 CRC cell line (which expresses low levels of Cten) caused changes in cell morphology and increased cell motility (both migration and invasion). Conversely, the reciprocal Cten knock-down experiments in SW620 CRC cell line (which expresses high levels of Cten) resulted in inhibition of both cell migration and invasion. Since Cten is in complex with integrins at focal adhesions, its interactiosn with integrin-linked kinase (ILK), focal adhesion kinase (FAK) and CD24 were tested. Cten was shown to regulate ILK, FAK and CD24. Moreover, inhibition of CD24 after forced expression of Cten abrogated the Cten-mediated effects on both cell motility and protein levels of ILK and FAK. The studies were expanded and Cten expression was tested by IHC in another cancer model i.e. breast cancer (BC).Consistent with the data from CRC, increased Cten expression in BC was found to be associated with poor prognostic variables and shorter disease free survival. In conclusion, Cten expression is associated with poor prognosis in CRC and BC. This may be consequent to an ability to enhance metastasis which is related to promotion of cell motility. The activities of Cten are probably consistent across different tumour models.
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21

Brown, Matthew. "The expression of Toll-like receptors-2 and-4 by human crypt intestinal epithelial cells, intestinal myofibroblasts and putative intestinal stem cells in inflammatory bowel disease." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/13437/.

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Host-microbial interactions are of major importance in the pathogenesis of inflammatory bowel disease (IBD). Toll-like receptors (TLR) are pattern recognition receptors which recognise conserved molecular patterns derived from micro-organisms. Crypt intestinal epithelial cells (IEC) were isolated form mucosal specimens of healthy controls and patients with IBD (ulcerative colitis, UC, and Crohn's disease). A population of IEC enriched for intestinal stem cells (ISC) were identified using Hoechst dye exclusion and by their adherence to cultured primary intestinal myofibroblast cell monolayers. Compared to healthy control colon, TLR2 and TLR4 mRNA and surface protein were significantly up-regulated in crypt IEC isolated from the inflamed mucosa of UC and Crohn's colitis. Compared to healthy control ileum, TLR4 mRNA was significantly up-regulated in crypt epithelial cells isolated from the inflamed mucosa of Crohn‟s ileitis. TLR2 and TLR4 mRNA expression from histologically normal and inflamed colonic mucosa in UC did not significantly differ, and expression of TLR4 transcripts was significantly greater in crypt IEC isolated from histologically normal proximal colonic mucosal samples compared to healthy controls. Myofibroblast-adherent crypt cells expressed TLR2 and TLR4 protein to a greater level than the underlying myofibroblasts. Hoechst-effluxing putative intestinal stem cells expressed both TLR2 and TLR4 transcripts and protein, and TLR3 and TLR5 transcripts. In conclusion, crypt intestinal epithelial cells up-regulated TLR2 and TLR4 expression in UC and Crohn's colitis and up-regulated TLR4 expression in Crohn's ileitis. TLR2 and TLR4 was expressed constitutively in crypt IEC from histologically normal mucosa, suggesting differential TLR expression may in part be a primary event in UC. This provides further insights into the pathogenesis of IBD. Putative intestinal stem cells expressed TLR2, TLR3, TLR4 and TLR5, suggesting that direct microbial sensing by ISC may be important in maintaining intestinal homeostasis and in regulating ISC function.
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22

Mirams, Gary R. "Subcellular phenomena in colorectal cancer." Thesis, University of Nottingham, 2008. http://eprints.nottingham.ac.uk/10567/.

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The Wnt signalling pathway is involved in stem cell maintenance, differentiation and tissue development, and in so doing plays a key role in controlling the homeostasis of colorectal crypts. In response to an external Wnt stimulus, the intracellular levels of the protein beta-catenin are regulated by the proteins which make up the Wnt signalling pathway. Abnormalities in the Wnt signalling pathway have been implicated in the initiation of colorectal and other cancers. In this thesis we analyse and simplify existing models of the Wnt signalling pathway, formulate models for Wnt's control of the cell cycle in a single cell, and incorporate these into a multiscale model to describe how Wnt may control the patterns of proliferation in a colorectal crypt. A systematic asymptotic analysis of an existing ODE-based model of the Wnt signalling pathway is undertaken, highlighting the operation of different pathway components over three different timescales. Guided by this analysis we derive a simplified model which is shown to retain the essential behaviour of the Wnt pathway, recreating the accumulation and degradation of beta-catenin. We utilise our simple model by coupling it to a model of the cell cycle. Our findings agree well with the observed patterns of proliferation in healthy colon crypts. Furthermore, the model clarifies a mechanism by which common colorectal cancer mutations may cause elevated beta-catenin and Cyclin~D levels, leading to uncontrolled cell proliferation and thereby initiating colorectal cancer. A second model for the influence of the Wnt pathway on the cell cycle is constructed to incorporate the results of a recent set of knockout experiments. This model reproduces the healthy proliferation observed in crypts and additionally recreates the results of knockout experiments by additionally including the influence of Myc and CDK4 on the cell cycle. Analysis of this model leads us to suggest novel drug targets that may reverse the effects of an early mutation in the Wnt pathway. We have helped to build a flexible software environment for cell-based simulations of healthy and cancerous tissues. We discuss the software engineering approach that we have used to develop this environment, and its suitability for scientific computing. We then use this software to perform multiscale simulations with subcellular Wnt signalling models inside individual cells, the cells forming an epithelial crypt tissue. We have used the multiscale model to compare the effect of different subcellular models on crypt dynamics and predicting the distribution of beta-catenin throughout the crypt. We assess the extent to which a common experiment reveals the actual dynamics of a crypt and finally explain some recent mitochondrial-DNA experiments in terms of cell dynamics.
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Chen, Liqiong. "The development of a novel in vitro model of human liver for the study of disease pathogenesis." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11020/.

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The development of systems for the long term in vitro culture of functional liver tissue is a major research goal. The central limitation of experimental systems to date has been the early de-differentiation of primary hepatocytes in cultures. Several factors including cell-cell interaction, cell-matrix interaction, soluble factors and 3D structures have been identified as the keys to overcome this limitation. The first aim of this project is to compare the established 3D model, co-culture of hepatocytes and hepatic stellate cells (HSCs) on PDLLA coated surfaces, to other best available systems using collagen and Matrigel. The hypothesis is that hepatocytes functionalities, established by cell-cell interaction, 3D structures and soluble factors, can be further enhanced by introduction of cell-matrix interaction. In order to test the hypothesis, rat hepatocytes were cultured in five different systems, including monoculture of hepatocytes on collagen gel, in collagen-Matrigel sandwich, co-culture of hepatocytes and HSCs on collagen gel, in collagen-Matrigel sandwich and on PDLLA coated surface. Hepatocyte specific function assays, namely albumin secretion, urea secretion, testosterone metabolism by HPLC and CYP activities by LC-MS-MS, were used to analyze cell functionalities. Homo-spheroids were only formed in monoculture on collagen gel, but hetero-spheroids were developed in all the co-culture systems. The results of function assays showed hepatocytes in collagen-Matrigel sandwich configuration had the best secretion of albumin and urea and best CYP activities during the culture period. These data demonstrated the hypothesis that hepatocyte functions of the established model can be further improved by introduction of cell-matrix interaction. In addition to establishment of rat hepatocyte culture systems, hetero-spheroids of primary human hepatocytes and primary human HSCs on PDLLA coated plates were developed successfully, due to the great improvements of isolation and culture of primary human HSCs. However, hepatocyte function assays have not been applied yet. Hepatic cell lines have several advantages that are not applicable to primary cultured human hepatocytes, namely unlimited lifespan and stable phenotype. The immortalized Fa2N-4 cell lines have recently been assessed as replacements of primary human hepatocytes in CYP induction studies. The second aim of this study was to simultaneously characterize CYP1A2, CYP2C9, CYP3A4 and CYP2B6 induction in Fa2N-4 cells through assessment of mRNA, protein and activity endpoints for a range of prototypical compounds (previously assessed in human hepatocytes) with known positive and negative induction potential. LC-MS-MS and RT-PCR were used for assessment of activity and mRNA endpoints respectively. As a result, it is considered that Fa2N-4 cells offer a substitute for primary human hepatocytes for CYP1A2 and CYP3A4 induction but not for CYP2B6 due to lack of cytosolic CAR expression.
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24

Fleming, Catherine Mary. "The epidemiology of cirrhosis and abnormal liver function in the general population of the UK." Thesis, University of Nottingham, 2010. http://eprints.nottingham.ac.uk/11371/.

