PhD, EngD and MSc by research theses (Cranfield Health)
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Item Open Access Development of an affinity sensor for prostate cancer diagnosis(2010-11) Uludag, Yildiz; Tothill, Ibtisam E.Prostate carcinoma is a fatal malignancy and is a major cause of death in men in the population aged 55 and over. Early diagnosis of prostate cancer is very important for successful treatment of the disease. The increase in prostate specific antigen (PSA) levels in serum above the normal limits is the primary indication of prostate malignancy; therefore, PSA is used as a biomarker for the diagnosis and prognosis of prostate cancer. To date PSA testing still dominate as the best biomarker for prostate cancer detection even though it is not prostate specific. This project aims to develop a rapid, sensitive and specific biosensor to detect prostate cancer biomarkers in order to reduce the number of people who undergo invasive examinations for diagnosis. As part of the work, alternative surface chemistry techniques were investigated to optimise the sensors immobilisation capacity, such as the use of dendrimer monolayers and surface grafting polymers. It was found that dendrimer monolayers can be used to increase the sensor surface capacity by 1.5 times for surface plasmon resonance (SPR) sensor chips but not for quartz crystal microbalance (QCM) chips. A novel polymer (UVlink) surface was also fabricated and successfully used for biomolecule immobilisation for SPR and QCM chips. The QCMA-1 prototype biosensor was applied to develop PSA detection assays. Initially an immunoassay was constructed and optimised on the QCM sensor chip. A new buffer was then formulated to eliminate 98% of non-specific binding due to human sera proteins and this enabled the PSA detection assay to be performed in 75% human serum. Since QCMA-1 instrument is a prototype, to confirm the results the PSA assay was repeated using a commercial biosensor, Biacore 3000. This newly developed method showed a limit of detection of 0.29 ng mL"1 (in 75 % serum) with a linear dynamic detection range up to 150 ng mL"1 with Au nanoparticles employed for sensitivity enhancement using both instruments. With the achieved detection limit, it is possible to use the developed QCM and SPR assays for cancer detection in patient samples. PSA detection binding data fitted to 1:1 Langmuir binding model and KD calculated as 9.46 x IQ"10 M for the assay performed with Biacore 3000 instrument and using the QCMA-1 biosensor 5.56 x 10"10 M. These results were comparable and show that the affinity between PSA and PSA-capture antibody is in the region of 10'10 M and that QCMA-1 assay results are compatible with Biacore 3000 assay results. In the study incorporation of fPSA, cPSA and PSMA immunoassays to the prostate cancer detection test was also considered. However, the commercial available antibodies were formed to have low affinity for these markers. Patient samples collected from Bedford Hospital NHS Trust and control samples collected from Cranfleld University staff were tested and it was found that the developed QCMA-1 test clearly discriminates the PSA level of patients from the controls. A second biosensor platform based on label-free interdigitated capacitive sensing principle was also employed in the study. Interdigitated electrode (IDE) arrays were fabricated with conventional microelectronics-micromachining technologies on silicon wafers. Later IDE arrays were utilised for the detection of PSA using an LCR meter. This new prototype enabled capacitance reading of individual capacitors in turn and also enabled the detection to be performed in buffer solution. Antigen binding assays were developed to capture the PSA molecule in buffer solutions achieving a detection limit of 15.6 ng mL"1 with a linear dynamic detection range of 15.6 - 250 ng mL"1 PSAItem Open Access Ecological factors affecting growth and ochratoxin A production of Aspergillus section Nigri species on grapes(2006-05) Mitchell, D.; Magan, NareshThis study investigated the effects of interacting environmental factors on the ecophysiology of Aspergillus carbonarius and ochratoxin A (OTA) production, to facilitate the building of a database of knowledge for risk prevention. Spore germination and germ tube extension was faster on grape flesh than in vitro on grape- based media, or grape skin. Under optimum conditions (25-35°C; 0.90-0.99 water activity (aw)) 50% of conidia germinated in < 4.5 hours. Two dimensional profiles in relation to aw x temperature were constructed for rate of germination and germ tube extension for the first time. Mycelial growth was also influenced by aw, temperature, pH and modified atmospheres. Two dimensional temporal profiles of growth and OTA production were constructed based on information on strains from four different European countries. This showed that the optimum aw and temperature, and range were 0.98 aw, 30°C and 0.99-0.88 aw and 15-40°C, respectively. In contrast, for OTA production these were 0.95 aw, 15-20°C, and 0.99-0.93 aw and 15-35°C respectively. Environmental factors also influenced mycelial interactions between A. carbonarius and nine other common grape fungi. An Index of Dominance showed that A. carbonarius was very competitive against these species over a range of aw levels and temperatures. OTA production was generally suppressed by interactions at 0.95 aw with some inhibition of OTA production at 30°C at 0.99 and 0.95 aw. At 0.99 and 20°C all strains except two species produced a stimulation in OTA production. The efficacy of fungicides (5) and anti-oxidants (2) on growth and OTA production was limited at the concentration used. The only two fungicides that did suppress growth were Switch (cyprodinil and fludioxonil) and carbendazim. OTA production was stimulated at sub-optimal concentrations regardless of aw level used. Anti-oxidants were ineffective under all conditions tested. Modified atmospheres (0.03, 25 and 50% CO2) at different aw levels showed that there was very little long