Masters Degrees (Physiological Sciences)

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Browsing Masters Degrees (Physiological Sciences) by browse.metadata.advisor "Durcan, Peter"

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    Exosome dynamics and changes in a selection of their microRNA cargo in response to an acute bout of eccentrically-biased exercise in healthy human volunteers
    (Stellenbosch : Stellenbosch University, 2017-03) Lovett, Jason; Myburgh, Kathryn H.; Durcan, Peter; Stellenbosch University. Faculty of Science. Dept. of Physiological Sciences.
    ENGLISH ABSTRACT: Background: Extracellular vesicles (EVs) are nanoscale (30 to 1000 nm diameter) mediators of intercellular information transfer, shuttling diverse cargos including functional nucleic acids, proteins and lipids. EVs are released by all cell types, are stable in circulation and are thus capable of transferring information between distant tissues. It has been established that circulating EV profiles adapt to differing physiological and disease states. However, a paucity of information on this type of communication arising from skeletal muscle is apparent, especially in the context of exercise. This is despite multiple opinion reviews highlighting the importance of EVs and their cargo (particularly microRNA) as biomarkers of muscle myopathies/dystrophies/damage and even as ameliorators of chronic diseases associated with a sedentary lifestyle. Aims: To determine whether circulating EVs, within the exosome size range, display size, number and cargo alterations in response to exercise-induced muscle damage (EIMD) from an acute bout of eccentrically-biased exercise. Specific cargo selected for analysis included: miR-1, 133a, 133b, 206 and 31. Methods: Blood samples were drawn from 9 human volunteers at baseline (BL), 2 and 24 hours after acute, sequential plyometric jumping and downhill running bouts. Leg muscle pain was assessed on a scale from 1 to 10. Serum creatine kinase (CK) activity was quantified spectrophotometrically. Plasma exosomes were isolated using size exclusion columns and visualised with transmission electron microscopy (TEM). Quantitative and qualitative information on exosome protein was determined by micro-bicinchoninic acid assays and Coomassie-stained gel visualisation respectively. Exosome sizes and numbers were quantified by nanoparticle tracking analysis (NTA). Selected microRNA (miR) cargo was reverse transcribed, and quantified using qPCR with normalisation to an exogenous control (cel-miR-39). Results: Perceived muscle pain and serum CK were elevated post-exercise, providing indirect evidence for EIMD. A simplified protocol for fast exosome visualisation using TEM was achieved, and revealed an abundance of exosomes of varying sizes. A concomitant abundance of exosomes was found using the more precise NTA technique (mean = 9 x 1010 exosomes/ml plasma). Mean exosome diameters were 127 ± 15 nm across all timepoints. No change in exosome size or number was seen over time. Similarly, no change in exosome total protein concentration was apparent between timepoints. Exosome enrichment of the skeletal muscle-specific miR-206 was detected in all but one participant. Neither miR-206, nor ubiquitous myomiRs-1, -133a and -133b changed across timepoints. However, exosome miR-31 decreased from BL to 24 hr post-exercise (p < 0.05). Grouping of participants according to increases (responders) or decreases (non-responders) in exosome number from BL to 24 hr post-exercise revealed that exosome miR-133b decreased between BL and 24 hr post-exercise (p < 0.05) in non-responders (n = 6), with no characteristic change present in responders. Conclusion: A profuse number of exosomes is present in human plasma. NTA analysis revealed that size exclusion was an effective exosome isolation method. Following EIMD the number or sizes of circulating exosomes did not change. Rather, exosome cargo profiles changed. Decreased exosome miR-31 following EIMD suggests the specificity of the response, with differences in response noted between responders and non-responders.