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Background Liver disease is a serious problem both in the UK and globally. While the incidence and mortality from several chronic diseases are decreasing, mortality from liver disease is increasing. As well as the medical sequelae for an individual with liver disease, in the UK the increase in chronic liver disease poses particular problems with respect to increasing hospital admissions, mortality and significant costs to the public both in terms of treatment and in loss of productivity. The increase in society of several risk factors for chronic liver disease, notably alcohol intake, obesity and type 2 diabetes, mean that these problems are likely to increase in the future. Despite these apparent problems there are surprisingly few reliable sources of data on the occurrence of chronic liver disease (cirrhosis) in the general population of the UK and the rate and consequence of disease progression particularly among ambulatory patients. Nor are their robust estimates of the prevalence of abnormal liver function tests (which may represent undiagnosed liver disease) and their associations with mortality. This thesis utilises two distinct datasets to examine separate areas of interest in the epidemiology of liver disease in the UK. The first three studies contained within this thesis are concerned with the epidemiology of cirrhosis in the general population of the UK. The second group of three studies focuses on the prevalence of elevated liver function tests in a population of older people in the UK, the demographic, clinical and lifestyle factors associated with such and the mortality following an elevated liver function test. Objectives 1. To estimate the incidence and prevalence of cirrhosis in the population of the UK 2. To describe the mortality associated with cirrhosis compared with the general population and the disease progression of cirrhosis 3. To estimate the prevalence of elevated liver function tests among people aged 75 and over in the UK 4. To describe the association between elevated liver function test and demographic, lifestyle, clinical characteristics and mortality among people aged 75 and over. Methods To examine objectives 1 and 2 I utilised the General Practice Research Database (GPRD) constructing a population based cohort of 4537 subjects with cirrhosis and 44,403 age, sex and practice matched controls. I used Poisson regression to estimate incidence rate ratios and describe trends in alcoholic and non-alcohol-related cirrhosis. Using Cox regression within an historical matched cohort design I estimated the absolute excess mortality rates and hazard ratios for mortality in people with cirrhosis compared to the general population. I described the probability of progressing from one disease state to another. To examine objectives 3 and 4 I accessed data from one arm of the Medical Research Council (MRC) Trial of Assessment and Management of Older People in the Community, a representative sample of community dwelling people aged 75 and over, totalling 15,308 participants. The prevalence of abnormal liver function was described as the proportion of study participants with elevated aspartate transaminase, alkaline phosphatase or serum bilirubin. Associations between elevated liver function and demographic, lifestyle and clinical factors were examined using multivariable logistic regression. I determined the absolute mortality rates and hazard ratios for all-cause and cause-specific mortality using a Cox proportional hazards model. Findings Epidemiology of cirrhosis (GPRD) These studies have shown an increasing trend in both the incidence and prevalence of cirrhosis in the UK with an estimated 45% increase in incidence of cirrhosis in the 10-year period studied. I estimate that 76 per 100,000 people were living with cirrhosis in 2001. Just over half of all cirrhosis was associated with alcoholism. Disease progression with cirrhosis among this mainly ambulatory population was rapid with a rate of decompensation in people with compensated disease of 5% per year and 1 in 10 dying in the first year following diagnosis. This figure increased to 25% of people dying within one year for those with decompensated disease. Mortality in subjects with compensated and decompensated cirrhosis was 93.4 and 178.0 per 1000 person years compared with only 19.2 per 1000 person years in the general control population. Following adjustment for age and sex people with compensated and decompensated disease were respectively 5 and 10 times more likely to die than the general population. Epidemiology of abnormal liver function tests (MRC cohort) Abnormalities in liver function were common with roughly 1 in 6 people aged 75 and over having at least one elevated liver enzyme, although most of these elevations were mild. A single elevated measurement of aspartate transaminase was associated with an increased consumption of alcohol and a lower age in contrast with that of a single measurement of alkaline phosphatase which showed an association with higher age and lower alcohol consumption. An elevated bilirubin measurement was strongly associated with being male. Having a single elevated liver function test was associated with a modest increase in the hazard of death compared with people with normal liver function tests (adjusted hazard ratio for death 1.27 (95% CI[1.19, 1.36]). As well as an unsurprising increase in the hazard ratio for death from liver disease, elevated aspartate transaminase or alkaline phosphatase were both associated with modest increases in the hazard of death from cancer (adjusted hazard ratios of 1.56 (95%CI[1.21, 2.01]) and 1.61 (95%CI[1.39, 1.86]) respectively). Elevated alkaline phosphatase was additionally associated with increases in the hazard of death from respiratory disease (adjusted hazard ratio 1.58 (95%CI[1.32, 1.90])) and cardiovascular disease (adjusted hazard ratio 1.34 (95%CI[1.17, 1.55])). Conclusions From my work on the incidence and prevalence of cirrhosis I estimate that a minimum of 31,000 people in the UK are living with cirrhosis, a figure which is likely to rise given increasing trends in the incidence of cirrhosis described in this thesis. The significant mortality and disease progression associated with cirrhosis means that more needs to be done to combat both the incidence and progression of this disease both on an individual and population level. Elevations in enzymes regarded as reflecting liver function are common in people aged 75 and over and in most people these abnormalities are less than 2x the upper limit of normal for the assays used. These elevations I observed are associated with both a modest increase in all-cause mortality and also with an increase in death due to specific causes. Rather than simply a marker of liver function the investigation of people with elevated liver function tests, particularly those with severely elevated tests, may lead to the identification of potentially treatable conditions that underlie death.
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25

Crooks, Colin J. "The epidemiology of upper gastrointestinal bleeding." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/13394/.

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Background There have been many conflicting changes in the prevalence of the risk factors for upper gastrointestinal bleeding and therefore it is not clear what the current trends in mortality or incidence are, nor which factors are important in driving these trends. As populations in many countries are ageing with an increasing burden of co-morbidity, this thesis investigates whether the relationship between non gastrointestinal co-morbidity and upper gastrointestinal bleeding might be an explanation for current trends. I hypothesised that non gastrointestinal co-morbidity was responsible for a large proportion of bleeds in the population and the deaths that occur following a bleed. Methodology Large scale routine population based data records were used to assess the current incidence and mortality trends of upper gastrointestinal bleeding in England, as well as more in depth studies of predictors of its occurrence and subsequent mortality. The databases were examined and compared to external sources to assess their representativeness, and methods for defining cases in linked primary and secondary care were developed. The specific questions addressed in the studies were: 1. What are the current trends and variations in occurrence of upper gastrointestinal bleeding? Incidence rates and adjusted incidence rate ratios were calculated by quintiles of socioeconomic status, age group, sex, region, and calendar year. 2. Has there been an improvement in 30 day mortality following upper gastrointestinal bleeding? A nested case control study using Hospital Episodes Statistics from England 1999-2007 examined mortality trends by age, sex, co-morbidity and type of bleed. 3. Does non gastrointestinal co-morbidity predict upper gastrointestinal bleeding? A matched nested case control study used the linked Hospital Episodes Statistics and General Practice Research Database to examine non gastrointestinal co-morbidity as a risk factor adjusted for other known risk factors for bleeding. Sequential population attributable fractions were calculated to estimate what each risk factor contributed to the disease burden. 4. What are the excess causes of death following upper gastrointestinal bleeding? Causes of death by ICD 10 category were extracted following a bleed from the linked Office for National Statistics death register. Crude mortality rates and excess cumulative incidence functions were calculated; the latter adjusted for the competing risks between different causes of death. Results 1. A higher incidence of upper gastrointestinal bleeding was observed in the north of England, but this variation was dwarfed by the variation associated with deprivation. Areas of greater deprivation had 2-3 fold higher rates of hospitalisation for upper gastrointestinal bleeding than areas of less deprivation suggesting that strong modifiable risk factors exist. 2. Over the last decade there was a 20% improvement in 28 day mortality following upper gastrointestinal bleeding, and those admitted with bleeding were increasingly older and had more co-morbidity. 3. A combined measure of non gastrointestinal co-morbidity was a significant independent predictor of upper gastrointestinal bleeding and explained a greater proportion of the burden of bleeding (19%) than any other risk factor in the population, including medications such as aspirin and NSAIDs. 4. More than half the absolute excess risk of death was due to co-morbidity not related to the upper gastrointestinal tract. Conclusions Non gastrointestinal co-morbidity both strongly predicts an event of upper gastrointestinal bleeding, and is responsible for a large proportion of the subsequent long term mortality. The magnitude of the association in the population explains both why its incidence had not decreased, and why the improvements in mortality were observed irrespective of endoscopic management or bleed type. Furthermore a bleed can be an indicator for a re-assessment of the severity of co-existing non gastrointestinal morbidity.
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26

Parsons, Caron Saeko. "The development of a patient reported outcome measure and imaging modalities in the evaluation of haemorrhoidal disease." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/28466/.

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With the advent of DGHAL and PPH, treatments that purport to work by disrupting the arterial supply of haemorrhoids, there has been resurgence in interest in the vascular theory of pathogenesis of haemorrhoids. Despite uncertainty surrounding recurrence and complication rates there has been significant uptake of the new surgical approach due to decreased post-operative pain. However this has not been matched by discussion or evaluation of how haemorrhoidal disease and successful outcome should be evaluated. This thesis evaluates different approaches to the measurement of the burden of haemorrhoidal disease to the patient. A patient reported outcome measure was designed, administered and evaluated by the investigator. Reliability, reproducibility, validity, responsiveness and acceptability have been demonstrated. Three-dimensional ultrasound was used to acquire volumetric data and power Doppler angiography from the anal canal, which was shown to be reliable. Measures of power Doppler angiography were shown to be significantly lower in healthy volunteers than in patients. This technique represents promising value as an outcome measure of haemorrhoidal disease. A dual isotope-surgical nuclear probe technique attempted to measure change in volume of haemorrhoids following rubber band ligation, however consistent results were not obtained. Magnetic resonance imaging was able to demonstrate anal cushions and haemorrhoids, and the feasibility of this method has been demonstrated.
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27

Mannath, Jayan. "Endoscopic multimodal imaging in Barrett's oesophagus." Thesis, University of Nottingham, 2013. http://eprints.nottingham.ac.uk/28688/.