term effect on spore germination and germ tube extension, even at 50% CO2. Regardless of aW) OTA production was influenced more by the aw itself than CO2 concentrations. The efficacy of Sodium metabisulphite (NaMBS) at different aw levels (0.99, 0.97, 0.93 aw) on grape juice-based media at 25°C on germination and germ tube extension showed inhibition by >500 mg kg' 1 of NaMBS. Low concentrations (100, 250 mg kg'1) stimulated growth and OTA production. LD50 and LD90 ranges were determined for both growth and OTA production for the first time. Field sampling found that black aspergill! were present on grapes from setting to harvest with a significant increase in the population with grape ripening, and higher temperatures. The most commonly isolated OTA species from grapes was A. carbonarius with no Pénicillium verrucosum and only a few Aspergillus ochraceus isolated. In all the field sampling found no OTA present in any of the grape musts.Item Open Access Extraction of genetic network from microarray data using Bayesian framework(2007-04) Kumuthini, Judit; Bessant, Conrad; Setford, S.The aim of the work described in this thesis was to develop novel methods for the extraction of gene regulatory networks (GRN) from gene expression data, and use these methods to capture previously unknown relationships between genes in specific biological applications. This has been accomplished through the application of Bayesian Networks (BN) through minimum description length (MDL) and taboo search for parameter and structure learning respectively to three large scale microarray datasets from Saccharomyeces cerevisae, Escherichia coli and human stem cells. The application of BNs for modelling the well characterised yeast cell cycle demonstrated the efficacy of the techniques employed. Using the cDNA microarray data from the yeast cell cycle project by Spellman et a l (1998), this study succeeded in extracting many biologically plausible genetic relationships, which were supported by evidence from publicly available genome and literature databases. Two novel knowledge extraction techniques were applied; Target Node (TN) analysis and learning through simulation. Further, it was demonstrated how the addition of prior knowledge to the extracted network can improve the network structure extracted purely from experimental data. The second part of this thesis demonstrated how the BN approach could be adapted to a data set of very high dimensionality, specifically data from a 54,634 probe array used to monitor human adipose tissue. Genetic networks extracted included insulin receptor (IR) and Fatty acid binding proteins (FABP) families that play key roles in fatty acid uptake, transport, and metabolism In the final part of this thesis, the genome-wide GRNs of a prokaryotic expression system were extracted from novel oligo cDNA microarray data from E-coli K12 to identify metabolic stress responsive genes during recombinant protein production. Also, detailed analysis of known metabolic stress related genes and the genes that are directly or indirectly associated in the GRN were used to establish possible markers for host system exhaustion. In conclusion, the BN methods developed proved to be a powerful and effective means of extracting GRNs in a variety of applications.Item Open Access An investigation into the effects of bacterial fermentation in autism(2006-11) Bilbao Montoya, Maria Pilar; Ritchie, Laurie; Hunter, John O.Gastrointestinal symptoms have been observed to be present very often in autistic children. These symptoms are very similar to those observed in Irritable Bowel Syndrome (IBS). An investigation to investigate whether there is a similar cause for autism as well as IBS was done. Methods for the analysis of breath VOCs and urine organic acids were developed to be applied afterwards in the research of biomarkers of autism and IBS. Bacterial metabolites in urine and breath were identified by restricting the diets of healthy volunteers to each of two enteral feeds. The way enteral feeds work in the treatment of Crohn’s disease (CD) was investigated. Enteral feeds work by reducing bacterial activity in the gut, measurably reducing their metabolic products. The urine of IBS patients was analysed and bacterial compounds investigated. Some gastrointestinal symptoms are frequently misdiagnosed as IBS. According to the symptoms and treatment that is successful for these different groups of patients three subgroups can be made: “retention and overflow”, “musculoskeletal” and “abnormal fermentation” IBS. When the urine of these subgroups was observed, different biomarkers for the various subgroups could be identified. Patients with abnormal fermentation IBS have increased bacterial products in urine, suggesting an increase in bacterial activity in the colon. These biomarkers decreased after an exclusion or fibre-free diet and symptoms improved. In a further study, potential biomarkers for autism were identified. The origin of the majority of these compounds is unknown, although some of them are of known bacterial origin. A conclusion of this is that autistic children may exhibit abnormal fermentation since GI symptoms manifest at almost the same time as the autistic behavioural symptoms start and may be present from birth. Autistic children share many symptoms with IBS patients, although the biomarkers are different, showing that the GI symptoms observed in autistic children are not IBS in the children studied. More research needs to be done to determine the origin of the autism biomarkers discovered in urine.Item Open Access Towards the development of an electrochemical immunosensor for the identification of transient ischemic attack via the labeless detection of biomedical markers.