PhD, EngD and MSc by research theses (Cranfield Health)

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Browsing PhD, EngD and MSc by research theses (Cranfield Health) by Supervisor "Carter, David"

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    Molecular ecology of aspergillus section flavi species : approaches to understand the role of aflatoxin genes in aflatoxin biosynthesis
    (Cranfield University, 2011-02) Abdel-Hadi, Ahmed; Magan, Naresh; Carter, David
    This is the first study to integrate and correlate the effect of ecophysiological factors on the life cycle of Aspergillus flavus by carrying out complementary work on gene expression of the aflatoxin gene cluster, with growth, sporulation and phenotypic toxin production. This information was used to understand the role of ecological factors on key biosynthetic genes and examine the use of such information for control of aflatoxin production using RNA interference. Ecological studies showed the profiles for growth, sporulation and aflatoxin B1 (AFB1) production with optimum ranges of water activity (aw) and temperature for AFB1 production being identified. A. flavus grew faster at 0.99 aw at all temperatures, but optimally at 30-35°C. The highest amount of asexual conidia was produced at 0.95 aw followed by 0.90 aw and then 0.99 aw at all temperatures examined. Interestingly, the partitioning of AFB1 into biomass, medium and spores showed that at 0.99 aw, about 50% of the mycotoxin was present in the biomass and the medium, with very little present in the spores. However, as water stress was imposed there was a switch to a significantly higher channelling of AFB1 (about 45%) into the spores, especially at 0.95 and 0.93 aw levels. A microarray analysis was used to examine the effect of aw x temperature interactions on the relative expression of the aflatoxin gene cluster for the first time using A. flavus NRRL 3357. This showed that under mild stress conditions (20°C/0.99 aw) several of the cluster genes, in particular aflS and aflJ, were highly induced concomitant with high levels of phenotypic AFB1 production. Highest amounts of AFB1 were produced in all conditions where aflS expression was elevated. When the ratio between the normalised expression data of the aflS/aflR genes was generated, high ratios were obtained at 25°C and 30°C at 0.99 and 0.95 aw and low ratios at 25°C and 30°C at 0.90 aw. This is in agreement with the AFB1 production profile. Cont/d.
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    The role of non-coding RNAs in haemoglobin regulation
    (Cranfield University, 2009) Trujillano Lidon, Daniel; Carter, David
    Non-coding RNAs appear to play a role in gene regulation by modulating chromatin structure. There is mounting evidence suggesting an essential role for non-coding RNAs in the complex process of the genetic regulation of the β-globin locus. Preliminary observations indicate that the BGL3 non-coding transcript may be involved in an RNA-protein interaction and may be interacting with chromatin in the β-globin locus as part of a regulatory function within the locus. However, the expression profile of this non-coding transcript has not yet been characterized and nothing is known about its mode of action. Here it is shown that the BGL3 transcript is dynamically up-regulated upon haemin induction of the K562 cell line (a human erythroleukemic cell line). To determine whether there is a correlation between the BGL3 transcript expression and the expression of the γ- and β-globin genes, the levels of the BGL3 transcript in K562 cells were perturbed by knocking it down using the RNA interference pathway. The effect of the knockdown of the BGL3 transcript was tested on the expression levels of the γ- and β-globin genes, which were quantified using qRT-PCR. Our results are the first, to our knowledge, that describe a developmentally regulated expression of the BGL3 non-coding transcript in haemin-induced K562 cells, and provide evidence that suggests that this transcript may be involved in the silencing of the β-globin gene.