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The incidence of oesophageal adenocarcinoma (OA) has increased exponentially in the western world over the past few decades. Barrett's oesophagus (BO) is a well known precursor of OA with a risk approximately 20 times more than that of background population. Regular endoscopic surveillance in patients with BO is recommended by most of the national gastroenterological societies. The advantage of Barrett's surveillance is to identify early subtle lesions which could then be managed early to avoid symptomatic and advanced cancers. The detection of such early lesions are challenging as they could be flat and inconspicuous on routine endoscopic examination. In the absence of any lesions, four quadrant biopsies every 1-2 cm of the whole length of Barrett's oesophagus is advised. This technique would map only 5-10% of the surface area of Barrett's segment and hence it is associated with significant sampling error. The improvement in electronics over the past decade has led to the production of endoscopes with better charged coupled devices and image enhancement techniques by altering the spectrum of light. This thesis examines the role of multi modal imaging in Barrett's oesophagus with a focus on detecting dysplasia and early cancer (EC). Firstly, the role of high definition (HD) imaging in routine clinical setting was studied using data from patients who have undergone Barrett's· surveillance. The yield of dysplasia by HD endoscopy was compared to standard definition (SD) endoscopy in this study. The role of narrow band imaging (NBI) with magnification in characterising abnormal lesions detected during BO surveillance was evaluated by performing a meta- analysis of clinical studies. The role of autofluorescence imaging (AFI) in Barrett's oesophagus was examined in detail with a view to understand the biological basis of autofluorescence and to improve the specificity of this technique as it is associated with significant false positive results in clinical studies. A meta-analysis was performed to identify whether AFI has a clinical advantage over white light endoscopy in detecting Barrett's dysplasia and the inter-observer reliability of this technology was studied using AFI expert and AFI non-expert endoscopists. An objective method of measuring the autofluorescence intensity was proposed as a ratio of the red to the green colour tone (AF ratio) of the area of interest. When the AF ratio of the lesion was divided by the AF ratio of the background mucosa, an AF index is obtained. A pilot study was performed to identify a cut-off value of AF index to differentiate high grade dysplasia (HGD) and EC from non-dysplastic BO. Finally, the biological basis of AF intensity was examined using APCmin mouse colonic models. This study looked into the AF ratio of the colonic mucosal lesions and correlated it with the amount of collagen and elastin in the submucosal tissue. Collagen and elastin are known to be the strongest fluorophores of the gastrointestinal tract and the question addressed is whether the low AF intensity associated with dysplastic lesions is due to the thickening of mucosa or to a reduction of collagen and elastin.
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28

Hammond, John Stotesbury. "Scaffolds for liver tissue engineering : in vitro co-culture & in vivo release." Thesis, University of Nottingham, 2009. http://eprints.nottingham.ac.uk/12556/.

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This thesis presents the development and evaluation of two applications for scaffolds in the field of liver tissue engineering. In the first study a poly (D,L lactic acid) (PDLLA) scaffold is used as a three-dimensional template for hepatocyte–hepatic stellate cell (HSC) co-culture. To enhance PDLLA ligand binding capacity scaffolds are surface modified using allylamine plasma deposition and treatment with NaOH. Primary adult rat hepatocytes and HSC are then seeded onto these scaffolds and cultured in static conditions. Scanning electron microscopy (SEM) is used to assess mono-culture and co-culture morphology whilst synthetic and cytochrome P450 function are measured using albumin and testosterone assays. The second study explores the potential for intrahepatic growth factor and extracellular matrix (ECM) delivery from a biodegradable polymer scaffold to promote liver growth and to enhance regeneration. The study is undertaken in rats. The scaffold design and implantation technique are first piloted in a short survival study. Hepatocyte growth factor (HGF), epidermal growth factor (EGF), fibroblast growth factor (FGF)1, FGF2 and liver derived ECM (L-ECM) are then loaded into poly(lactic-co-glycolic acid) (PLGA) + 5% poly(ethylene glycol) (PEG) scaffolds and implanted into normal and partially hepatectomised liver. Implant morphology is assessed by micro-CT reconstruction. Growth factor bioactivity and release are confirmed by in vitro profiling. Liver growth and volume redistribution are assessed by liver weight analysis. Parenchymal injury and function are quantified by measuring serum aspartate aminotransferase (AST) & bilirubin. 5-bromodeoxyuridine (BrdU) inclusion & MIB-5 immunohistochemistry (IHC) are used to identify hepatocyte and non-parenchymal cell proliferation. Liver-scaffold interaction is characterised by H&E and Masson’s trichrome staining. Non-parenchymal cell migration is assessed by ED-1 and desmin IHC. All histology is then subjected to image analysis.
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Stevenson, Diane J. "P2X7, inflammation and gastrointestinal disease." Thesis, University of Nottingham, 2008. http://eprints.nottingham.ac.uk/28897/.

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The inflammatory bowel diseases, ulcerative colitis and Crohn's disease are characterised by spontaneously relapsing and remitting inflammation, associated with increased mucosal levels of the inflammatory cytokine, interleukin-1 (IL-1)β. IL-1β processing and release is mediated by ATP stimulation of the purine receptor, P2X7. P2X7 is a membrane ion channel highly expressed in immune cells. Signal transduction occurs via rapid cation exchange, plasma membrane depolarisation and increased intracellular calcium. Additionally, prolonged or repeated P2X7 stimulation leads to formation of a non-selective membrane pore permeable to small molecules, and ultimately to cell death. The aim of this project was to investigate the properties of the P2X7 receptor in mononuclear cells, to show that it is associated with IL-1β release in the colon, and that this release can be modified by P2X7 antagonists. Studies of ethidium bromide uptake, a functional assay, showed that P2X7 receptors are present on LPMCs and displayed properties similar to those of PBMCs and THP-1 cells. P2X7 receptor-stimulation released mature IL-1β from LPMCs in a dose-dependent manner that, in IBD patients, matched the severity of their inflammation, and could be markedly reduced by P2X7 antagonists. P2X7 stimulation also results in increased exposure of phosphatidylserine on the outer cell membrane (PS flip), often considered to be a marker of apoptotic cell death. P2X7-stimulated PS flip however is reversible and is not associated with cell death following brief stimulation times. Cell death caused by longer stimulation did not have features of apoptosis, was more evident in monocytes than lymphocytes, with LPMCs being less susceptible than PBMCs and THP-1 cells. These studies have shown that the P2X7 receptor is intimately involved in the release of IL-1β from human colonic mononuclear cells, that the release is greater in cells from IBD tissue and can be markedly inhibited by P2X7 antagonists.
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30

Crusz, S. A. "Lectin-mediated biofilm maturation, quorum sensing and Pseudomonas aeruginosa infections in cystic fibrosis." Thesis, University of Nottingham, 2009. http://eprints.nottingham.ac.uk/10734/.

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Chronic infections with Pseudomonas aeruginosa are primarily responsible for the decline in lung function and ultimate mortality in cystic fibrosis patients. The overall aim of this project was to elucidate some of the molecular mechanisms governing the pathogenesis of P. aeruginosa in the cystic fibrosis lung. This was with particular reference to (a) quorum sensing, a cell-to-cell communication system controlling the production of virulence determinants in a population density dependent manner using diffusible signal molecules and (b) the pseudomonas lectins, LecA and LecB, which are known to contribute to biofilm formation. Serial sputum samples were collected from adult and paediatric patients with cystic fibrosis and a cohort of clinical P. aeruginosa isolates was assembled. Using bioreporters, these isolates were shown to synthesise a range of quorum sensing signal molecules. Furthermore, the direct detection of these P. aeruginosa products from infected sputum samples in conjunction with patient clinical data implied an association between sputum quorum sensing signal molecule level and cystic fibrosis disease status, response to intravenous antibiotics and the presence of non-culturable P. aeruginosa. Quorum sensing also makes an important contribution to P. aeruginosa biofilm maturation, antibiotic tolerance and resistance to host defences. There is evidence that the quorum sensing regulated lectins LecA and LecB contribute to biofilm development and this was investigated using different biofilm assays, including the flowchamber biofilm system. This work demonstrated that LecA contributed to biofilm maturation in both laboratory and clinical strains and hydrophobic galactosides were shown to be able to inhibit biofilm development. The putative biofilm target ligand for LecA was tentatively identified as the Psl exopolysaccharide. Mutants deficient in either lecA or lecB produced defective biofilms, which could be inhibited and/or dispersed by galactosides or furanosides respectively, including novel synthetic furanoside dendrimers. The latter proved inhibitory to both laboratory and clinical P. aeruginosa isolates and constitute a potential novel therapeutic.
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31

Royal, E. L. "Interplay between hypoxia and gastrin in gastrointestinal cancer." Thesis, University of Nottingham, 2009. http://eprints.nottingham.ac.uk/10805/.

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Tumour hypoxia has been linked to increased resistance to both radiotherapy and chemotherapy, especially in solid metastatic GI tumours. Under hypoxic conditions, genes that promote tumour growth and survival are up-regulated, via the transcription factor hypoxia-inducible factor-1 (HIF-1). The digestive hormone gastrin, which is often over-expressed in GI cancers, has also been shown to act as a pro-survival factor, up-regulating processes such as tumour proliferation, angiogenesis and migration, and down-regulating apoptosis. Due to the high level of similarity between the downstream events mediated by the two proteins, the relationship between gastrin and HIF-1 was investigated. HIF-1α nuclear protein expression was inducible under hypoxic conditions, which led to an expected increase in VEGF gene expression, followed by a 12-50 fold increase in hypoxic gastrin mRNA expression. HIF-1α expression and transcriptional activity were not consistently affected by exogenous gastrin. RNA-interference-mediated knockdown of HIF-1α resulted in a 40-60% down-regulation of gastrin gene expression under hypoxic conditions suggesting that HIF-1α is partially responsible for gastrin up-regulation in hypoxia. Potential hypoxia-response elements (HREs) were identified within the gastrin promoter, but were only partially responsive to hypoxic incubation in GI carcinoma cells in luciferase-reporter assays. Other possible mechanisms that may account for the increased gastrin gene expression induced under hypoxic conditions include interactions of gastrin with other transcriptional regulators, either in synergy with or independent from HIF-1, or the sequestration of gastrin within the cell by ‘P’-bodies or RNA-binding proteins. These findings may indicate that the addition of anti-gastrin agents such as CCK-2 receptor antagonists or gastrin immunogens to the treatment regime of patients with solid GI tumours may be clinically beneficial, especially if combined with agents used to reduce radiotherapy and chemotherapy resistance.
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32

Abdul, Rahim Roslina. "Molecular mechanism underlying the pathogenesis of NAFLD and NASH." Thesis, University of Nottingham, 2011. http://eprints.nottingham.ac.uk/12074/.