(2006) Barton, Andrew Charles; Higson, Seamus P. J.The thesis describes the development of generic platforms for labeless transduction of binding events. A particular application of the project is the labeless detection of biochemical markers for stroke, specifically transient ischemic attack, to facilitate rapid diagnosis and subsequent introduction of appropriate therapy within the required three-hour time frame. Novel biocomponents are under development for detection of these biochemical markers, and further demonstration of the generic nature of the technologies being developed will be realised via the use of a library of monoclonal antibodies as capture biocomponents. This work forms part of an investigation into labeless affinity immunosensor detection via impedimetric transduction utilising the technique of electrochemical impedance spectroscopy (EIS). Novel microelectrode arrays were fabricated by the sonochemical ablation of a homogeneous insulating layer of poly(o-phenylenediamine) exposing discrete areas of an underlying screen-printed conductive substrate. The receptor biocomponents are then subsequently coupled to the transducer by either electropolymerised entrapment or non-covalent affinity within and/or at the surface of conducting polymer ‘mushroom’ shaped protrusions for site-specific immobilisation. Upon exposure to a range of concentrations of antigen analyte solution, complex plane impedance analyses are used to relate the differing redox states of the polymer to possible charge transfer and / or related mechanisms between the immobilised antibody / antigen analyte and the polymer. Results show that signal differentiations were obtained, for a number of antibody / antigen species over a range of concentrations of clinical relevance (ng-pg/mf1), suggesting that the techniques employed offer a viable approach towards the labeless quantification of biochemical markers of transient ischemic attack (TIA).Item Open Access A study of the cyber security awareness and use of protective cyber security practices in defence settings(2018-07) Sherchan, Sundar; Smy, VictoriaThe UK Strategic Defence and Security Review (2015), places ‘cyber’ in the highest category tier-one risk. The threats from cyberspace is ever increasing as UK Armed Forces is becoming increasingly dependent on its’ information systems and networks for daily business processes. Hardware and software technological defences are effective tools to protect our systems and networks, nonetheless these defences are useless if humans operators allows attackers to maliciously exploit our systems through use of social engineering techniques. There is currently no measurement framework in the R SIGNALS or the Army to assess basic cyber awareness and behaviour of soldiers and officers and benchmarking user cyber awareness maturity state. In this study, the author creates an innovative measurement framework that is utilised to measure cyber security awareness and behaviour in the R SIGNALS. The framework is an extension and adaptation of the government NCSC infographics for basic cyber security protective practices which in this study is split into five themes for measuring awareness (device safety, device backup, phishing, password and malware) and one theme for behaviour. The research adopts a quantitative positivist approach with using a questionnaire to measure human cyber awareness and behaviour. Study of human psychology models in the literature indicates that factors such as awareness and subsequent attitudes have direct influences on human behaviour. Results after codification and statistical analysis confirmed that technical trades in the R SIGNALS has better awareness of device safety, malware and phishing while cyber training was directly related to user behaviour and awareness of device safety. Overall user awareness in the R SIGNALS was found to be at Integrated level out of the five levels in the Community Cyber Security Maturity Model. The measurement framework is not limited to application to R SIGNALS and has the utility for other corps and organisations within the Army. Key future research recommendations included adding an attitude scale to the framework and having the correct sample to represent population variationItem Open Access The hydraulic lung(2004-10) Prime, David; Turner, Anthony P. F.; Barr, H.A Hydraulic Lung has been designed and constructed. The Hydraulic Lung can inhale through a Dry Powder Inhaler (DPI) with a pre-determined level of inspiratory effort, and the characteristics of the inhalation profile generated, such as the peak pressure drop, peak flow rate and the flow acceleration are determined by the resistivity of the inhaler. The Hydraulic Lung has been used to explore the relationship between the level of inspiratory effort, the DPI resistivity and the resultant profile characteristics. A simple empirical equation has been found to describe the peak pressure drop achieved for any given level of inspiratory effort and device resistivity. This equation can be adapted to provide the equivalent peak inspiratory flow rate. A second simple empirical equation was found to describe the flow acceleration rate achieved under defined conditions of inspiratory effort and device resistivity. A clinical study has been performed to generate equivalent human inhalation data. A comparison between the relationships derived from the Hydraulic Lung data and the human inhalation data has demonstrated the validity of the key equation for pressure drop as a tool for predicHng human inhalation characteristics. The equation for flow acceleration rate was found to underestimate the flow accelerations achieved by human volunteers, but with slight modification could be used for this purpose. Correlations were established between the findings from this study and the work of earlier researchers in this area, which was based on clinical data alone. The Hydraulic Lung was also used as a practical tool for the evaluation of DPI performance in-vitro, including the behaviour of devices with variable resistivity which cannot easily be assessed using either standard pumps or sophisticated apparatus such as the Electronic Lung.Item Open Access Nanofunctionalised orthopaedic implants(2018-01) Mirza, Farhan A.; Ge, YiDue to an aging population and younger patients presenting with musculoskeletal disorders, there is a need