Browsing by Author "Warner, P."
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Item Open Access Characterization of a novel virus associated with the MVX disease of Agaricus bisporus(Cranfield University, 2007-07) Maffettone, Eliana; Challen, M. P.; Warner, P.; Mills, P.‘Mushroom Virus X’ (MVX) disease of the cultivated mushroom Agaricus bisporus first arose in UK during the 1990’s. This disease resulted in devastating crop losses in the UK and gradually became more widespread (e.g. Netherlands and Eire). Up to twenty-six, non-encapsidated, double stranded RNA (dsRNA) elements have been found to be associated with diseased mushrooms, and these are believed to be the result of a complex of viruses. Although considerable data has accumulated on the symptoms of infection, aetiological sources, epidemiology and molecular characterization of the MVX dsRNA elements are limited. Research described in this thesis focused principally on sequence characterization of a frequently occurring dsRNA element ( MVX 14.4), which was shown to be a novel Endornavirus. Assigned ‘Agaricus bisporus endornavirus 1’ (AbEV1), this represents the first endornavirus known to infect edible mushrooms. AbEV1 is the first MVX element to be fully sequenced. Putative domains for RNA-dependent RNA polymerase (RdRp), helicase and glycosyltransferase were identified and used in comparisons with other viruses. Characterization of an AbEV1- type dsRNA found in a culture sample derived from a wild Agaricus bisporus collection indicates a possible source of the MVX dsRNA infections. Epidemiological studies were used to demonstrate that the AbEV1 dsRNA was transmissible both vertically through spores and horizontally by mycelial anastomosis between infected donor and MVX free acceptor strains. As a first step in the effort to understand the role of AbEV1in MVX infections and to investigate possible host defence mechanisms, dsRNA hairpin sequences were introduced into A. bisporus by Agrobacterium-mediated transformation. Both helicase and RdRp sequences were able to confer resistance to the uptake of MVX dsRNA elements in transformants. These observations suggest that homology-dependent gene silencing pathway(s) may be present in A. bisporus and represent a residual antiviral defence mechanism. Advances and approaches developed in this project open new opportunities to characterize the other dsRNA elements from the MVX complex, to further our understanding of mycovirus infections and host responses, and to investigate the origins of infectious dsRNA elements.Item Open Access Contaminant interaction and remediation in soil microcosms and pilot-scale studies(Cranfield University, 2003-04) Herbath, Y.; Warner, P.; Patel, D.; Butcher, M.The contamination of soil, groundwater and ultimately potable water sources, is a quantifiable risk associated with the sub-surface release of cable oil into the environment. This research has provided the National Grid Transco (NGT) with an evaluation of the feasibility of biological systems as a means of re mediating soil and groundwater contaminated with cable oil. This has been achieved through treatability studies undertaken at three levels of process sophistication; laboratory-scales micro-cosms, a pilot-study to develop full-scale design criteria and a full-scale pilot-study under semi-controlled field conditions. The full-scale pilot-scale study was undertaken together with a simple temporal and spatial model of oil distribution through two contrasting soil blocks. Experimental data obtained in this thesis has shown, for the first time that anaerobic degradation processes are able to offer an effective alternative to aerobic in situ bioremediation for cable oil. Each level of process in this three-phase study has demonstrated anaerobic soil organisms capable of survival on cable oil and mineral salts ~lone. Sulphate reducing micro-organisms are also suggested as playing an important role in the degradation process. Biodegradation of the cable oil of up to 41 % was achieved, in some cases reducing the concentration to below 50ppm threshold required by the Dutch Intervention Values (1994). The study of pollutant migration found that the temporal and spatial distribution of cable oil is specific to soil type and is influenced by the soil structure, particle size distribution and water suction potential. The extent of oil migration in both soils is a function of the volume of cable oil present and is time dependent. The significant outcome of this work is that prior to this study, there have been no reports of higher alkylbenzenes being degraded anaerobically. Consequently, monitored natural attenuation may now be considered by NGT as a feasible remediation option under certain conditions, providing an acceptable, non-intrusive technique for sites contaminated with cable oil.Item Open Access Development of simulation tests to assess the fate of Unilever ingredients under untreated discharge conditions(Cranfield University, 2007-12) Finnegan, Chris J.; Warner, P.Unilever product ingredients are discharged into the environment via a number of routes, in many regions of the world there is a lack of municipal waste water treatment and