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Pioglitazone (PGZ) is a peroxisome proliferator-activated receptor (PPAR)-γ agonist that improves peripheral insulin sensitivity and reduces hepatocellular injury/ inflammation in non-alcoholic steatohepatitis (NASH). However, the underlying hepatic mechanism of action is not clearly understood with PGZ treatment. Therefore, liver biopsies were used to study genes, protein and immunohistochemistry expression of hepatocyte and hepatic stellate cell markers. PGZ decreased both αSMA and PPAR β expression in stellate cell and PPAR α hepatocyte expression, hence inhibit both stellate cell activation and β-oxidation in hepatocytes. PGZ also inhibit cell proliferation by reducing both PCNA and Ki67 in the liver. Up regulation of PPAR β, PXR, LXRα, IkBα and TNFRSF1B were observed (using Taqman Low density array gene expression), which indicates that PGZ exerts an anti-inflammatory and anti-fibrotic effect. PPAR β, PGC1α, ACADVL and UCP2 up regulation lead to increase β-oxidation and reduced reactive oxygen species (ROS) production. LXRα, ChREBP and SREBP1C activation leads to lipogenesis in the liver was also observed in PGZ treatment group. In vitro study was also conducted in freshly isolated human hepatic stellate cells, where these cells were incubated in vehicle and 5μM of PGZ for 72 hours consecutively. After 24 hours, 15ng/ml of PDGF-BB was incubated for 48 consecutive hours, followed by cell proliferation and gene expression analysis. PGZ up regulate the adipogenic genes expression followed by reduction in stellate cells marker expression and cell proliferation induced by PDGF-BB. PPAR β elevation after PGZ treatment in human liver and HSCs culture are novel findings in this study, but the role of PPAR β is clearly unknown in the liver and stellate cells. Therefore, similar treatment was performed using PDGF-BB on freshly isolated human HSCs, followed by treatment with PPAR β agonist (GW0742). GW0742 restores the adipogenic genes expression maintaining the quiescent phenotype of the stellate cell in contrast to previous study where PPAR β has been reported to cause stellate cells activation and proliferation. Overall, PGZ improved injury and fibrosis, inhibiting cell proliferation, increased both lipogenesis and β-oxidation in NASH patients. PGZ also inhibit stellate cells activation and proliferation and up regulated the adipogenic genes.
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33

Maxwell-Armstrong, Charles Alan. "Studies using the anti-idiotypic monoclonal antibody 105AD7 in patients with advanced and primary colorectal cancer." Thesis, University of Nottingham, 1998. http://eprints.nottingham.ac.uk/14137/.

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Introduction. The anti-idiotypic monoclonal antibody 10SAD7 mimics the tumour associated antigen 791T/ gp72, present on approximately 80% of colorectal cancer cells. A Phase I study using 10SAD7 in 13 patients with advanced colorectal cancer has shown that it is nontoxic, and conferred a survival advantage on patients who received it [Denton GWL 1994]. Aim. There were two aims of this work. The first was to assess whether. the survival advantage seen in the Phase I study was reproducible in a Phase II study. The second was to immunise patients with primary colorectal cancer, in a non-randomised adjuvant study, and explore further the immune responses generated. Materials and Methods. Patients with advanced colorectal cancer were recruited to a randomised, double-blind, placebo controlled survival study. The first patient was recruited to this Phase II study in April 1994, and the last in October 1996. Four trial centres were used- Nottingham, Hull, Leeds, and Newcastle. Eligible patients had a life expectancy of 3 months, and none had received radiotherapy or chemotherapy in the preceding 1 and 3 months, respectively. Patients attended on 3 occasions, 6 weeks apart, receiving 10µg of 10SAD7/alum i.d. followed by 100µg i.m. Venous blood was assayed for blood count and differential, liver function, urea and electrolytes, and CEA. Chest X-rays and CT scans were performed at trial entry and week 12 where possible. Dates of death were recorded following consultation with General Practitioner or referring clinician. In addition, patients with primary colorectal cancer were recruited to a non-randomised adjuvant study, whereby they received 10SAD7 before surgery. Venous blood samples were taken between immunisation and operation, and assayed for lymphocyte subsets. Samples taken from resection specimens were analysed immunohistochemically. Fresh tumours were in addition disaggregated, and separated TIL labelled with a panel of monoclonal antibodies, and analysed by flow cytometry. Control tumours were similarly labelled. All analysis was performed blind. Results. 162 patients were randomised to the Phase II study, between April 1994 and October 1996. 85 received 105AD7 and 77 placebo. The mean ages and sex-ratios of the two groups were comparable, as was the time from diagnosis of advanced disease to trial entry (172v179 days). Median survival from date on study was 124 and 184 days, in 105AD7 and placebo arms, respectively (p=O.38). Survival from date of diagnosis of advanced disease was 456 and 486 days (p=O.82). Chemotherapy and radiotherapy all prolonged survival in a multivariate analysis. Only one serious adverse event was seen in the 105AD7 arm, and this was felt unlikely to be attributable to the vaccine. Twenty-four patients were recruited to the adjuvant study. Immunohistochemical analysis of tumour sections from 16 patients showed increased infiltration of CD4 and CD8 expressing lymphocytes, relative to a well matched control group (p
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Card, Timothy R. "A contribution to knowledge of the aetiology and indirect impact of inflammatory bowel diseases : (based upon analysis of routinely and semi-routinely available data)." Thesis, University of Nottingham, 2004. http://eprints.nottingham.ac.uk/12445/.

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The incidence of the idiopathic inflammatory bowel diseases ulcerative colitis and Crohn’s disease appears to have risen markedly during the 20th century. These diseases now account for a considerable proportion of the workload of gastroenterologists in the developed world, and may affect as much as 1% of the population at some point in their lives. The aetiology of these diseases has been subject of much research over a number of decades and it is clear that both genetic and environmental factors are involved. The certain knowledge of environmental risk factors however remains scant. Similarly although inflammatory bowel diseases cause considerable morbidity and a small amount of mortality for their sufferers directly there is little agreement as to their overall impact once indirect effects are accounted for. This thesis contains studies contributing to the knowledge of both these areas using routinely or semi-routinely collected data. It examines two hypotheses relating to the aetiology of IBD (that risk is related to the season of birth, and that it is related to antibiotic use), and two areas of the impact of the diseases (overall mortality and fracture risk). With regard to aetiology the studies described show no variation in the risk of IBD with season of birth. They do show an increase in risk associated with the use of antibiotics, but since this is not specific (it is seen to occur with other groups of drugs also) it is far from clear that the association is causal. With regard to the indirect impact of the diseases a significant excess in overall mortality is demonstrated which is greater in Crohn’s disease than in ulcerative colitis, and is greatest in relative terms in the young but in absolute terms in the elderly. An excess is also shown for hip fractures in those with inflammatory bowel diseases, which is only partially explained by the use of corticosteroids.
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Gemmill, Elizabeth H. "Minimally invasive gastro-oesophageal surgery for cancer : current evidence and practice." Thesis, University of Nottingham, 2012. http://eprints.nottingham.ac.uk/55424/.

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Background Since its introduction in the early 1990s, minimally invasive gastro-oesophageal surgery for cancer has been growing in popularity. Despite this, published evidence on this type of technique is weak and its role in the management of gastric and oesophageal cancer remains controversial. Aims The aim of this thesis was to test the hypothesis that: minimally invasive gastro- oesophageal cancer surgery has superior outcomes compared to control studies of conventional open surgery; but current studies are methodologically inadequate to confirm this. Methods The first study (chapter 3) is a systematic review of the literature on minimally invasive gastro-oesophageal cancer surgery, outlining the differences between literature published in Eastern and Western countries. The following 3 chapters outline and use a phase II surgical study to obtain data on minimally invasive gastro-oesophageal cancer (MIGOCS.) The MIGOCS group was set up in 2005 amongst UK surgeons. An online database was developed to enable data collection and comprises 5 sections: demographics; pre-operative staging and assessment; surgical intervention; post-operative course; pathology and clinical outcome. The first study is retrospective collecting data up to December 2006; the second study is prospective with data obtained between December 2006- July 2008 from centres around the UK utilising the MIGOCS database. Chapter 7 involves analysis of the learning curve in laparoscopic gastro-oesophageal cancer surgery using CUSUM (continuous surveillance monitoring) assessment. By studying operative time at each centre, improvement or deterioration in quality were detected. Results The systematic review of minimally invasive gastro-oesophageal surgery consists in the majority of case reports, with no randomised controlled trials of oesophagectomies and 4 (low quality) randomised controlled trials of gastrectomies. It demonstrates a mortality and morbidity of 2.3% and 46.2% respectively for oesophagectomies; 0.1% and 12.7% respectively for gastrectomies. Data from this review suggests that the minimally invasive approach is beneficial compared to open surgery in terms of reduced mortality, respiratory complications, blood loss and quicker return to a good quality of life (but not reduced hospital stay as expected.) There are currently 60 MIGOCS member consultant surgeons from over 40 UK centres. The retrospective study obtained data from 7 UK centres with an overall mortality and morbidity of 6.0% and 57% respectively for oesophagectomies and 7.7% and 13% respectively for gastrectomies. The prospective study collected data from 7 UK centres, comprising a total of 258 minimally invasive oesophagectomies and 33 minimally invasive gastrectomies. Overall mortality and morbidity were 2.5% and 56.6% respectively for oesophagectomies and 10.8% and 27.3% respectively for gastrectomies. CUSUM analysis varied considerably between centres. The two larger volume centres however demonstrated an improvement in their operative time with experience, with a possible pateau at around 30 procedures. Conclusions Published data suggests that the minimally invasive approach to gastro-oesophageal cancer has advantages over conventional open surgery. Data collected in this thesis does not overwhelmingly support published evidence, but does demonstrate that this technique is both safe and feasible even during the early part of a surgeon's learning curve. It is the first study to provide an insight into outcomes of this type of surgery in a multicentre setting in the UK; and has made progress towards a randomised controlled trial.
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36

Ingram, Richard J. M. "Duodenal ulcer promoting gene : effects on the pathophysiology of Helicobacter pylori infection and the host immune response." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/42105/.