for orthopaedic implants with improved healing rates and longer implant life. Numerous research has developed implant surfaces with micro-topography and biomolecules to imitate the native extra cellular matrix (also known as biomimetic surfaces). This research has utilised such a biomimetic approach by immobilising the cell adhesive peptide, RGD (Arginine-Glycine-Aspartic Acid), to a titanium alloy Ti6Al4V surface. This research polymerised Hyperbranched Polyglycerol (HBPG) from the titanium surface using Ring Opening Multi-Branching Polymerisation (ROMBP). HBPG is a biologically compatible and non-toxic synthetic biopolymer, able to reduce non-specific protein adsorption, increase the titanium surface wetting (hydrophilicity), thereby limiting foreign body reactions. Extensive hydroxyl groups at the periphery of HBPG provides conjugation sites for biomolecule attachment. In this work the RGD peptide was conjugated to the polymer via a siloxane layer. This research developed a novel passivation solution for the preparation of the titanium alloy surface, using a mixture of hydrogen peroxide and nitric acid (a passivation mixture not used in the literature). This novel mixture was shown to etch the titanium surface, producing micro and nano surface features, both of which have been shown to improve cellular function in the literature. The hydrogen peroxide/nitric acid solution showed extensive oxidising ability on titanium, leading to the formation of reactable hydroxyl groups. Contact-angle measurements showed that the novel passivating solution produces a hydrophilic surface similar to that of peroxidation for 12-hours, but achieved in only 2-hours. In conjunction with the etching and oxidising abilities of hydrogen peroxide, the nitric acid reacts with the titanium surface, leading to the formation of a protective titanium oxide layer, enhancing corrosion resistance and improving biocompatibility. Biological investigations with the pre-osteoblast cell line MC3T3-E1 showed greater osteoblast cell attachment and adhesion strength, as well as improved bone matrix mineralisation on the passivated titanium surface functionalised with HBPG and the RGD peptide, compared to the raw and passivated titanium surfaces. Antibacterial testing of HBPG revealed substantially reduced bacterial cell colonies on the passivated/polymerised titanium surface, possibly arising from electrostatic and hydrophobic repulsion. This research has successfully developed a new titanium passivation solution (hydrogen peroxide/nitric acid) that can yield a contact-angle of around 35° in just 2-hours, rivalling the Piranha solution. The successful immobilisation of a cyclic RGD (cyclic-RGDfc) to a titanium surface functionalised with HBPG, has been shown in this research to drastically improve mineralised bone matrix production from the MC3T3-E1 cell line. This indicates earlier osseointegration of the implant may be possible, thereby improving patient healing times.Item Open Access Biosensor stabilization using hypersolutes(Cranfield University, 2009-06) Loose, Elizabeth; Setford, Steve; Newman, Jeffrey D.Introduction A biosensor may be described as a sensor incorporating a biological element such as an enzyme, antibody, nucleic acid, microorganism or cell. A biosensor should exhibit both shelf-stability and operation stability. Compatible solutes from hyperthermophilic bacteria, called hypersolutes, are very efficient for the preservation of the performance of a wide variety of biomaterials; ranging from proteins to whole cells and artificial tissues. The overall objectives of this work have been to investigate the application of hypersolutes to enhance the performance of biosensors based on the stabilization properties offered by hypersolutes compounds, particularly with respect to storage and operational lifetime. Materials and Methods The stabilizing agents considered for this study were firoin, firoin A, ectoine®, hydroxyectoine, diglycerol phosphate (DGP) and potassium mannosyl-lactate (PML), provided either by Bitop AG (Witten, D) or StabVida (Oeiras, P).The following enzymes were selected due to their commercial importance: Glucose oxidase (GOx), alcohol oxidase (AOx), acetylcholinesterase (AchE) and lactate dehydrogenase (LDH). On immunosensors, a model system was first designed using ELISA tests. The influence of hypersolutes was then studied using BIAcore. The antibody test system selected for examination of the effect of stabilizing agents on immunosensor performance was based on an anti-human immunoglobulin G (IgG) primary antibody, grown in goat, and an anti-goat secondary antibody conjugated to horseradish peroxidase enzyme. A model DNA binding system was sought: The poly-A strand was tethered to the sensing surface within the BIAcore system via a biotin-streptavidin linkage whereas the complimentary poly-T strand contained a fluorescent Cy3 label, that offered the possibility to also use more conventional detection techniques to ensure thathybridization between the two complimentary strands had been achieved, as well as add a significant weight to the strand, increasing its visibility on the BIAcore signal.Item Open Access In vitro toxicity of new engineered nanoparticles through their life cycle(Cranfield University, 2013-11) Irfan, Muhammad Adeel; Zhu, Huijun; Njuguna, JamesThe rapid development of nanotechnology has caused concerns about nanoproducts on human health throughout their lifecycle. As part of the consortium NEPHH (nanomaterial related environmental pollution on human health through their life cycle, funded by EU-FP7), this project aimed to assess the potential effect of novel polymer-silicon composites on human health from a lifecycle perspective, focusing on in vitro toxicity of raw silica nanoparticles (SiNP) and dust nanoparticles (NP) released from silicon-based polymer composites. The main objectives were to characterise a group of amorphous SiNP and dust NP in water and cell culture medium; assess NP toxicity potential in in vitro models; and establish mode of SiNP