| Background: Exposures of cells to very short (less than 1 microsecond) electric pulses in the megavolt/meter range have been shown to cause a multitude of effects, both physical and molecular in nature. Physically, nanosecond electrical pulse exposure can disrupt the plasma membrane, leading to a phenomenon known as nanoporation. Nanoporation is the production of nanometer sized holes (less than 2 nanometers in diameter) that can persist for up to fifteen minutes, allowing the flow of ions into and out of the cell. Nanoporation can lead to secondary physical effects, such as cellular swelling, shrinking and blebbing. Molecularly, nanosecond electrical pulses have been shown to activate signaling pathways, produce oxidative stress, stimulate hormone secretion and induce both apoptotic and necrotic death. The mechanism by which nanosecond electrical pulses cause molecular changes is unknown; however, it is thought the flow of ions, such as calcium, into the cell via nanopores, could be a major cause. The ability of nanosecond electrical pulses to cause membranes to become permeable and to induce apoptosis makes the technology a desirable modality for cancer research; however, the lack of understanding regarding the mechanisms by which nanosecond electrical pulses cause nanoporation impedes further development of this technology. This dissertation documents the genomic and proteomic responses of cells exposed to nanosecond electrical pulses and describes in detail the biophysical effects of these electrical pulses, including the demonstration for the first time of the generation of acoustic pressure transients capable of disrupting plasma membranes and possibly contributing to nanoporation.;Methods: Jurkat, clone E6-1 (human lymphocytic cell line), U937 (human lymphocytic cell line), Chinese hamster ovarian cells and adult primary human dermal fibroblasts exposed to nanosecond electrical pulses were subjected to a variety of molecular assays, including flow cytometry, fluorescent confocal microscopy, microarray analysis and or real time polymerase chain reaction. To investigate the physical interaction(s) of the electrical pulse with the aqueous environment, optical techniques such as pump-probe imaging, schlieren imaging, and probe beam deflection were used. Finally, electrochemistry was employed to modify the electrical parameters of the exposures such that different biophysical phenomena could be detected.;Results: Approximately 500 genes were selectively up-regulated in each of the assayed cells. Validation of the microarray data indicated genes such as the putative transforming gene of avian sarcoma virus 17, commonly known as jun proto-oncogene, and the Finkel--Biskis--Jinkins murine osteosarcoma viral oncogene homolog were significantly up-regulated in response to the exposure. Many of the genes selectively up-regulated in each cell type are biomarkers of mechanical stress. Proteomic analysis indicated proteins responsible for mitigation of reactive oxygen species were produced in response to nanosecond electrical pulse exposure. Analysis using the Probe Beam Deflection Technique identified the generation of an acoustic pressure transient emanating from the electrodes immediately after the application of the pulse. This acoustic pressure transient traveled at approximately 1500 meters per second, had a frequency bandwidth of 2.5 megahertz and was capable of delivering 13 kilopascals of pressure at 5 millimeters distance from the generating electrodes. Visual confirmation of the acoustic pressure transients was accomplished using pump-probe, schlieren and ultrasonic imaging techniques. Modification of the bathing media in which the cells were exposed indicated that acoustic pressure transient formation was directly dependent on the amount of electrical current induced by the exposure. Confocal microscopy revealed that, in the absence of the acoustic pressure transients, nanoporation, as detected by a green fluorescent carbocyanine nucleic acid stain, was greatly enhanced.;Conclusions: We found several genes, some of which are mechanosensitive, were selectively up-regulated due to nanosecond electrical pulse exposure. The source of this apparent mechanical stress was likely the acoustic pressure transients generated by the nanosecond electrical pulse exposure interacting with the plasma membrane of exposed cells. Contrary to our original hypothesis that these acoustic pressure transients enhance nanoporation, it appears that the opposite is true. Acoustic pressure transients generated by nanosecond electrical pulses inhibit nanoporation (or at least are negatively correlated with nanopore formation). This finding is substantiated by other reports in the literature, which indicate shock waves produced by electrical exposures inhibit gene transfection.;General Significance: This work provides strong evidence that cells exposed to nanosecond electrical pulses experience a mechanical stress which by some unknown mechanism inhibits nanoporation. The findings in this dissertation are not only poised to cause a paradigm shift in how researchers understand electrical pulses cause electropermeabilization, but also will help fill in a gap in the knowledge concerning this technology, thus enabling its further development as a potential cancer therapy. |