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Duodenal ulceration remains a significant problem. Helicobacter pylori infection is the major cause of duodenal ulcers (DU). Worldwide, around 1 in 2 people are chronically colonised by H. pylori, most of whom will not develop gastroduodenal disease, though some will develop DU whilst others will develop gastric ulcer or gastric carcinoma. The risk of disease is related to bacterial virulence factors, host and environmental factors. Disease-specific bacterial risk factors have not been established. Duodenal ulcer promoting gene (dupA) was proposed as a disease-specific bacterial virulence factor for DU. This study aims to examine the influence of dupA on clinical outcomes and whether these are epidemiologically consistent, if the dupA status of clinical isolates correlates with the key pathogenic features of DU in vivo, and if there is a biologically plausible role in disease through effects on host immune responses. Results showed that the influence of dupA status on clinical outcomes was not specific to DU, but rather that epidemiological associations link dupA with an increased risk of gastroduodenal diseases in general for some populations. The dupA status of clinical isolates did not correlate with the key pathogenic features of DU in vivo, though there was some evidence of increased gastric mucosal inflammation in association with dupA+ strains. Experimental results using a human blood immune cell model show that monocyte-derived cells mediate a more pro-inflammatory response through interaction with the dupA system. This might be the mechanism that explains the in vivo associations of dupA with inflammation and disease. In this study population, dupA did not satisfy the criteria of a true virulence factor that promotes duodenal ulceration. The assertion of this thesis is that dupA is misnamed.
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Ham, Karom Hersh Abdul. "Investigate tumour heterogeneity and genetic pathways in colorectal cancer." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/42487/.

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Background: Colorectal cancer is a major global public health problem that has predominantly been considered a genetic disease following a precise series of molecular events. These are characterized by sequential accumulation of genetic and epigenetic alteration in several oncogenes and tumour suppressor genes. Understanding of the genetic mechanisms that explain the initiation and evolution of colorectal cancer are key to improving risk prediction, prognostication and treatment. Aims: The aim of this study was to understand the basic principles of the molecular biology of colorectal cancer based on genomic, transcriptomic, and proteomic profiles analyses. Methods: DNA extracted from 147 formalin fixed paraffin embedded (FFPE) samples from 83 patients with colorectal cancer (first cohort) including (83 primary colorectal cancer (CRC), 22 matched liver metastases, 25 matched biopsies and 17 normal colon tissue) were screened for mutation in 26 genes (Trusight tumour kit) using Targeted Next Generation Sequencing. Additionally, exonuclease domain region of POLE and POLD1 were also screened using High Resolution Melting and Sanger Sequencing methods. These data used to: Investigate mutation profiles of CRCs among 83 primary samples. Investigate the difference between chromosomal instable-CRC (46 primary sample) and chromosomal and microsatellite stable-CRC (35 primary sample). Compare mutations in 26 genes of 25 paired biopsy samples and corresponding resection specimens. Investigate genetic discrepancies between 22 primary colorectal cancers and their respective metastases. Additionally, expression of a panel of six miRNAs (miR-20a, miR-21, miR-29a, miR-31, miR-92a and miR-224) was measured using RT-qPCR and protein expression of 20 genes was measured using Reverse Phase Protein Array (RPPA). In a second cohort including 81 primary CRC and their matched normal samples, expression of the six miRNAs and mRNA of six genes (SMAD4, PTEN, BCL2, TGFBRII, KLF4 and RASA1) targeted by the six miRNAs were measured using RT-qPCR. Additionally, expression of proteins of the targeted six genes was also measured using immunohistochemistry (IHC). Cell-free DNA (cfDNA) extracted from 16 blood samples (third cohort), which were taken from 5 CRC patients at different time points (pre- and post-surgery) were screened for mutations in KRAS, TP53, PTEN, SMAD4, BRAF and PIK3CA genes. Additionally, expression of the six miRNAs was measured using RTqPCR. Results: In the first part; investigating mutation profile of the first cohort 83 CRC showed high frequency of mutation in TP53 (75%), APC (57%) and KRAS (53%). Approximately 93% CRCs have mutation in at least one of APC/TP53/KRAS/BRAF/SMAD4/PIK3CA/PTEN/FBXW7 genes. Moreover, mutations were found in the exonuclease domain regions of POLE in 9.6% and POLD1 in 2.4%. Regarding biopsy vs resection, the mutant allele frequency was 1.03-fold higher in resection specimens than biopsies and there was no mutation in the biopsy specimens that were not seen in the resection specimens. In the second part; Comparison of CIN-CRC vs MACS-CRC, which were included in the first cohort CRCs showed similar mutation frequencies of mutation in all 28 genes except KRAS (41%CIN vs 68%MACS), POLE (15%CIN vs 2%MACS), GNAS (0%CIN vs 11%MACS). Statistically there was a significant difference (each p=0.01) which was lost following multiple testing correction. In the third part; comparison of primary CRC vs matched metastasis showed that a total of 61 non-synonymous somatic variations in 12 genes were found in primary 22 specimens whereas 60 were found in metastasis cases. The mutant allele frequency was 1.01-fold higher in primary than metastasis CRCs. Evaluated expression levels of six miRNAs and protein expression of other 20 genes, did not show any significant differences between primary CRC and matched metastasis. In the fourth part; Expression of the six miRNAs and mRNA and protein of the six targeted genes were tested in the second cohort 81 samples. Statistical analysis revealed significant increase in the expression level of miR-20a (p=0.04), miR-21 (p=0.01) miR-29a (p=0.03) and miR-31 (p=0.01) and decrease in the mRNA expression level of TGFBRII and RASA1 in tumour samples compared to normal tissues. IHC staining showed low expression level of SMAD4 in 51 (63%), PTEN 67 (83%), TGFBRII 65 (80%), RASA1 61 (75%) BCL2 47 (58%) and high expression of KLF4 36 (44%). High miR-21 and miR-224 expression were associated with low expression of TGFBRII. In addition, over expression of both miR-29a and miR-31 inversely correlated with RASA1. In the fifth part; Mutation in the cfDNA was detected in 5 cases. Two of these showed a loss of the mutant signal post-operatively. Whereas the mutant signal was persistent in the rest 3 of the cases for all the samples taken post-operatively. Although miRNAs expression was fluctuated between these time points, paired test showed a non-significant difference when comparing pre- and post-surgical miRNAs level. However, level of the cfmiRNAs is changed by more than 2 folds (upregulated) in the day of surgery compared to normal plasma as follow, miR- 20a in 1/5 (20%), miR-21 in 4/5 (80%), miR-29a in 3/5 (60%), miR-92a in 4/5 (80%) and miR-224 in 2/5 (40%). Conclusion: Investigation profiles of CRCs from both cohorts indicated that, different mutated genes and upregulated miRNAs, which are involved in different signalling pathways, may have roles in CRC carcinogenesis. Significant difference was neither noticed between MACS and CIN group and nor between primary and metastasis tumour. miRNAs from tissues and cfmiRNAs from plasma, can differentiate CRC from healthy group and have potential clinical value in early CRC detection. In addition to the resection specimens, the study found that it is acceptable to use biopsy material for predictive testing and cfNAs can be used for a variety of clinical and investigational applications.
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Onyido, Emenike K. "Mechanisms and roles of FLYWCH1 in colorectal cancer." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/42558/.

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Human colorectal cancer (CRC) is the fourth most common cause of cancer-related death in the UK and worldwide. Defects in conserved signalling pathways play key roles in the development of almost all cancers and, in CRCs, over 80% of tumours show hyper-activation of the canonical Wnt signalling pathway. This pathway, through the transcriptional activity of β-catenin (and its binding partner, TCF4), maintains the stem cell compartment of colon and intestinal-crypts as well as cancer-initiating cells. Whilst the role of β-catenin/TCF4 in the development of both normal and neoplastic colon/intestinal tissues is well documented, the molecular basis of these functionally distinct nuclear-transcriptional programs is still under investigation, hence the functional elucidation of nuclear cofactors that interact with nuclear β-catenin contribute to further unravel the mechanisms involved in the β-catenin-mediated nuclear transcription. In addition to LEF/TCFs, interaction of β-catenin with a plethora of other transcriptional co-activators and/or co-repressors remains vital for gene regulation. To this end, our lab have been dedicated to identifying -catenin/TCF4 interacting partner proteins (CIPs) capable of fine-tuning the Wnt-level in CRC cells. Among other CIPs, my proposed project was focused on FLYWCH1, a totally novel protein with a FLYWCH/Zn-finger DNA-binding domain, called “FLYWCH”. Previous data in our lab demonstrated that FLYWCH1 preferentially binds the nuclear/ un-phosphorylated--catenin whilst -catenin is still bound to TCF4 (Muhammed et al., submitted). Muhammed et al., found that FLYWCH1 is able to modulate transcription of many -catenin target genes including the stem cell marker (Lgr5) and genes that are associated with migration and invasion of CRC cells. They also showed that FLYWCH1 mRNA expression is restricted to a subpopulation of tumour cells in both human CRCs and ApcMin model mouse for intestinal cancer via in-situ hybridization (ISH). However, prior to these almost nothing was known about the FLYWCH1. In my research project it was proposed, to build on these advances in FLYWCH1,Wnt and CRC, and to undertake a cell & molecular research program on the role of the FLYWCH1-transcription regulator in potential suppression of colon cancer via direct regulation of microRNAs. However, commercially available FLYWCH1-antibodies worked endogenously only for immunocytochemistry/immunofluorescent (IF), but not for immunohisto-chemistry (IHC) and Western blotting analysis. Here we provide evidences via FLYWCH1 overexpression and shRNA knockdown in cultured fibroblast (TIG119) and CRC cell lines that FLYWCH1 possess tumour suppressor functions mainly by; i) Inhibition of cell migration via modulating actin cytoskeleton re-modelling and stress fibre formation and by targeting E-cadherin suppressor ZEB1. ii) Inhibiting cancer stemness in-vitro (colonosphere assays), by modulating the Wnt/β-catenin signalling pathway and possibly in a GSK-3β dependent manner. iii) The localization of FLYWCH1 speckled nuclear with splicing factor SC-35 foci, a potential mechanism involved in regulation of miRNA expression. iv) Positively regulating the expression of let-7 miRNA expression via modulating the LIN28A and LIN28B subcellular distribution. We also showed that FLYWCH1 expression is correlated positively with let-7 miRNA expression in primary colorectal cancer samples and matched metastases from patients. While we are currently striving for obtaining substantial knowledge about FLYWCH1 function in-vivo and mechanistic insight into the regulatory circuitry of FLYWCH1/miRNAs, collectively, our data suggest that FLYWCH1 possesses tumour suppressor activity and may exert its influence on cancer cells homeostasis through miRNA regulation.
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Shaheen, Sameerah. "Roles & mechanisms of NANOG-mediated drug resistance in human colorectal cells." Thesis, University of Nottingham, 2017. http://eprints.nottingham.ac.uk/43364/.