action. The selection of SiNP of size 7-14 nm was based on their wide use in developing polymer nanocomposites. Dust NP were generated from mechanical processing of polymer composites made of polyamide-6 (PA6), polyurethane (PU) and polypropylene (PP), each incorporated with SiNP or 3 other different silicon reinforcement materials. The dispersion and size of NP in water and in cell culture medium were characterized using dynamic light scattering, scanning electron microscopy and transmission electron microscopy. The chemical composition of NP was assessed by infra-red spectroscopy. NP were assessed in vitro for induction of membrane damage, intracellular reactive oxygen species (ROS), loss of cell viability, and cellular uptake by flow cytometry and confocal microscopy. In order to identify potential biomarkers for toxicity prediction, miRNA array and extracellular metabonomic assays were performed. The size of SiNP (10-100 µg/ml) ranged from ~200-500 nm in water and ~20- 500 nm in culture medium, indicating the presence of aggregates. The infra-red spectrum of SiNP dried from culture medium showed a slight difference as compared with that dried from water, indicating protein adsorption. SiNP induced acute ROS increase, cell membrane damage, and reduction in cell viability after 48 h in human lung carcinoma epithelial A549 cells, lung fibroblast MRC-5 cells and skin HaCaT keratinocytes. SiNP were up taken by all 3 cell types, and located in the cytosol. Six early (<48h) SiNP responsive miRNAs were identified in A549 cells. SiNP also induced early changes in metabolites including glucose, lactate, ethanol, phenylalanine, histidine and tyrosine. Dust NP generated from PA6 group materials were more toxic than those from other polymer composites when assessed at 25-100 µg/ml at 72 h in A549 cells. The results obtained from this study suggest that 1) both small and larger SiNP aggregates are taken up into the target cells; 2) conventional cytotoxicity assays combined with miRNA and metabonomic assays provide insight into the molecular mechanisms of the nanotoxicity; 3) metabonomics and miRNA assays can serve as robust tools for recognising sub-toxic dose-effect relationships; 4) the toxicity of dust NP from polymer composites depends on polymer type but not reinforcement materials. This study demonstrated the importance of lifecycle analysis as opposed to single stage analysis of novel materials. Further studies need to improve study design to enable interpretation of cytotoxicity in relation to NP size, physiochemical property and intracellular dose, and to simulate the health effect of polymer-silicon composites under more realistic scenarios.Item Open Access Study of phenolic content and antioxidant activity of Greek red and white wines by means of classical methods and FTIR(Cranfield University, 2015) Strataridaki, Argiro E.; Aldred, David; Lydakis, DimitriosPhenolic and antioxidant contents of wines are very important in terms of both flavour attributes and health benefits. Changes occur during ageing of wine in containers (e.g. wooden barrels) in relation to their antioxidant activity and phenolic content. Vilana, Dafni, Kotsifali and Mandilari single variety Cretan wines, were vinificated to determine their antioxidant activity and phenolic content Wines were aged in different containers after two vinifications. Changes in the above characteristics were determined every three months for a twelve month period. Stainless steel with and without oenosticks containers, American oak, French oak, Acacia and Chestnut barrels were used for wine ageing. As far as phenolic and antioxidant contents are concerned, ageing of wine in chestnut barrels, Kotsifali and Mandilari (red wines) and in Acacia barrels for Vilana and Dafni (white wines), gave the best results, achieving the highest phenolic content and antioxidant activity after 12 months of ageing. The phenolic fingerprints of Vilana, Dafni, Kotsifali and Mandilari wines were determined for the first time. The phenolic fingerprint of wines has been recently used for the authentication and discrimination of red wines. In this study, attempt has also been made to use the phenolic fingerprint of white wines, for authentication. Differences were observed in Kotsifali and Mandilari (red wines) and in Vilana and Dafni (white wines) directly after vinification, allowing their discrimination. Also the changes in their phenolic fingerprints were monitored during ageing in different containers for a 12 month period. The effect of hydroxytyrosol and oleuropein on wine spoilage induced by acetic acid bacteria was also determined. Hydroxytyrosol was better than oleuropein in controlling the increase of volatile acidity, causing wine spoilage. Wines treated with 0.5mg/l hydroxytyrosol showed control of volatile acid production, and may be a promising alternative to sulphites in wine production in the future. Finally, extracts obtained from olive oil mills and winery by-products were used in Vilana vinification instead of sulphites and antioxidant activity and phenolic content of the wines determined.Item Open Access Vertebrate somite development and neural patterning(Cranfield University, 2013-09) De Sousa, C. A. Ferreira; Tannahill, David; Toro, Carla T.The segmentation of the axial skeleton and peripheral nervous system involves a complex integration of multiple patterning molecules. For the latter, axon-repelling molecules in the posterior half-sclerotome are particularly important. This study built on a previously performed mouse microarray screen for novel candidate genes in the posterior half-sclerotome. Multiple candidates were selected for whole-mount in situ hybridization in chick. Two were expressed in the posterior half-sclerotome: thrombin receptor (F2R) and fibronectin leucine rich transmembrane protein-2 (Flrt2). Flrt2 was selected for siRNA-mediated knockdown and a new in ovo transfection technique for somites successfully developed. Scrambled siRNA-transfection did not affect morphogenesis, somite patterning or axon guidance. However, Flrt2 