Expression of tumour necrosis factors during chick lens development

During development of the lens, epithelial cells at the lens equator begin a differentiation process to become secondary fibre cells. The differentiating cells elongate and migrate towards the centre of the lens where they envelop the older, central fibre cells. Differentiation into fibre cells is accompanied by the breakdown of all organelles, such as the mitochondria. All organelle degradation is completed and denucleation occurs at the border of the organelle free zone (OFZ) which contains the central, terminally differentiated, fibre cells. The differentiation pathway is not well characterised, though it is believed to have similarities to an attenuated form of apoptosis supported by the identification of apoptosis related genes, such as TNF, in the lens. This study continues the search for and characterisation of apoptosis related genes expressed during lens development, focusing on TNFs and their extended family. Reverse Transcriptase-(RT-) PCR was carried out, identifying a number of TNF and extended family member genes in the chick lens, expression studies established novel, statistically significant differential expression for TRAF2 and TRAF3. TRAF2 protein expression from western blotting, similar to RT-PCR expression was found to decline as the lens developed. TRAF2 localisation studies showed limited expression in the equatorial region but there was extensive signalling found in the developing iris, a region in the corneal-scleral boundary and some staining was also detected in the ciliary body. TRAF3 protein and RT-PCR expression were similar, with increasing expression as the lens developed. Western blotting identified two bands and subcellular fractionation confirmed different localisation for the two isoforms. Immunofluorescence identified increasing TRAF3 staining in the cortical fibre cells, this staining was found to be similar to proteins that were reported to be involved in lens fibre cell remodelling and maintenance, suggesting a possibly similar role for TRAF3. Following interest in TRAIL as a gene therapy for Posterior Capsule Opacification (PCO) its expression was examined using RT-PCR and Western blotting which showed low, similar levels of expression throughout the stages of lens development studied. Peroxidase staining showed interesting staining in the equatorial epithelial cells and those just beginning to differentiate at the transition zone. Novel nuclear staining was identified at all time points in both epithelial and fibre cells containing nuclei. Characterisation of whole lens culture was undertaken to discover the optimum culture system for the whole chick lens. Of the published research using whole chick lens culture none stated the basic morphology of the developing lens in organ culture, though each lab had their preferred methodology. The characterisation resulted in the preference of E10 chick lenses being grown with vitreous attached in medium containing glucose. Understanding the morphology of lenses in culture will be invaluable when undertaking the functional studies required to clarify the roles in the lens of the newly identified genes, specifically TRAF2 and TRAF3.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Capital and culture : an investigation into New Labour cultural policy and the European Capital of Culture 2008

This thesis is an investigation into the relationship between culture in New Labour policy and within the competition for the European Capital of Culture 2008. The study interrogates a policy paradigm which it identifies as a 'creative city/urban planning' approach to urban regeneration. It locates this approach within a wider New Labour 'Third Way' politics, in that it attempts to reconcile economic instrumentalism with a rhetorical commitment to a politics of the social. Based on elite interviews and documentary analysis, this thesis argues that this approach to urban regeneration draws on a misappropriation of the work of cultural theorist Raymond Williams. It demonstrates how this misappropriation results in an unbounded anthropological definition, whereby culture colonises all areas of economic and social life. Within this template, culture becomes a surrogate economic and social policy. This is illustrated in the case-study of Liverpool's bidding for, winning of and plans for Capital of Culture 2008. This analysis shows how culture without parameters is usurped within both a neo-liberal economic agenda, and a policy template which recasts social inequality as a personal cultural deficit. Within Liverpool's urban strategy, culture is conceived as a social and economic panacea. However, when culture comes to mean everything, it invariably means nothing. This thesis attempts to put Raymond Williams' 'vague and baggy monster' back in its theoretical cage.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Study of nuclear targets of phosphatidylinositol-3 kinase in lymphocytes