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NANOG is a transcription factor that functions as a central regulator of pluripotency and determines the cell fate in embryonic stem cells (ESCs). Dr Nateri’s laboratory and others have reported the expression of embryonic NANOG in a small subpopulation of cells in several human cancers including human colorectal cancer (CRC). NANOG positive CRC cells express the normal intestinal stem cell marker LGR5, as well as the induced pluripotent stem (iPS)-linked transcription factors SOX2, c-MYC, and high β-catenin. These cells strongly resemble a small subpopulation of poorly differentiated cancer stem cells (CSCs). Recent studies have reported that, in CSCs, the self-renewal and survival signals are dominant over the differentiation signals. This suggests that NANOG is an essential modulator of drug resistance complexity and tumour heterogeneity in cancer cells. Dedifferentiation is an established hallmark of carcinogenesis and is accompanied by key signalling pathways mediated in drug resistance, such as the mitogen-activated protein kinase (MAPK) and glycogen synthase kinase-3β (GSK-3β)/β-catenin pathways, via epithelial–mesenchymal transition (EMT). However, the gain of stemness and the concomitant loss of differentiation might affect and alter the signalling pathways exclusive to NANOG-expressing cells that are required for drug resistance, not fully studied. Therefore, we aimed to characterise the mechanism by which CSCs gain self-renewal ability and lose differentiation ability through NANOG. Hence, this study was initially focused on testing the role of NANOG in chemotherapy drug resistance in human CRC cells, using 5-fluorouracil (5-FU)–treated HCT116 cells stably overexpressing exogenous human NANOG (HCT116GFP/NANOG) and control GFP expressing cells (HCT116GFP). We show that NANOG overexpression promotes 5-FU resistance in HCT116GFP/NANOG cells versus HCT116GFP cells (Chapter 3). To define the possible downstream regulatory pathways directly or indirectly mediated by NANOG, we examined the MAPKs; JNK (Jun-N-terminal kinase), ERK1/2 (extracellular signal–regulated kinase) and Wnt/β-catenin signalling pathways through GSK-3β association with β-catenin in HCT116GFP/NANOG cells versus HCT116GFP cells, and if NANOG mediated in activation of the EMT (epithelial–mesenchymal transition). Our data show that overexpression of NANOG protein in the CRC cells mimics previously reported ESC differentiation mechanism mediated by induction of the phosphorylated ERK1/2 (p-ERK1/2) expression and phosphorylated GSK-3β (p-GSK-3β) at Ser9 (Chapter 3). Consequently, NANOG overexpression increases the β-catenin and represses the E-cadherin, while significantly increases the vimentin level, leading to EMT activation (Chapter 4). In this study, we have also demonstrated NANOG induction of the EMT signature, results increasing activity of symmetrical cell division, while reducing differentiation of HCT116 derived colonosphere formation (Chapter 5). Taken together, this study describes the mechanisms of NANOG induction of EMT and NANOG sustainment of CSC-like traits via activation of the ERK/GSK-3β/β-catenin pathways in CRC cells. These findings highlight the specific mechanism of action of the NANOG-CSC signalling pathways in human CRC tumour heterogeneity, in which it might eventually identify promising approaches to cancer treatment via selectively targeting of CSCs.
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40

Poghosyan, Anna. "Molecular mechanisms of enhanced expression of the chemokine interleukin 8 (CXCL8) in cystic fibrosis (CF) airway epithelial cells." Thesis, University of Nottingham, 2014. http://eprints.nottingham.ac.uk/27801/.

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Cystic fibrosis (CF) is a fatal disease caused by a mutation of the CFTR gene and severe inflammation of the lungs. The inflammatory process is characterised by increased production of the potent neutrophil-attracting chemokine interleukin 8 (CXCL8), but the mechanism responsible is poorly understood. We tested the hypothesis that altered epigenetic regulation is responsible for the basal and cytokine-induced CXCL8 upregulation in CF airway epithelial cells. We found that CXCL8 protein levels and mRNA expression were higher in CF as compared to normal cells both basally and following cytokine stimulation. The difference in the expression was independent of increased mRNA stability or increased transcription factor activation and/or expression in CF cells. We found increased basal, but not cytokine-induced transcription factor binding to the CXCL8 promoter in a chromatin environment in CF cells in comparison with normal cells, increased histone H3 lysine 4 trimethylation, hypomethylation of CpG sites and increased binding of BRD3 and BRD4 to the CXCL8 promoter. Disruption of BRD4 association with chromatin using the selective BET bromodomain inhibitor JQ1 decreased CXCL8 protein release from CF cells to the levels observed in normal cells. Our observations suggest that epigenetic alterations are responsible for the upregulation of CXCL8 in CF and could become potential targets in the development of new therapeutic strategies.
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41

Saini, Francesca. "Role of the Hedgehog pathway in the pancreatic tumour microenvironment." Thesis, University of Nottingham, 2014. http://eprints.nottingham.ac.uk/27738/.

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Pancreatic cancer is a solid tumour with poor prognosis and ineffective therapeutic approaches. The role of the tumour microenvironment in supporting pancreatic tumour growth and metastasis has been demonstrated. In particular, a new concept of the stem cell niche as a mixed population of mesenchymal stem cells and niche cells (myofibroblast cells) involved in promoting these processes is emerging. Paracrine transmission of the Hedgehog (Hh) pathway in the pancreatic tumour microenvironment has previously been reported. This project aimed to identify the stromal pancreatic cancer cells able to respond to Hh pathway exogenous stimuli and to investigate their relationship to tumour progression, in order to define new targets for pancreatic cancer therapies. Gene and protein expression analysis in pancreatic primary tumours demonstrated overexpression of Shh ligand not only at the epithelial but also at the stromal level in advanced stages of pancreatic cancer. In vitro modelling of the mesenchymal stem cell niche (mSCN) using human bone marrow-derived mesenchymal stem cells (MSCs) co-cultured with cancer-associated fibroblasts (CAFs) or myofibroblast-like cells (MF-like, obtained by treating MSC with TGFβ) showed up-regulation of Shh gene and protein expression in comparison to single stromal cell populations (MSCs; CAFs and MF-like cells). The investigation of Hh paracrine signalling in pancreatic tumour microenvironment using different 2D in vitro assays (transwell and direct co-cultures, NShh treatments and tumour condition media cultures) demonstrated the inability of CAFs and MSCs, when grown in culture conditions that prevent their activation (increase of αSMA), to respond to Hh exogenous stimuli and suggested the mSCN as the stromal context in which paracrine induction of the Hh pathway takes place. Taken together, these results suggest a new concept: Shh expression is an indicator of the mSCN in the pancreatic cancer microenvironment and that its role as a possible target for chemotherapy should be explored.
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Garsed, Klara C. "Predicting the response to Ondansetron, a 5HT3 receptor antagonist, in irritable bowel syndrome with diarrhoea : the utility of clinical features and objective biomarkers." Thesis, University of Nottingham, 2014. http://eprints.nottingham.ac.uk/14332/.

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Background: Patients with diarrhoea predominant irritable bowel syndrome (IBS-D) suffer from loose frequent stools with associated urgency and fear of incontinence. Relief from these symptoms is an important unmet need. The 5-HT3 receptor antagonist Alosetron has been shown to increase stool consistency, decrease urgency and reduce abdominal pain leading to a global increase in satisfaction with treatment [1]. Its use is restricted following an increased incidence of ischaemic colitis and this agent is not available in Europe. The serine proteases family of proteolytic enzymes have been identified as the source of increased faecal proteolytic activity in patients with IBS. These enzymes may be mechanistically important via their action on the Protease activated receptor (PAR) -2, inducing increases in permeability and hypersensitivity. Aims: To assess the efficacy of the commonly prescribed 5-HT3 antagonist Ondansetron, in patients with IBS-D and to identify biomarkers that might allow us to predict response defining an Ondansetron responsive endophenotype of IBS. To structurally characterise faecal serine proteases and define the impact of treatment with Ondansetron. Methods: 120 patients meeting Rome III criteria for IBS-D entered a randomised, double-blind, placebo controlled, cross-over study of 5 weeks of Ondansetron 4mg versus placebo with dose titration allowed up to two tablets thrice daily in the first 3 weeks. Patients completed daily bowel symptom diaries documenting stool consistency using the Bristol Stool Form Score (BSFS). Gut transit and small bowel water content were measured in the last week of each treatment. The primary endpoint was average stool consistency in the last 2 weeks of treatment. Faecal samples were obtained from 30 healthy volunteers (HV) and 79 IBS-D patients participating in a trial of Ondansetron versus placebo. Colonic transit was measured using radio-opaque markers. Faecal serine proteases (FSP) were purified from faecal extracts using Benzamidine-Sepharose affinity chromatography. SDS-PAGE profiled components were identified using trypsinolysis and tandem-mass-spectrometry. Functional protease activity in faecal extracts was measured using a colourimetric assay based upon the proteolysis of azo-casein. Results: Ondansetron significantly improved stool consistency In the intention to treat analysis n= 101, with a 1.39 (95% CI1.20-1.58)point decrease on the Bristol stool form scale whilst taking Ondansetron compared to a 0.51 (95% CI 0.32-0.72) point reduction whilst taking placebo p=<0.0001. Compared to placebo patients on Ondansetron experienced fewer days with urgency (p=0.01), lower urgency scores (P<0.001), reduced frequency of defecation (p=0.001) and less bloating (p=0. 25) although pain scores did not change significantly. Protein analysis identified the most abundant FSPs as being of human origin and likely pancreatic juice derived. Functional assays showed increased FSP and faecal amylase in IBS-D compared to HV. Those with higher amylase had significantly higher FSP and greater anxiety. FSP activity correlated negatively with whole gut transit in IBS-D (Spearman r=-0.32, p=0.005) and HV (r=-0.55, p=0.014), but was not affected by treatment with Ondansetron. Conclusions: Ondansetron is an effective and well tolerated treatment in patients with IBS-D with a low number of side effects. It slows whole gut transit, but without a demonstrable difference in small bowel water content. Clinical rather than biochemical indicators predicted responsiveness to Ondansetron best. Patients with less severe symptoms are more likely to respond well to Ondansetron which should prove a useful addition to the current rather limited therapies available for this important group of patients. Previous reports that FSP activity is elevated in some patients with IBS-D has been confirmed. We have increased our understanding of this phenomenon by characterising the proteins responsible for the serine protease activity, showing that most of this activity is likely due to human pancreatic enzymes.
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Dainty, Andrew David. "The cognitive behavioural treatment of irritable bowel syndrome : feasibility of a nurse delivered model of guided self-help." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/35537/.

