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Stratton, Dan (2012) Biochemical and physiological analysis of microvesicle subtypes. Doctoral thesis, London Metropolitan University.
The research presented in this thesis encompasses a detailed biophysical and biochemical analysis of extracellular vesicles: in particular microvesicles (MVs). The characterisation utilised novel applications of existing protocols, demonstrated for the first time within this thesis, resulting in an up-to-date and greatly improved series of techniques for the analysis of microvesicles.
Application of these techniques revealed two distinct subtypes of MVs that can be collected from the same parent cell types in culture, originating via different pathways of biogenesis. cMVs are constitutively released and sMVs stimulated for release. They express distinct biochemistries leading to the potential to perform different physiological functions. These subtypes exhibit distinct FACs morphologies, distinct protein, non-specific carbohydrate and nucleic acid profiles. The techniques used demonstrated in this thesis also show that the MVs have remarkable differences in size as shown for the first time by TEM, flow cytometry, DLS and Nanosight particle tracking.
FT-IR spectroscopy was used for the first time to profile similarities and differences between cell lines by analysis of the fingerprint region [1800cm-1 to 900cm-1). The MV subtypes derived from parent cells were analysed using FT-IR to demonstrate similarity in non-disclosed protein expression. BCA protein analysis, modified schiffs test, spectroscopic and quartz crystal microbalance techniques demonstrated cMVs to be smaller, denser and more slowly released from cells than the sMVs are larger, lighter and released sMVs rapidly in response to a stress stimuli.
Analysis of calcium homeostasis has revealed that MVs contain high calcium levels, that are deliverable to cells via MVs as cargo. Furthermore, the MV subtypes express different arrays of deliverable proteins and receptors that were transiently expressible upon the recipient cell.
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