Pathways regulated by Phosphotidylinositide-3-kinase (PI3K) have emerged as important mediators of cell proliferation and survival. When altered, several components of this pathway have been identified to contribute towards a wide range of human malignancies. PI3K has been implicated in the development of several EBV-associated malignancies of both lymphoid and epithelial origin. These include Burkitt's lymphoma, Hodgkin's disease and nasopharyngeal carcinoma. Although progress has been made in dissecting the pathways regulated by PI3K, the key components contributing to lymphocyte transformation have not been fully characterised. This study sought to investigate downstream targets of PI3K in lymphocytes in order to further our understanding of the contribution of PI3K signalling to lymphocyte proliferation and survival, particularly within the context of EBV-associated B-cell lymphomas. Initial work in this study revealed that a component of the mammalian ribosome, S6-ribosomal protein, is a major target for PI3K activation in transformed lymphocytes. In order to study PI3K and EBV regulated proteins on a larger scale, the technology of two-dimensional electrophoresis (2DE) was employed. The use of 2DE in combination with a PI3K inhibitor did not allow the identification of PI3K regulated proteins. However, three EBV regulated proteins were detected in the B-lymphocyte nucleus using this technology. The technology was further developed to study the post-translational modifications of DNA bound transcription factors. This detected multiple isoforms of the cAMP-response element binding protein (CREB), signal transducers and activators of transcription 1 (STAT1) and forkhead box O (FOXO) transcription factors in the nuclei of EBV immortalized lymphocytes. More detailed analysis of the PI3K regulated pro-apoptotic transcription factor, FOXOl, revealed that this protein is downregulated in EBV positive cells at both the transcriptional and translational levels. This downregulation was shown to directly correlate with the protein expression of a known target gene activated by FOXOl, Bcl-6, and to inversely correlate with protein levels of Cyclin D2, a target transcriptionally repressed by FOXOl. Further investigations into the mechanisms by which EBV downregulates FOXOl implicated a role for two EBV encoded proteins, Latent membrane protein-1 (LMP1) and LMP2A in the downregulation of both FOXOl, and its target gene, Bcl-6. In conclusion, this work has explored the use of antibody detection and proteomic techniques for the identification and analysis of nuclear proteins and transcription factors regulated by PI3K and EBV. Together, these investigations have deepened our understanding of the molecular changes that occur in lymphocytes in response to EBV infection, and how EBV may influence malignancy.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Hierarchical modelling of multiphase flows using fully resolved fixed mesh and PDF approaches

Fully–resolved simulations of multiphase flow phenomena and in particular particulate flow simulations are computationally expensive and are only feasible on massively parallel computer clusters. A 3D SIMPLE type pressure correction algorithm is implemented and extensively tested and parallelized to exploit the power of massively parallel computing clusters currently available. Domain decomposition and communication schemes applicable to a general unstructured or structured multi–block CFD codes are discussed and algorithms are proposed, implemented and tested. Several high–performance linear solvers and a multi–grid strategy for the current framework are implemented and the best types of solvers are identified. A 2D CFD code is developed by the author to test several possible fixed–mesh strategies. Variations of immersed boundary (IB) and fictitious domain (FD) methods are implemented and compared. FD methods are identified to have better properties especially if other transport phenomena are also considered. Therefore an FD method is adapted by the author for the SIMPLE type flow solvers and is extended to heat transfer problems. The method is extensively tested for the simulation of flow around stationary in addition to freely moving particles and forced motion where both natural and forced convection are considered. The method is used to study the flow and heat transfer around a stationary cylinder and a new high resolution correlation is devised for the estimation of the local Nusselt number curves. Free fall problem for a single circular cylinder is considered and the effects of internal heat generation and also long term behavior of single cold particle subject to natural convection are also studied in detail. A particle collision strategy is also adapted and tested for the particle–particle collision problems. The FD algorithm is extended to the 3D framework and the flow around single stationary sphere and also free fall of a single sphere are used to validate the FD algorithm in 3D. A unique polydispersed fluid-particle turbulent modelling process is reviewed and the closure problem for this framework is studied in detail. Two methods for the closure of the non–integer moments which results from the polydispersity of the particles are proposed namely PDF reconstruction using Laguerre polynomials and a unique direct method named Direct Fractional Method of Moments (DFMM). The latter is derived using the results of the fractional calculus by writing an equation for the fractional derivatives of the moment generating function. The proposed methods are tested on a number of problems consisting of analytical, experimental and DNS simulations to asses their validity and viability which shows that both methods provide accurate results with DFMM having more desirable properties.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Studio di trattabilità di un sito contaminato da arsenico mediante Pteris vittata

Il presente studio riporta i risultati di una sperimentazione in campo riguardante la fattibilità dell’impiego della specie vegetale Pteris vittata L. per il fitorimedio di un suolo contaminato da arsenico, la Rotonda di San Giuliano a Mestre (VE), caratterizzata da particolari condizioni microambientali e pedologiche. La felce P. vittata è, infatti, una nota iperaccumulatrice di arsenico ed è particolarmente indicata per la fitoestrazione, essendo molto efficiente nella traslocazione di As verso le parti aeree; è una pianta perenne, versatile, resistente, con un’elevata velocità di crescita ed un esteso network di radici e peli radicali. Il sito in esame presenta un suolo di natura limo-argillosa e carenza di sostanza organica umificata. Di conseguenza si ha formazione di crepe e croste superficiali nei periodi secchi, mentre in quelli piovosi si ha rigonfiamento del suolo, perdita di porosità, ristagno d’acqua e l’instaurarsi di condizioni asfittiche locali. Le analisi chimiche condotte sul suolo hanno evidenziato un inquinamento superficiale da arsenico diffuso in tutta l’area, con valore di circa 45 mg∙kg-1, e hanno messo in luce che tutto il contenuto di arsenico è associato agli ossidi/idrossidi di Fe e Mn e quindi potenzialmente biodisponibile in condizioni riducenti. La sperimentazione in campo si è svolta tra l’estate e l’autunno 2007. Nonostante le difficoltà di crescita e sopravvivenza delle piante, dovute alle caratteristiche avverse del suolo e accentuate dalle particolari condizioni microambientali, P. vittata ha mostrato un’elevata capacità di accumulare arsenico. Infatti, a fronte di concentrazioni iniziali pari a circa 4 mg∙kg-1, dopo 105 giorni le concentrazioni misurate nelle fronde erano comprese tra 200 e 900 mg∙kg-1 p.s. con fattori di bioaccumulo compresi tra 4.8 e 20 e fattori di traslocazione circa uguali a 10. I risultati hanno evidenziato la necessità di stabilizzare la struttura del suolo nei riguardi degli sbalzi idrici e migliorare la resistenza di P. vittata mediante l’ottimizzazione delle pratiche agronomiche, anche attraverso l’eventuale utilizzo di piante micorrizate