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Background: Irritable bowel syndrome is a medically unexplained phenomena relating to the lower gastrointestinal tract with symptoms such as altered bowel habit and abdominal pain. Patients experience poor quality of life and consume significant healthcare resources. Mechanisms for the delivery of evidence based psychological interventions for irritable bowel syndrome within the National Health Service are lacking and the feasibility of these interventions is poorly understood. Methods: A novel, low-intensity, nurse-led psychological intervention has been developed and trialled within a mixed methods feasibility study. Twenty participants were randomly allocated across four treatment conditions consisting of; a treatment as usual control (n = 5), self-help (n = 5), low-intensity (n = 5) and high-intensity (n = 5) cognitive behavioural therapy interventions. A total of ten participants took part in post-intervention interviews analysed using a group thematic analysis. Results: Recruitment to this feasibility study was a significant challenge with 22 participants recruited of which, 20 were randomised to the feasibility interventions. Of the 104 patients approached within secondary care gastrointestinal clinics, 27.7% of patients volunteered to enrol into the study. Reasons provided relate to difficulties with committing to taking part and personal circumstances. Themes derived from post-intervention interviews suggest participant’s valued face-to-face therapist interaction and described their perceived treatment utility along with a variety of barriers and facilitators to engagement in CBT interventions. Conclusion: Low-intensity and self-help cognitive behavioural therapy may be feasible mechanisms for the delivery of evidence based psychological interventions for patients with IBS, although significant concerns regarding recruitment of participants to future trials will need to be addressed. Further development of these lower-intensity interventions in collaboration with service users is required in order to improve the acceptability and relevance of the interventions.
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Al-Masmoum, Hussain. "An investigation of chromosome 20q13 amplification in colorectal cancer." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/36938/.

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Background: Colorectal cancer (CRC) is the third most prevalent and second most fatal type of cancer in the Western world. 20q amplification has been identified in approximately 50% of CRC cases. 20q amplification and may be associated with liver metastasis. In our lab, a previous array comparative genomic hybridization (aCGH) study of a series of primary CRCs and corresponding liver metastases, found that the 20q13 was frequently amplified in both groups. Several known oncogenes are located at Chromosome 20q (for example SRC, TPX2 and CSE1L) suggesting that 20q amplification may be an important event in the development and metastasis of CRC. Hypothesis and Aim: Although there are many genes located in 20q identified to have role in developing CRC, other genes with potential role in tumour progression have not been studied in CRC. We hypothesized that 20q harbouring genes have a potential role in CRC progression. Therefore, we aimed to (i) study the frequency of 20q amplification in CRC by validating our aCGH data by alternative method and screen larger sample set for 20q amplification. (ii) Protein expression of candidate genes was investigated. Then, candidate genes were evaluated in several cancer related processes including proliferation and cell motility. Materials and Methods: The evaluation of 20q amplification was performed in DNA from FFPE of micro-dissected 20 CRCs and their matched liver metastasis, using comparative quantitative PCR (qPCR) based on 6 genes (PTPN1, CSE1L, ADNP, PREX1, ELMO2 and PTGIS), which are located in the same region. It was found that quantification of two genes was sufficient to evaluate amplification and thus separate series of 103 cases were screened for 20q amplification based on qPCR of two genes (ELMO2 and PTPN1). For studying the functional activity, PTPN1, CD40, PREX1, ELMO2 and PTGIS were studied in CRC cell lines. To evaluate gene function, small interference RNAs (siRNAs) were used to knockdown genes of interest in CRC cell lines. Knockdown was validated by western blot and qPCR and the effect of knockdown was evaluated on cellular functions such as proliferation, cell migration, cell invasion and wound healing. Immunohistochemistry was used to investigate protein expression in primary CRCs. Results: The amplification of 20q in CRC sample was studied by qPCR and aCGH. High concordance was found between the two methods aCGH, and high correlation coefficient between the qPCR results of the genes demonstrated the utility of this method for measuring amplification. In the combined data set, 20q amplification was seen in both primary tumours (51%) and metastases (60%). TP53 mutation was associated with 20q amplification (p =0.010). Then, we investigated the functional activity of 5 genes located on 20q, which might be has a role in CRC progression. (1) PTPN1 has been reported as tumour suppresser and oncogene. Therefore, we studied protein expression and its functional role in CRC cell lines. PTPN1 was overexpressed in 59% of primary tumours. Knockdown of PTPN1 reduced cell motility but did not affect cell proliferation. (2) CD40, previously, has been identified as a tumour suppresser in CRC. However, high CD40 expression was detected 25% of tumours. Functional assays showed CD40 promotes proliferation by altering subG1 phase of the cell cycle. Therefore, CD40 might promote tumour growth by inhibiting apoptosis. (3) PREX1 was expressed in 61% of primary tumours. Functional analysis showed that it promotes both cell proliferation and motility. (4) ELMO2 was expressed in 38% of primary tumours and functional analysis showed it promotes proliferation and cell migrations. (5) Finally, PTGIS is highly expressed in CRC and its expression associated with liver metastasis. However, PTGIS effect on tumour progression has not been studied. PTGIS knockdown had no effect on CRC cell lines growth and motility. Conclusion: Using alternative methods to evaluate the 20q13 amplification, we have confirmed our previous aCGH and found a frequency similar to that reported in the literature. Amplification of 20q appears frequently in primary tumours but it not positively associated with metastasis. Many genes located in 20q have oncogenic effects which would support the hallmarks of cancer. Understanding their role in CRC development could reveal new avenue in understanding cancer biology and treatment.
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Mahdi, Adnan Hammad. "The structure and innervation of the sphincters in the large intestine of the domestic duck (Anas platyrhynchos)." Thesis, University of Edinburgh, 1989. http://hdl.handle.net/1842/29864.

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Findlay, John Mitchell. "Precision staging and management of Barrett's oesophagus and oesophageal cancer : genomic, imaging and pathological biomarkers." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/38037/.

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Barrett’s oesophagus and oesophageal cancer represent two of the most important and challenging oesophageal disease processes globally, combining increasing incidences with high morbidity treatments, often with poor clinical outcomes. A major contributory factor is that disease susceptibility, progression and response to therapy are largely unpredictable, due to inherent biological complexity and variability. At present, just staging groups are used routinely as thresholds for guiding the use of therapies such as ablation, resection, and oncological therapies. However, these represent blunt tools that do not necessarily reflect patients’ experiences, or appropriately select from the range of treatments available. The aim of this thesis was to explore the potential of genomic, imaging, and pathological biomarkers in guiding more tailored and personalised therapy. The first half of this thesis explores the role of genomic markers. The first results chapter describes the identification of new loci and gene pathways associated with susceptibility to Barrett’s oesophagus, dysplasia and oesophageal adenocarcinoma, by further replication and analysis of a genome-wide association study. In addition, all reported genomic markers of these endpoints were identified and criticised by systematic review, and synthesised by meta-analysis. Validation of these was then attempted, and lessons for markers and future research drawn. The second results chapter describes a similar appraisal and synthesis of genomic markers of oesophageal cancer prognosis, response to therapy, and stage. The third describes the first next generation sequencing study performed in oesophageal adenocarcinoma (and indeed any gastrointestinal cancer as far as the author is aware), before and after neoadjuvant chemotherapy. Using whole exome sequencing a new model of genomic tumour response was developed, and the implications for biomarkers explored. The second half of this thesis follows a large cohort of patients with oesophageal cancer, from nearly 1000 undergoing staging, to more than 300 undergoing neoadjuvant chemotherapy, restaging and resection. In the fourth results chapter, the first application of decision theory to cancer staging identified the potential for routine imaging data to personalise and optimise oesophageal cancer staging. In the following chapter, positron emission tomography-computed tomography was found to be more sensitive for identifying disease progression during neoadjuvant chemotherapy than computed tomography alone. Two factors were identified that could stratify risk of progression to incurable disease, including that encountered at surgery. These included 18F-FDG avid nodes, with new concepts of metabolic nodal stage and response developed in conjunction with predictive models. Thereafter, a number of conventional and experimental metrics of metabolic tumour response were compared and refined as predictors of pathological response. Existing metrics of metabolic tumour response were found to be suboptimal, and these new concepts and classifications of metabolic nodal stage and response were found to have independent utility for clinical practice. Again, predictive models were generated. Finally, the prognostic utilities of these markers were explored. Metabolic tumour response was found to be an imperfect surrogate of pathological response. However, metabolic nodal response demonstrated independent utility in identifying patients at high risk of early recurrence and death, both when used before surgery and afterwards. Indeed, a number of analyses demonstrated the additive utility of considering the primary tumour and nodal metastases as separate entities. Finally, prognostic models were generated, and a simple risk score was generated, using the four independent prognostic markers identified to stratify prognosis.
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Canavan, Caroline. "Using real world data to generate health economic models : a worked example assessing the cost-effectiveness of referral to gastroenterology for irritable bowel syndrome in the UK." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/32666/.