siRNA-transfection resulted in defects in notochord, dermomyotome and neural tube morphogenesis, and in the de-fasciculation and mis-targeting of spinal axons into the posterior half-sclerotome and dermomyotome. Hence, Flrt2 may be a chemorepellent for spinal axons. An unidentified peanut agglutinin (PNA)-binding glycoprotein in the posterior half-sclerotome was previously shown to repel spinal axons. In this project, the expression of a family of mucin-type O-glycosylation enzymes (which could glycosylate the PNA-binding protein) was investigated by whole-mount in situ hybridization in chick, but none was differentially expressed in the posterior half-sclerotome. One candidate for the PNA-binding glycoprotein, Presenilin1, was investigated because of previously published loss of spinal nerve segmentation in Presenilin1 mutants. However, analysis of Presenilin1-hypomorphic mutant mouse embryos showed this was not the PNA-binding molecule. Live-immunostaining for a second candidate, prolyl 4-hydroxylase, beta polypeptide (P4HB), showed its expression coincided with PNA-binding at the surface of posterior half-sclerotome cells. P4HB siRNA-transfection into somites reduced PNA binding and disrupted spinal axon segmentation and expression of a posterior sclerotome marker, Uncx4.1. Overall, these results suggest that P4HB is a strong candidate to be the key PNA-binding glycoprotein in the posterior half-sclerotome that repels spinal axons.Item Open Access Temporal changes in vase water(Cranfield University, 2013-02) Salih, Magdi; Lambert, R. J. W.This study investigated the influence of flower food on vase water quality with the attempt to correlate this with the flowers’ appearance and microbial growth occurring in the vase water. A mixed bouquet of different cut flowers was used in this study for the first time instead of the common practice in the literature of using a single cut flower or a single cultivar. Different combinations of vase solutions; standard water and reverse osmosis water with or without added flower food were used as initial vase solutions and also as the topping up water. The effect of vase solution’s pH on microbial growth and therefore flowers vase quality was also examined. Moreover the analysis of sugar content of vase water was conducted using HPLC and LC/MS. The analysis of vase water in the Cranfield Health laboratory has shown that: Sugar presumably plays a central role in energy for both microbes and plants but the concentration levels present in flower food seems to have no subsequent effect on the growth or otherwise of the microbes even when diluted with top up water. Water uptake by the flowers is little influenced by the presence of flower food or the microbial population. Flower food reduces the pH of Standard water, but not sufficiently enough to inhibit the growth of common pathogens or spoilage organisms. If microbial growth begins, addition of further flower food in the top-up does not inhibit further growth. If reverse osmosis water (ROW) is used with flower food the initial pH is lower than the pH minimum for all common pathogens and the majority of common spoilage organisms. Topping up with ROW with flower food maintains the low pH environment. If growth is initiated due to the presence of microbes capable of growth in the low pH environment, then growth will continue regardless of topping up solution. Microbial growth in ROW with flower food is confined to acidophilic organisms. Addition of weak acid preservatives such as benzoic acid or sorbic acid could control or prevent the growth of such acidophilics, whilst allowing a pH compatible with the flowers to be maintained.Item Open Access Effect of controlling ethylene on ripening of avocado cv. hass fruit(Cranfield University, 2010-05) Meyer, Marjolaine D.; Terry, Leon A.Rigorous control of ethylene inside storage atmosphere is cardinal to maintain quality of climacteric fruit, including avocado cv. Hass. This can be achieved using the ethylene action inhibitor, 1-methylcyclopropene (1-MCP). The recent development of a novel palladium (Pd)-based ethylene scavenger, e+® Ethylene Remover, provides a new opportunity to delay avocado fruit ripening. A new method was developed to sequentially extract and quantify both lipids and sugars from the same avocado mesocarp tissue sample. Extraction by homogenization with hexane yielded slightly less oil than the standard Soxhlet technique whilst the fatty acid profiles of the oil extracts were similar. Extraction of the resulting filter residue with methanol (62.5%, v/v) better recovered sucrose, perseitol and mannoheptulose as compared to ethanol (80%, v/v). The new method has a shorter extraction time, lower extraction temperature and requires less solvent. Presence of e+® Ethylene Remover in storage atmosphere removed all ethylene and accordingly delayed the ripening of avocado cv. Hass stored at low temperature. 1-MCP also inhibited ripening, yet, unlike e+® Ethylene Remover it impaired subsequent ripening. It was possible to slow down the ripening rate after the climacteric has been induced by removing ethylene below 1 μL L-1 in presence of e+® Ethylene Remover, and the scavenger was effective in combination with modified atmosphere packaging (MAP). Blocking ethylene action or removing ethylene did very slightly affect the fatty acid composition of the mesocarp oil. Depending on the origin and maturity of the fruit, 1-MCP and e+® Ethylene Remover better maintained seven-carbon sugars concentrations in mesocarp. Results support the view that mannoheptulose and perseitol could be important features of the avocado ripening process but more research is necessary to elucidate their exact function. Mesocarp abscisic acid (ABA) was quantified using a newly developed LC-ESIMS/ MS method. ABA increased as fruit ripened but appeared to be at least partly regulated by ethylene. Whether ABA influences the ethylene-associated ripening in avocado cv. Hass remains to be determined in future.Item Open Access The clinical utility and cost-effectiveness