the discharge of chemicals directly into the environment in the presence of untreated sewage is a major pathway. An absence of data on the behaviour of the fate and effects of chemicals under such conditions requires overly stringent and unrealistic assumptions when assessing risk (e.g. no biodegradation is assumed). Traditional risk assessment fails since water quality is compromised by pollutants associated with raw sewage (e.g. BOD and ammonia) and the relevance of the ‘standard’ risk assessment approach has thus been questioned. An alternative risk assessment model, based on the ‘impact zone’ concept, has been proposed for direct discharge conditions. In this model, chemicals are assessed in terms of their predicted environmental concentration (PEC) at the end of an impact zone, within which the ecosystem is impacted by the pollutant, free ammonia, and beyond which it is not. Linear alkylbenzene sulphonate (LAS) was used a model compound to understand the fate of materials classified as readily biodegradable in this scenario. Batch and dynamic test systems simulating conditions associated with untreated discharge, confirmed that LAS was degraded quicker than the general organics present in settled sewage and that beyond the defined ‘impact zone’ it is extensively removed. Predicted no effect concentrations (PNECs) can also be generated for chemicals on the inhibition of key microbial processes (biological oxidation and nitrification) which are essential in rivers for self purification. A variety of detergent ingredients (ranging from readily biodegradable to anti-bacterial) were investigated in short term toxicity tests. The tests produced a range PNECs and confirmed that these ingredients can show selective inhibition towards heterotrophic or autotrophic bacterial populations. All of the PNECs generated were above the PEC for these ingredients.Item Open Access Effects of 1-MCP on storage of "Queen cox" and "Bramley" apple fruit(2007) Dauny, Paul Trevor; Warner, P.; Joyce, Daryl C.Better maintenance of firmness and suppression of ethylene production in 'Queen Cox' and 'Bramley' apple [Ma/us sylvestris (L.) Mill. var. domestica (Borkh.) Mansf.] fruit was achieved by prestorage applications of 1-MCP. 1-MCP concentration, exposure time and exposure temperature ranges of 0.1 to 10.0 µl r1 1-MCP, 6 to 48 h and O to 20°C, respectively, were effective on fruit subsequently stored for 2 ('Cox') and 3 ('Bramley') months in air at 3 to 4°C. However, 1-MCP had little effect on either firmness or ethylene production after 4 ('Cox') or 6 ('Bramley') months storage. Nonetheless, 1-MCP treated 'Bramley' fruit had reduced rot and superficial scald incidence compared with untreated control fruit. 1-MCP application was most effective when applied within 24 h of harvest · compared to 14 d later. Earlier-harvested 'Cox' and 'Bramley' apple fruit showed better response to 1-MCP-treatment than those harvested towards the end of the picking season. 1-MCP-treatment was shown to improve apple storage alone and in combination with controlled atmosphere (CA) storage. Furthermore, 1-MCPtreatment maintained fruit quality during shelf-life better than CA storage alone. Chlorophyll fluorescence was not demonstrated to be an effective method to determine 'Cox' and 'Bramley' apple fruit quality. There was no recorded correlation between the concentration of five antifungal compounds and 1-MCP-treatment after inoculation with Penicillium expansum or Botrytis cinerea. 1-MCP treatment for apple storage was developed for AgroFresh Inc., the holder of the 1-MCP patent. Part of this research was used for the UK efficacy trials for registration of 1-MCP as an apple storage treatment. On the 18th July 2002 the US Environmental Protection Agency (EPA) granted approval for 1-MCP to be applied to food crops. Approval was granted in the UK in time for the 2003 apple harvest, and for 2004 across Europe.Item Open Access Plasma homocysteine, measurement and clinical application(Cranfield University, 2006-01) Hill, D. M.; Warner, P.; Kenney, A. C.Raised plasma homocysteine (Hcy) levels have been cited as a major risk factor for several vascular disorders. Yet hyperhomocysteinaemia is easily treated through dietary intervention and vitamin supplementation. Commercial assays have facilitated routine plasma Hcy analysis. However, the problem faced by clinicians is stabilisation of Hcy in whole blood samples prior to delivery to the laboratory. Following blood collection, erythrocytes continue to produce and excrete Hcy increasing plasma concentrations by up to 10% per hour. This thesis describes the investigation of stabilising plasma Hcy in whole blood, allowing wide scale screening for hyperhomocysteinaemia. The most effective method appears to be inhibition of the enzyme responsible for Hcy production, Sadenosylhomocysteine hydrolase (SAHH), using a competitive inhibitor 3- deazaadenosine (3DA). Clinical trials were conducted on a pilot batch of evacuated blood tubes. Samples were stored in EDTA whole blood in the presence and absence of 3DA, at ambient temperatures (20 to 25ºC), and under refrigerated conditions (2 to 8ºC). Only samples that were collected into EDTA plus 