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Introduction: Irritable bowel syndrome (IBS) has substantial impact on Quality of Life (QoL) and patients have high healthcare utilization. Guidelines recommend diagnosis and management within primary care, yet around 25% of patients are referred to gastroenterology. These studies aimed to assess the incidence of organic gastrointestinal disease in patients diagnosed with IBS, the cost of healthcare utilization and the QoL in patients with IBS before and after seeing a gastroenterologist and to estimate the cost-effectiveness of a gastroenterology appointment. Methods: Patients with IBS were identified within the UK Clinical Practice Research Dataset. Incidence rates of coeliac disease, colorectal cancer (CRC) and inflammatory bowel disease (IBD) were calculated. Individual-level healthcare utilization data were extracted for IBS patients who first visited a gastroenterologist in 2008 or 2009. Mean costs of total healthcare utilization were calculated before and after gastroenterology attendance. A questionnaire study of patients with IBS attending a gastroenterology outpatient clinic for the first time measured QoL and utility before and after the appointment. Quality Adjusted Life Years (QALYs) were modeled from these utility values. Cost-effectiveness of a referral to gastroenterology in IBS was assessed using mean cost per QALY. Results: Fifteen years after IBS diagnosis, the combined cumulative excess incidence of coeliac disease, IBD and CRC in IBS is 3.7%. Over one year following gastroenterology appointment, the expected QALY gain compared to no appointment was 0.03 and the expected extra total healthcare costs were £657. The incremental cost-effectiveness ratio was £27865.64/QALY. Referral for patients younger than 30, men, and increasing the time horizon, reduces the expected cost effectiveness. Conclusions: My findings provide reassurance that non-specialists are unlikely to be missing an organic condition in the majority of IBS patients. Referral to a gastroenterologist for IBS might be cost-effective for the NHS but more data, especially on potential QALY gains, are needed.
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48

Keeler, Barrie D. "Investigation of patient blood management in colorectal surgery." Thesis, University of Nottingham, 2016. http://eprints.nottingham.ac.uk/33457/.

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Introduction: Perioperative allogeneic red blood transfusions (ARBT) are associated with impaired short and long term outcomes. Consequently, perioperative ARBT should be avoided, yet preoperative anaemia increases this need. The study aimed to compare the efficacy of preoperative intravenous (IVI) and oral iron (OI) in reducing ARBT use in anaemic patients undergoing colorectal cancer (CRC) surgery. Methods: 116 anaemic patients with non-metastatic CRC adenocarcinoma were recruited preoperatively and randomised to receive either OI (ferrous sulphate) or IVI (ferric carboxymaltose). Perioperative changes in Haemoglobin (HB) and ARBT were recorded across groups. Parametric data was compared with 2 tailed T-test and non-parametric paired data with Wilcoxon Rank test, and Mann-Whitney U test. Nominal data was compared with 2-tailed Chi squared test. Results: There was no difference in demographic data between groups. HB levels at recruitment were comparable (OI 10.4g/dL 95%CI 10.1-10.7; IVI 10.2g/dL 95%CI 9.8-10.5, P=0.24), as was median treatment duration (OI 21 days IQR 15-33; IVI 21 days IQR 15-34, P=0.75). However, HB levels were higher on the day of Surgery in IVI (11.9g/dL 95%CI 11.5-12.3 vs OI 11g/dL 95%CI 10.6-11.4, P<0.01). Median preoperative HB change in patients not transfused preoperatively was higher in IVI (1.5g/dL IQR 0.9-2.6 vs OI 0.5g/dL IQR-0.1-1.3, P<0.01). There were fewer anaemic patients at surgery in the IVI group after treatment (75% vs 90%, P<0.05). OI patients received a mean 0.63u (95%CI 0.26-1) from recruitment to day 28 postoperatively vs mean 0.47u (95%CI 0.1-0.84) for IVI. Neither number of patients transfused (P=0.33) nor mean units transfused (P=0.54) differed over this period. When patients with heavy intraoperative losses (>1.5L) were excluded in subgroup analysis, a significant difference in mean units of blood transfused was seen up to 7 days post operatively (n= 108; OI 0.6u 95%CI 0.23-0.96; IVI 0.16u 95%CI 0.01-0.3, P< 0.05) and significantly less IVI patients were transfused (10% vs 25%, P<0.05) Conclusions: In patients undergoing CRC surgery, IVI appears more efficacious than OI at treating preoperative anaemia. It does not appear to minimise overall ARBT requirement, but may reduce ARBT use in the immediate perioperative period when the implications of ARBT are probably at their greatest.
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Varadhan, Krishna Kumar. "The mechanistic basis of metabolic response to surgery and postoperative insulin resistance in patients having abdominal surgery." Thesis, University of Nottingham, 2015. http://eprints.nottingham.ac.uk/29095/.

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Postoperative insulin resistance (POIR) is a hallmark feature in patients having major abdominal surgery. Surgical stress may induce changes in metabolic pathways that perturb glucose homeostasis, resulting in stress hyperglycaemia. The studies described in the present thesis set out to examine the evidence behind (1) the ‘Enhanced Recovery After Surgery’ pathway (2) preoperative carbohydrate drinks (CHO), in attenuating the surgical stress response and (3) to investigate the mechanistic basis of metabolic response to surgery and the development of postoperative insulin resistance in patients having major abdominal surgery. Meta-analysis of randomised studies in patients having major abdominal surgery showed that ERAS pathway was associated with a significant reduction in length of hospital stay and postoperative complications. Meta-analysis of randomised studies using CHO was also associated with reduced length of stay and decreased POIR in support of reduced fasting times and CHO, before surgery. The study in patients having major abdominal surgery showed that surgical trauma was associated with increased skeletal muscle interleukin-6 and pyruvate dehydrogenase kinase isoform-4 (PDK4) mRNA and protein expression. Increased PDK4 was associated with a concomitant reduction in pyruvate dehydrogenase complex (PDC) activity that controls the rate of muscle carbohydrate oxidation in mitochondria, and contributed to impaired glucose tolerance and decreased mitochondrial ATP production postoperatively. One hypothesis is that by maximising the contribution of carbohydrate derived oxidative ATP regeneration by activating the PDC with the synthetic PDK4 inhibitor dichloroacetate (DCA) and/or by increasing muscle glucose uptake using CHO may reverse the changes in carbohydrate oxidation seen postoperatively. The results from the study showed that administration of either CHO or CHO with DCA attenuates the impairment of CHO oxidation and the development of POIR, induced by surgical stress. Furthermore, DCA increased mitochondrial CHO oxidation above that achieved by CHO alone, which waned by 48 hours after surgery.
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50

Molokwu, Anuri Joy. "Assessment of dysphagia in people with Parkinson's disease, multiple sclerosis and muscular dystrophy." Thesis, University of Nottingham, 2015. http://eprints.nottingham.ac.uk/29048/.

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Introduction: Previous research has shown that patients with Parkinson’s disease (PD), multiple sclerosis (MS) and muscular dystrophy (MD) are known to be at risk of dysphagia and could benefit from dysphagia screening. The aim of this study was to describe the use of dysphagia screening and assessment procedures amongst patients with neurological conditions when they have an unplanned admission to hospital. Methods: Two methods of data collection were used in this study. The first method was a prospective observational study to determine the use of dysphagia screening and assessment procedures amongst patients with Parkinson’s disease, multiple sclerosis and muscular dystrophy. The second, qualitative study examined clinicians’ perceptions of the factors that influenced the decision to screen for dysphagia in people with neurological conditions and the difficulties experienced. Data were collected from clinicians using semi-structured in-depth interviews. Potential interventions to improve the management of dysphagia in these conditions were identified. Results: Two hundred patients were recruited to the observational study. Thirty four percent (n=68) of this group underwent a swallow screening assessment (SSA) during the first week of admission and 93% (n=63) of these were judged to have dysphagia. Amongst those who were not assessed initially (n=132), a further 77% (n=101) were found to have dysphagia. Twenty people took part in the interview study including doctors, health care assistants, nurses and therapists. Clinicians reported that the factors which underpinned their decision to screen for dysphagia included pre-existing swallowing difficulties, recognition of symptoms, staff/relative anxiety, communication difficulties and the presenting complaint and diagnosis. However, clinicians reported that their limited knowledge, clinical competencies in swallow screening, a lack of confidence and resources, affected their practice and use of dysphagia screening and assessments. Clinicians noted a number of interventions that could improve the management of dysphagia when patients are admitted to hospital and these included: training in dysphagia screening; development of dysphagia pathways or guidelines; provision of an alert system, introduction of on-call speech and language therapy services; and research funding. Discussions and Conclusions: The findings of this study suggest that screening for dysphagia does not occur routinely when patients with neurological conditions are admitted to hospital for an acute condition. This means that opportunities to detect treatable causes of potentially life-threatening complications are being missed. Many inter-related factors were reported to account for this practice and these related primarily to limited knowledge and confidence and the limited accessibility of speech and language therapists outside usual working hours. Interventions to improve routine dysphagia screening should help to reduce the incidence of avoidable complications and perhaps shorten length of stay. Dysphagia pathways or guidelines are needed to support effective management in acute hospital settings.
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