Date of Award
Doctor of Philosophy (PhD)
Andrei G. Pakhomov
R. James Swanson
Exposure of cells to a pulsed electric field (PEF) is the basis of multiple techniques and treatments. Nanosecond pulsed electric field (nsPEF) poses unique characteristics to induce subtle cellular effects while preserving cell integrity. Improving understanding of the mechanisms triggered by nsPEF in cells inspires new applications for the nanosecond pulse technology. Although many effects of nsPEF remain unknown, they can be inferred from morpho-physiologic changes, or cell reshaping, that accompany nsPEF exposure. During the exposure cells undergo reshaping that is manifested in swelling and diffuse blebbing. Recently we identified two new distinct forms of reshaping, pseudopod-like blebbing and microvesiculation, which are present in cells exposed to long trains of nsPEF with high pulse repetition frequency (PRF). Microvesiculation is known in activated and damaged cells, while pseudopod-like blebbing has not been described previously. The objective of this dissertation is to characterize and establish the key mechanisms involved in these new nsPEF-induced phenomena. Two specific aims of the dissertation are 1) to establish the factors involved in nsPEF-induced microvesiculation; and 2) to define pseudopod-like blebbing as a function of pulse parameters and establish the mechanisms underlying the formation of pseudopod-like blebs (PLBs). These aims are fulfilled through the analysis of microscopy data obtained from the nsPEF-exposed cells using fluorescent labeling and pharmacologic inhibition. The adapted labeling techniques take advantage of the nsPEF-induced cell permeabilization to induce staining of microvesicles and pseudopod-like blebs (PLB). The results show that microvesiculation develops in HL60 and U937 cells in response to Ca2+ presence during nsPEF exposure. Microvesiculation does not depend on colloid-osmotic swelling (COS). PLBs are produced in U937 cells due to active formation of actin cortex and require the absence of Ca2+. Extension of PLBs is triggered and guided by nanosecond pulses while bleb growth is fueled by water uptake through a COS mechanism. PLB retraction is produced by myosin contractility and can be coupled to cell translocation.
Rassokhin, Mikhail A..
"New Types of Morpho-Physiological Changes in Cells Exposed to Nanosecond Pulsed Electric Field"
(2013). Doctor of Philosophy (PhD), Dissertation, , Old Dominion University, DOI: 10.25777/brmj-kt68