Phytoremediation potential of the arsenic hyperaccumulator Pteris vittata: preliminary results from a field study

Phytoextraction is a promising technique for the remediation of soils contaminated by metals and metalloids and is proposed as a green alternative to conventional remediation methods. This paper reports the preliminary results of a field study carried out to evaluate the potential of the fern Pteris vittata for the phytoremediation of arsenic polluted sites. P. vittata is a known arsenic hyperaccumulator and its properties have been assessed in a number of studies, mainly at laboratory or glasshouse scale, while few field investigations are reported in the literature. The experimental activity was planned to compare and evaluate the effect of different conditions on plant growth and on as uptake by P. vittata. The study area is located in northeastern Italy. During the experimental period, pedoclimatic conditions were shown to affect strongly plant growth as well as As bioaccumulation. The results of two consecutive field trials confirm the phytoextraction ability of P. vittata under field conditions, but indicate also that the optimization of agronomic practices is crucial for the success of a phytoextraction application at fullscale. The inoculation of ferns with arbuscular mycorrhizal (AM) fungi seems to have a positive influence on plant growth, while its role on phytoextraction efficiency still remains unclear

Decellularization of pericardial tissue and its impact on tensile viscoelasticity and glycosaminoglycan content

Bovine pericardium is a collagenous tissue commonly used as a natural biomaterial in the fabrication of cardiovascular devices. For tissue engineering purposes, this xenogeneic biomaterial must be decellularized to remove cellular antigens. With this in mind, three decellularization protocols were compared in terms of their effectiveness to extract cellular materials, their effect on glycosaminoglycan (GAG) content and, finally, their effect on tensile biomechanical behavior. The tissue decellularization was achieved by treatment with t-octyl phenoxy polyethoxy ethanol (Triton X-100), tridecyl polyethoxy ethanol (ATE) and alkaline treatment and subsequent treatment with nucleases (DNase/RNase). The quantified residual DNA content (3.0 ± 0.4%, 4.4 ± 0.6% and 5.6 ± 0.7% for Triton X-100, ATE and alkaline treatment, respectively) and the absence of nuclear structures (hematoxylin and eosin staining) were indicators of effective cell removal. In the same way, it was found that the native tissue GAG content decreased to 61.6 ± 0.6%, 62.7 ± 1.1% and 88.6 ± 0.2% for Triton X-100, ATE and alkaline treatment, respectively. In addition, an alteration in the tissue stress relaxation characteristics was observed after alkaline treatment. We can conclude that the three decellularization agents preserved the collagen structural network, anisotropy and the tensile modulus, tensile strength and maximum strain at failure of native tissue

Assessment of phenolic herbicide toxicity and mode of action by different assays

A phytotoxicity assay based on seed germination/root elongation has been optimized and used to evaluate the toxic effects of some phenolic herbicides. The method has been improved by investigating the influence of experimental conditions. Lepidium sativum was chosen as the most suitable species, showing high germinability, good repeatability of root length measurements, and low sensitivity to seed pretreatment. DMSO was the most appropriate solvent carrier for less water-soluble compounds. Three dinitrophenols and three hydroxybenzonitriles were tested: dinoterb, DNOC, 2,4-dinitrophenol, chloroxynil, bromoxynil, and ioxynil. Toxicity was also determined using the Vibrio fischeri MicrotoxA (R) test, and a highly significant correlation was found between EC50 values obtained by the two assays. Dinoterb was the most toxic compound. The toxicity of hydroxybenzonitriles followed the order: ioxynil > bromoxynil > chloroxynil; L. sativum exhibited a slightly higher sensitivity than V. fischeri to these compounds. A QSAR analysis highlighted the importance of hydrophobic, electronic, and hydrogen-bonding interactions, in accordance with a mechanism of toxic action based on protonophoric uncoupling of oxidative phosphorylation. The results suggest that the seed germination/root elongation assay with L. sativum is a valid tool for the assessment of xenobiotic toxicity and can be recommended as part of a test battery