Browsing by Author "Espach, Yolandi"
Now showing 1 - 2 of 2
Results Per Page
Sort Options
- ItemThe detection of mycoviral sequences in grapevine using next-generation sequencing(Stellenbosch : Stellenbosch University, 2013-03) Espach, Yolandi; Burger, J. T.; Maree, H. J.; Mostert, Lizel; Stellenbosch University. Faculty of AgriSciences. Dept. of Genetics.ENGLISH ABSTRACT: Metagenomic studies that make use of next-generation sequencing (NGS) generate large amounts of sequence data, representing the genomes of multiple organisms of which no prior knowledge is necessarily available. In this study, a metagenomic NGS approach was used to detect multiple novel mycoviral sequences in grapevine phloem tissue. Individual sequencing libraries of doublestranded RNA (dsRNA) from two grapevine leafroll diseased (GLD) and three shiraz diseased (SD) vines were sequenced using an Illumina HiScanSQ instrument. Over 3.2 million reads were generated from each of the samples and these reads were trimmed and filtered for quality before being de novo assembled into longer contigs. The assembled contigs were subjected to BLAST (Basic Local Alignment Search Tool) analyses against the NCBI (National Centre for Biotechnology Information) database and classified according to database sequences with which they had the highest identity. Twenty-six putative mycovirus species were identified, belonging to the families Chrysoviridae, Endornaviridae, Narnaviridae, Partitiviridae and Totiviridae. Two of the identified mycoviruses, namely grapevine-associated chrysovirus (GaCV) and grapevine-associated mycovirus 1 (GaMV-1) have previously been identified in grapevine while the rest appeared to be novel mycoviruses not present in the NCBI database. Primers were designed from the de novo assembled mycoviral sequences and used to screen the grapevine dsRNA used for sequencing as well as endophytic fungi isolated from the five sample vines. Only two mycoviruses, related to sclerotinia sclerotiorum partitivirus S and chalara elegans endornavirus 1 (CeEV-1), could be detected in grapevine dsRNA and in fungus isolates. In order to validate the presence of mycoviruses in grapevine phloem tissue, two additional sequencing runs, using an Illumina HiScanSQ and an Applied Biosystems (ABI) SOLiD 5500xl instrument respectively, were performed. These runs generated more and higher quality sequence data than the first sequencing run. Twenty-two of the putative mycoviral sequences initially detected were detected in the subsequent sequence datasets, as well as an additional 29 species not identified in the first HiScanSQ sequence datasets. The samples harboured diverse mycovirus populations, with as many as 19 putative species identified in a single vine. This indicates that the complete virome of diseased grapevines will include a high number of mycoviruses. Additionally, the complete genome of a novel endornavirus, for which we propose the name grapevine endophyte endornavirus (GEEV), was assembled from one of the second HiScanSQ sequence datasets. This is the first complete genome of a mycovirus detected in grapevine. Grapevine endophyte endornavirus has the highest sequence similarity to CeEV-1 and is the same virus that was previously detected in fungus isolates using the mycovirus primers. The virus was detected in two fungus isolates, namely Stemphylium sp. and Aureobasidium pullulans, which is of interest since mycoviruses are not known to be naturally associated with two distinctly different fungus genera. Mycoviral sequence data generated in this study can be used to further investigate the diversity and the effect of mycoviruses in grapevine.
- ItemAn investigation into the importance of the ATM protein in the myocardial pathology associated with insulin resistance and type 2 diabetes(Stellenbosch : Stellenbosch University, 2017-03) Espach, Yolandi; Huisamen, Barbara; Strijdom, Hans; Engelbrecht, Anna-Mart; Stellenbosch University. Faculty of Medicine and Health Sciences. Dept. of Biomedical Sciences: Medical PhysiologyENGLISH ABSTRACT : Introduction: Ataxia-telangiectasia (A-T) is an autosomal, recessive disorder that is caused by mutations in the ataxia-telangiectasia mutated (ATM) gene. The gene product, ATM, is a 350 kDa serine/threonine protein kinase with a large number of substrates in various pathways. Patients suffering from A-T display a high incidence of insulin resistance or type 2 diabetes mellitus (T2DM) and are more susceptible to ischaemic heart disease. Although it is known that the ATM protein is expressed in the heart and that structural and functional changes are observed in the hearts of ATM knock-out mice, very little research has been done on ATM and its role in insulin signalling in the cardiovascular context. This project aimed to identify and characterise an obese, insulin resistant animal model in which myocardial ATM expression is altered; to use this model to determine the effect of ATM manipulation on myocardial function and response to ischaemia/reperfusion injury (IRI); and to determine the effect of ATM manipulation on insulin signalling networks. Methodology: Male Wistar rats received a high caloric diet (HCD) or standard rat chow (control) for 16 weeks after which biometric data was collected. ATM was manipulated in perfusion studies prior to ischaemia using insulin (activator) and KU-60019 (inhibitor). Hearts perfused using the Langendorff balloon model were subjected to global ischaemia followed by reperfusion and Western blots were performed to evaluate insulin signalling intermediates. Hearts were also perfused using the working mode and subjected to regional ischaemia. Following reperfusion, infarct size (IFS) was determined. Aortic ring isometric tension studies were performed to determine the effect of KU-60019 on vasodilation. Results: The HCD resulted in significantly increased body mass, visceral fat mass, glucose levels, insulin levels and HOMA-IR index compared to the control diet and ATM expression was reduced in the HCD hearts. Cardiac function and IFS were comparable in the control and HCD hearts. In control hearts, insulin administration activated the insulin signalling network prior to ischaemia and cardiac function was improved during reperfusion. Insulin had no effect on the insulin signalling network or cardiac function in the insulin resistant HCD hearts. High concentrations of insulin increased IFS in both the control and HCD hearts. ATM inhibition improved cardiac function in control and HCD hearts during early reperfusion but had no effect on cardiac function during later reperfusion. ATM phosphorylation was increased by insulin and decreased by KU-60019 in control hearts, but could not be manipulated in HCD hearts. Insulin-stimulated PKB/Akt activation is not ATM-dependent in the heart. However, ATM inhibition appears to down-regulate insulin signalling via PI3K, PTEN and GSK-3β. ATM inhibition caused NO-dependent vasodilation in control hearts, suggesting a role for ATM in vasoconstriction. Conclusion: ATM is a complex signalling regulator with numerous substrates. In our study, we found that acute cardiac ATM inhibition did not result in significant cardiac dysfunction or complete abrogation of insulin signalling. However, we found that ATM possibly plays a significant role in vasoconstriction. More research needs to be done to fully understand the cardiac role of ATM in insulin signalling.