of non-bone mineral density based prognostic tests for osteoporotic fracture(Cranfield University, 2012-06) Poku, Ernest Kofi; Newman, Jeffrey D.This thesis investigated a number of aspects of the clinical utility and cost-effectiveness of clinical risk factors, online multivariate algorithms, DXA and a nail-based Raman spectroscopy test, BQT for fracture risk using archived nail samples, and questionnaire data from the Nurses’ Health Study which followed women for up to 23 years. The results showed that the BQT in combination with CRFs improved the results over CRFs alone, using logistic regression and Cox’s proportional hazards analysis. The improvement seen was larger using the Cox model, indicating that time is an important factor. The multivariate algorithms, FRAX and QFractureScores were compared in retrospective and cohort models and found to be predictive, but the relative performance of the two algorithms was highly dependent on the input data. Reclassification is an exciting new approach to evaluating the addition of new biomarkers in multivariate algorithms and was found in the Nurses’ Health Study to provide better discrimination than AUC. Cost-effectiveness analysis using Markov and decision tree approaches showed that the BQT with a low cut-off in combination with DXA was consistently on the cost-effective frontier, indicating that this new biomarker would be an integral part of any mass screening strategy. In conclusion, it is clear that the use of the BQT can enhance the performance of clinical risk factors and, with further improvements, the combination may offer a cost-effective alternative to the use of DXA for mass screening in multivariate algorithms.Item Open Access Elucidating ethylene-mediated physiology and biochemistry in selected climactic and non-climacteric fruits using e+® ethylene remover(Cranfield University, 2013-05) Elmi, Fardusa; Terry, Leon A.The presence of ethylene in a storage environment can undermine both quality and postharvest life of many fruits, often generating significant waste and associated economic losses. A demand for discovering alternative technologies capable of scavenging ethylene has led to the development of a new material, e+® Ethylene Remover, which has significant ethylene adsorption capacity. The material has been shown to remove ethylene to below physiologically active levels during fruit storage at 0-20 o C and consequently extend postharvest life for a variety of fresh produce types. Different formats incorporating e+® Ethylene Remover have been developed. Successful application of e+® Ethylene Remover in laboratory settings has created opportunities to test new formats of the product. To this end, work was conducted herein to attest whether e+® Ethylene Remover, which has been shown to maintain avocado (Persea americana cv. Hass) firmness in recent laboratory trials, could result in a meaningful extension of storage life in a commercial setting. It was shown that e+® Ethylene Remover coated sheets were a highly efficacious format for suppressing ethylene and extending storage life of imported avocado and pluot plums in a series of commercial trials. Moreover, the potency of the e+® Ethylene Remover treatment in retarding ethylene induced ripening was significantly enhanced when avocados were treated during the early stages of ripening. After a storage period of up to 31 days (5-6 o C), avocado fruits which had received an early treatment at source and then treated again in the laboratory were significantly more firm and greener compared to fruits treated following 5 weeks of transit alone . Strawberry is regarded as non-climacteric fruit; nevertheless, exogenous ethylene can negatively influence postharvest life. The low ethylene produced by non-climacteric fruits has been generally ignored and research reporting on the involvement of ethylene in these fruits is typically devoid in the literature. To this end, application of a highly sensitive laser based photoacoustic ethylene detector has revealed a possible role of ethylene in determining the postharvest life of strawberries. Moreover, fruit quality parameters including disease incidence, sugars, organic acids, phenolic compounds and plant phytohormones/metabolites were found to be profoundly affected by ethylene and likewise the removal of ethylene. Storage of strawberries in the presence of e+® Ethylene Remover was associated with lower disease incidence, significantly less red fruits and higher ascorbic acid content. In contrast, ethylene and 1-methylcyclopropene (1-MCP) treatments resulted in the higher postharvest disease. Ethylene-treated fruits were associated with lower level of sucrose and higher simple sugars (fructose and glucose) suggesting a role of ethylene in promoting the rate of senescence and concomitant reduced postharvest quality of strawberries. Changes in ABA, ABA metabolites and auxins within different tissues of ripe strawberry during storage were investigated. ABA was more abundant in the flesh than in the achenes, while auxins were undetectable in the flesh tissue. Auxins indole-3-acetic acid (IAA) and the conjugated form indole-3-acetylaspartic acid (IAAsp) were detected in high concentrations in the achenes and were affected by ethylene and storage length.Item Open Access Sonochemically induced reactions of oils(Cranfield University, 2012-10) Mercantili, Laura; Higson, Seamus P. J.This work will describe the use of the ultrasonic power for the modification of a wide range of oils from vegetable, through to mineral and synthetic oils. It will be shown that ultrasound is effective in cleaving the chains of carbon-based oils with the generation of products with a lower viscosity. The proposed mechanism through which these less viscous products are generated involves the formation of radical species which can be either oxygen or non-oxygen related. The process can be improved by the addition of nucleating agents, with the achieved lower viscosity being stabilised by the addition of radical scavengers. It will be also shown