3DA tubes and stored refrigerated showed stability over 72 hours (p = 0.2761). For wide scale screening, samples must be stable under ambient conditions. As the structure of SAHH is known a molecular modelling approach was adopted in an attempt to identify other potential inhibitors from screened databases. Interference of SAHH, in an immunochemical method for Hcy, was to be utilised for in vitro screening before any further clinical trials were conducted. The thesis also focuses on Hcy as a marker of vitamin deficiency and explores links between thiol metabolism and the development of cognitive decline eventually leading to dementia. Disruption of single carbon metabolism can lead to an increase in Hcy and a decrease in available methyl groups important in regulation of several metabolic pathways. Increased oxidative stress may also be a causative factor.Item Open Access The study of molecular markers for the progression of Barrett's Oesophagus to adenocarcinoma to identify markers that can be used as diagnostic tools.(Cranfield University, 2002-05) Cadd, Verity Anne; Warner, P.; Barr, H.; Shepherd, N. A.Barrett's oesophagus is a complication of gastro-oesophageal reflux disease and is the single most important predisposing factor for the development of adenocarcinoma of the oesophagus. New molecular markers are needed for early diagnosis and to monitor disease progression. Telomerase is a ribonuclear protein with reverse transcriptase activity, which uses its own RNA component as a template for the addition of telomeric repeats to the end of chromosomes. Telomerase activity has been studied during the neoplastic progression of Barrett's oesophagus using a TRAP based ELISA technique, which found telomerase to be reactivated early during . disease progression. A non-isotopic method of in situ hybridisation for the detection of the RNA component of telomerase has also been successfully developed. Plasminogen activation is an inducible extracellular proteolytic system involved in the regulation of cellular interactions and invasion. The components of the urokinase-type Plasminogen Activator system have been fully investigated during the progression of Barrett's oesophagus to adenocarcinoma utilising immunohistochemistry and ELISA techniques. Changes in the expression of this invasive phenotype were found to occur late during disease progression in malignant tissues. Two-oesophageal cell-lines have been characterised using molecular biological techniques to detect a range of molecular markers to produce ex vivo models of oesophageal adenocarcinoma and oesophageal squamous cell carcinoma. In order to assess the effects of bile salts and acidity on oesophageal tissues these celllines were then utilised as ex vivo models. Exposure to acidic conditions both alone and with bile salts altered the morphological appearance of the cells and disrupted adhesion molecules in the cellular membrane. Investigations into both telomerase reactivation and the plasminogen activator system have provided new information concerning the nature and timing of molecular changes during the Barrett's metaplasia/dysplasia/ adenocarcinoma sequence.Item Open Access Towards a low-cost clinical multiple mutation diagnostic: cystic fibrosis as a model(2002-05) Bull, Elizabeth E. A.; Cullen, David C.; Evans, S.; O'Donnell, J. O.; Warner, P.Cystic fibrosis is used in this work as an example of a genetic disease where early diagnosis and medical intervention can improve quality of life. Current methods of cystic fibrosis diagnosis rely heavily on the sweat test, a biochemical method of measuring the concentration of sodium and chloride in sweat. A repeat test is required for sodium levels between 40 and 70 mmol per litre, which may occur in babies with either mild or no cystic fibrosis. The advantage of a DNA test is that a cut off value is not necessary, this reduces false positive and negative results. A mutation is either present in a person’s DNA or it is absent. Cystic fibrosis has been associated with over 500 mutations, therefore a type of mutation detection was investigated which could potentially examine a large proportion of these in one test. A simple, low cost method of mutation detection is required for use within the National Health Service. The reverse dot blot hybridisation allows a known sequence of DNA to be placed on a membrane (mutant and wild type) and hybridised with an unknown sequence (patients’ DNA). Covalent coupling of oligonucleotides to membrane produced increased attachment over physical attachment as examined via radioactive labels. Polystyrene slides were chemically modified (using 5% potassium permanganate in 1.2M H2SO4) to provide carboxyl groups for the covalent attachment of amino terminated oligonucleotides. Human DNA was amplified by PCR, labelled with biotin and detected via chemiluminescence. Genomic DNA was extracted from blood samples with the Qiagen QIAamp system and both wild type and mutant sequences were amplified. Hybridisations were performed on nylon membranes and modified polystyrene slides. Hybridisation was specific at high stringency (0.1%SDS/0.1xSSC). Further work is required to produce a prototype diagnostic device.