Identification of RNA editing in the human exome and development of DARNED DatabaseSF

RNA editing is a biological phenomena that alters nascent RNA transcripts by insertion, deletion and/or substitution of one or a few nucleotides. It is ubiquitous in all kingdoms of life and in viruses. The predominant editing event in organisms with a developed central nervous system is Adenosine to Inosine deamination. Inosine is recognized as Guanosine by the translational machinery and reverse-transcriptase. In primates, RNA editing occurs frequently in transcripts from repetitive regions of the genome. In humans, more than 500,000 editing instances have been identified, by applying computational pipelines on available ESTs and high-throughput sequencing data, and by using chemical methods. However, the functions of only a small number of cases have been studied thoroughly. RNA editing instances have been found to have roles in peptide variants synthesis by non-synonymous codon substitutions, transcript variants by alterations in splicing sites and gene silencing by miRNAs sequence modifications. We established the Database of RNA EDiting (DARNED) to accommo-date the reference genomic coordinates of substitution editing in human, mouse and fly transcripts from published literatures, with additional information on edited genomic coordinates collected from various databases e.g. UCSC, NCBI. DARNED contains mostly Adenosine to Inosine editing and allows searches based on genomic region, gene ID, and user provided sequence. The Database is accessible at http://darned.ucc.ie RNA editing instances in coding region are likely to result in recoding in protein synthesis. This encouraged me to focus my research on the occurrences of RNA editing specific CDS and non-Alu exonic regions. By applying various filters on discrepancies between available ESTs and their corresponding reference genomic sequences, putative RNA editing candidates were identified. High-throughput sequencing was used to validate these candidates. All predicted coordinates appeared to be either SNPs or unedited.

Profiling phytochemical and nutritional components of potato

Potatoes (Solanum Tuberosum L.) contain secondary metabolites that may have an impact on human health. The aim of this study was to assess the levels of some of these compounds in a wide range of varieties, including rare, heritage and commercial cultivars. Vitamin C, total carotenoids, phenolics, flavonoids, antioxidant activity and glycoalkaloids were determined, using spectroscopy and chromatography, in the skin and flesh of tubers grown in field trials. Transcript levels of key synthetic enzymes were assessed by qPCR. Accumulation of selected metabolites was higher in the skin than in the flesh of tubers, except ascorbate, which was undetected in the skin. Differences were on average 2.5 to 3-fold for carotenoids, 6-fold for phenolics, 15 to 16-fold for flavonoids, 21-fold for glycoalkaloids and 9 to 10-fold for antioxidant activity. Higher contents of carotenoids were associated with yellow skin or flesh, and higher values of phenolics, flavonoids and antioxidant activity with blue flesh. Variety ‘Burren’ had maxima values of carotenoids in skin and flesh, variety ‘Nicola’ of ascorbate, variety ‘Congo’ of phenolics, flavonoids and antioxidant activity in both tissues, except antioxidant activity in the skin, which was higher in ‘Edzell Blue’. Varieties ‘May Queen’ and ‘International Kidney’ had highest glycoalkaloid content in skin and flesh respectively. The effect of the environment was diverse: year of cultivation was significant for all metabolites, but site of cultivation was not for carotenoids and glycoalkaloids. Levels of expression of phenylalanine ammonia-lyase and chalcone synthase were higher in varieties accumulating high contents of phenolic compounds. However, levels of expression of phytoene synthase and L-galactono-1,4-lactone dehydrogenase were not different between varieties showing contrasting levels of carotenoids and ascorbate respectively. This work will help identify varieties that could be marketed as healthier and the most suitable varieties for extraction of high-value metabolites such as glycoalkaloids.

Multi-parametric metabolic assessment of cells under stress conditions

Glycolysis, glutaminolysis, the Krebs cycle and oxidative phosphorylation are the main metabolic pathways. Exposing cells to key metabolic substrates (glucose, glutamine and pyruvate); investigation of the contribution of substrates in stress conditions such as uncoupling and hypoxia was conducted. Glycolysis, O2 consumption, O2 and ATP levels and hypoxia inducible factor (HIF) signalling in PC12 cells were investigated. Upon uncoupling with FCCP mitochondria were depolarised similarly in all cases, but a strong increase in respiration was only seen in the cells fed on glutamine with either glucose or pyruvate. Inhibition of glutaminolysis reversed the glutamine dependant effect. Differential regulation of the respiratory response to FCCP by metabolic environment suggests mitochondrial uncoupling has a potential for substrate-specific inhibition of cell function. At reduced O2 availability (4 % and 0 % O2), cell bioenergetics and local oxygenation varied depending on the substrate composition. Results indicate that both supply and utilisation of key metabolic substrates can affect the pattern of HIF-1/2α accumulation by differentially regulating iO2¬, ATP levels and Akt/Erk/AMPK pathways. Inhibition of key metabolic pathways can modulate HIF regulatory pathways, metabolic responses and survival of cancer cells in hypoxia. Hypoxia leads to transcriptional activation, by HIF, of pyruvate dehydrogenase (PDH) kinase which phosphorylates and inhibits PDH, a mitochondrial enzyme that converts pyruvate into acetyl-CoA. The levels of PDH (total and phosphorylated), PDH kinase and HIF-1α were analysed in HCT116 and HCT116 SCO2-/- (deficient in complex IV of the respiratory chain) grown under 20.9 % and 3 % O2. Data indicate that regulation of PDH can occur in a manner independent of the HIF-1/PDH kinase 1 axis, mitochondrial respiration and the demand for acetyl-CoA. Collectively these results can be applied to many diseases; reduced nutrient supply and O2 during ischemia/stroke, hypoglycaemia in diabetes mellitus and cancer associated changes in uncoupling protein expression levels.