that ultrasound is effective as a power source to drive organic chemistry reactions such as the alkaline hydrolysis of triglycerides (saponification reaction). Benefits of this work will be related to the possible production of more easily degradable oils and to the possibility of using such oils as alternative energy sources, with a particular interest in investigating the environmental and energetic benefits of this approach.Item Open Access Development of a database and its use in the Investigation of Interferences in SRM assay design(Cranfield University, 2013-04) Dokpesi, Oshiobugie; Bessant, ConradSelected Reaction Monitoring (SRM), is a form of mass spectrometry that guarantees high throughput and also a high level of selectivity and specificity. Performing SRM experiments requires the development of assays to aid in peptide identification. This is a time consuming and expensive process thus biological researchers have come up with bioinformatics solutions for the design of SRM assay. The accuracy of these bioinformatics methods is quite high and the next step is to optimise the process by tackling the interference issue. As various analytes may have the same signals within an SRM experiment and thus interfere with each other’s signals, different solutions are being derived to tackle the issue. This thesis describes the development of a SRM transition database to store peptide and transition data, software to populate the database and also software to retrieve the data from the database. Finally the database is tested with the MRMaid transitions for the human proteome which were mined from the PRIDE database and the results analysed to investigate the transition interference issue. The database currently contains data for 20220 proteins and approximately 870,000 tryptic peptides from the human proteome.Item Open Access The biochemistry and physiology of different hybrid and grafted eggplants in response to NaCL salinity in soil and hydroponic systems(Cranfield University, 2013-12) Panagiotakis, Giorgos D.; Dragassaki, Magdalene; Aldred, DavidThe major problem of salinity n regions of intensive cultivations such as Ierapetra, has started to affect the crop production. This project was designed to study the effects of salinity on several biochemical and physiological parameters in two hybrids of eggplant as grafted and non-grafted. The two hybrids were the Solanum melongena L hybrid Habana and Solanum melongena L hybrid Vernina. The concentrations that were chosen (12.5 25, 50, 100, 150 and 200mM) represent a scale of salt concentration and were cultivated as self rooted plants in soil, hydroponically and hydroponically as grafted. Grafting also seems to play an important role for salinity tolerance. To corroborate the different parameters were used as indicators of salinity affection and quantified. Plant height and number of leaves were affected in all three experiments and for all plant types whether grafted or not. Similar were the results for the dry and fresh weight of stem leaves and fruits. For the physiological function of the plants factors such as chlorophyll fluorescence and photosynthetic rate were affected especially at higher concentrations of NaCL. The chlorophyll content in the leaves, which was also measured, was negatively affected in all hybrids. Biochemical measurements the K and Na concentrations in dry leaves and the enzyme activity of APX, GPX and SOD were also made. The K and Na ion concentrations were similar with other studies presenting a reduction of K and raise of Na as the concentration of salt was rising. Unfortunately the enzyme activity was not affected at the specific experiment so it was not able to find exactly how the salinity affects this factor. Overall the results of grafted plants were better than as self rooted and the Solano torvum plant used as rootstock is more tolerant than the Tomato Resistar also used as rootstock.Item Open Access Electrochemical immunosensors for personal care product development applications(Cranfield University, 2011-09) Holford, Timothy Richard John; Higson, Seamus P. J.This thesis describes the fabrication, characterisation and validation of two novel immunosensors for the detection of the inflammation markers nerve growth factor-β (NGF) and psoriasin (S100-A7). The initial phase of sensor construction involves the electrodeposition of polyaniline onto commercial screen-printed carbon electrodes. A classical avidin-biotin link was then used to immobilise biotinylated antibodies. Fully fabricated immunosensors were incubated with their target antigen and interrogated with AC impedance to observe the change in real (Z‘) impedance (Ohms) with respect to antigen exposure. The Z‘ impedance was found to increase over physiologically significant concentration ranges of the target antigen. The effects of non-specific antigen binding were accounted for via controls using a non-specific antigen analogue for each immunosensor. The sensors reported here, after optimisation of the fabrication procedure using the BioDot AD3200 automatic dispensing system, are capable of detecting the presence and concentration of target antigen in a commercial sample within 35 minutes with a lower limit of detection (LLD) (a response observed that is more than three times the standard deviation of the immunosensors baseline impedance measurement) of 50pg ml- 1 for NGF and 250pg ml- 1 for psoriasin. An evaluation is also presented of the competitive advantage gained by companies such as Unilever by undertaking research projects in conjunction with universities and forming other such strategic networks. As part of this EngD project, a review paper concerning antibody based sensors in the past decade was written and is now submitted ready for publishing in the peer reviewed journal 'Biosensors and Bioelectronics‘. A further two papers describing the development, characterisation and validation of the NGF and psoriasin immunosensors described within this thesis have also been submitted for publishing in 'Biosensors and Bioelectronics‘.