Undergraduate & Postgraduate Theses
3D heart cell culture model from Zebrafish larvae for cardiac research
Author: Jake Ireland
Introduction: Zebrafish (ZF) provide an excellent platform for modelling human cardiac arrhythmias since they have comparable action potential rhythms and have high sequence identity to humans for a number of ion channels. Previous studies showed that fish heart cells can be spontaneously propagated from embryonic heart progenitor cells to a mature 3D myocardium in vitro, termed Zebrafish heart aggregates (ZFHAs). Although generated from ZF larvae, ZFHAs show similar cellular architecture to adult zebrafish in terms of sarcomeric structures and cell-to-cell connections. Numerous studies have assessed the contribution of adrenergic tone in maintaining resting heart rate in adult and larval ZF although little is known about the electrophysiology of ZFHAs. Method: To help further validate ZFHAs as a cardiac model we assessed the distinct differences in ECG pattern, contraction frequency and size under adrenergic stimulation with phenylephrine. These observations were compared to a control group of ZFHAs grown under normal culture conditions in high glucose DMEM. The development of ZFHAs involved the homogenisation of whole ZF larvae in accordance with schedule 1 methods stipulated by the home office. The first goal of this study was to distinguish the role of cardiac adrenergic receptor function in regulating contraction frequency (CF) and inotropic response in larval zebrafish compared to ZFHAs using the methods developed by Kopp et al., 2007. Cardiac function was observed using an inverted microscope and a high speed camera. Contraction frequency was measured by counting heart rate in a 20 second video and multiplied by three to get beats per minute (BPM). The size of aggregates was measured using imageJ calibrated against a 1mm graticule. Results: ZFHAs in the control group showed gradual decreases in size and a gradual increase in CF between days 1 and 6 post homogenisation (d.p.h) Day1(Size=8191.9um^2 +- 1083.1, CF=45+-3 BPM) Day6 (Size7614.6um^2 +- 1428.3um^2 CF= 67+-12BMP) while the addition of phenylephrine caused an increase in size of ZFHAs and an increased trend in CF compared to control, Day1(Size=8191.9um^2 +- 1083.1, CF=52+-14 BPM) Day6 (Size7614.6um^2 +- 1428.3um^2 CF= 75+-22BMP). At 13 (d.p.h) a positive chronotropic change was observed in ZFHA in the presence of phenylephrine (p=0.0026, n=12) which aligned with a significant increase in size seen at 12 (d.p.h) (p=0.0017, n=12). Finally a positive correlation between size and contraction frequency (r=0.2376) in the presence of phenylephrine is seen, which was significant (p=0.0415, n=74) while control groups show no correlation between CF and size (p=0.3333, n=253). The chromogenic patterns of ZFHAs in response to phenylephrine showed positive inotropic responses as well as more defined ECG patterns. Conclusion: These data suggest that ZFHA have similar developmental patterns of adrenergic receptors as larval ZF. It is also observed that adrenergic response to the agonist phenylephrine results in positive chrontropic and inotropic tone that maintains CF beyond 6 (d.p.h) and helps increase size. This response of ZFHAs to adrenergic stimulation could highlight important correlations between human and ZFHA response to chromptropic drugs developed for cardiac disease.
Genomic and Phenotypic comparison of Escherichia coli isolates from host and environmental sources
Author: Jake Ireland
BACKGROUND: Escherichia coli are human intestinal pathogens that are a frequent cause of food poisoning in humans. Studies into the genetic characteristics of E. coli shows they have a special ability to thrive in secondary habitats like soils and waters. There is also evidence to suggest that E. coli have the ability to specify pathogenicity in a few specific hosts. This has been shown in part to stem from the horizontal transfer of fitness and pathogenicity islands. AIM: Our aim is to apply comparative growing environments to determine phenotype responses of E. coli strains isolated from host and environmental sources, to determine if there are any preferences for certain environmental conditions. RESULTS: No difference was determined between host and non host but there was a comparative difference between two small groups of strains that showed preference for a particular growing condition. We identified 36 unique genes to a small set of E. coli strains that prefer to grow in endotherm mimicked conditions. In addition we have found 107 unique genes that are present in strains that prefer moderate temperate climates. We used multi genome analysis to show the presence of certain fitness and virulence factors that correlates with preferential growing conditions. CONCLUSION: From our analysis we have identified phenotypes of strains that show preferential growth based on the precedence or absence of certain virulence and fitness factors that might be relevant for distinguishing between commensal strains with enhanced growing abilities.