Electroporation-based treatments are motivated by the response of biological membranes to high- intensity pulsed electric fields. These fields rearrange the membrane structure to enhance the membrane's diffusive permeability, or the degree to which a membrane allows molecules to diffuse through it, is impacted by the structure, composition, and environment in which the cell resides. Tracer molecules have been developed that are unable to pass through intact cell membranes yet enter permeabilized cells. This dissertation investigates the hypothesis that the flow of such molecules may be used to quantify the effects of the electrical stimulus and environmental conditions leading to membrane electroporation. Specifically, a series of electrical pulses that alternates between positive and negative pulses permeabilizes cells more symmetrically than a longer pulse with the same total on-time. However, the magnitude of this symmetric entry decreases for the shorter alternating pulses. Furthermore, a method for quantitatively measuring the permeability of the cell membrane was proposed and validated. From data near the electroporation threshold, the response of cells varies widely in the manner in which cells become permeabilized. This method is applied to study the transient cell membrane permeability induced by electroporation and is used to demonstrate that the cell membrane remains permeable beyond 30 min following treatment. To analyze these experimental findings in the context of physical mechanisms, computational models of molecular uptake were developed to simulate electroporation. The results of these simulations indicate that the cell's local environment during electroporation facilitates the degree of molecular uptake. We use these models to predict how manipulating both the environment of cells during electroporation affects the induced membrane permeability. These experimental and computational results provide evidence that supports the hypothesis of this dissertation and provide a foundation for future investigation and simulation of membrane electroporation. / PHD / Electroporation is a biophysical process in which intense electric fields permeabilize bilayer membranes. The degree to which a membrane allows molecules to diffuse through it is called its diffusive permeability, and is impacted by the structure, composition, and environment in which the cell resides. This dissertation investigates the hypothesis that the flow of molecules into cells through their membranes may be used to quantitatively study the effects of the electrical stimulus and environmental conditions leading to membrane disruption. Here, I demonstrate that the cellular response to pulsed electric fields is affected by the waveform of the applied electrical stimulus. Specifically, a series of electrical pulses that alternates between positive and negative pulses permeabilizes cells more symmetrically than a longer pulse with the same total energized time. However, the total molecular uptake decreases for the shorter alternating pulses over the longer pulse. A method for quantitatively measuring the permeability of the cell membrane using a fluorescent tracer molecule is also developed and validated. This method is applied to show how cell membrane permeability changes following electroporation. To analyze these findings, computational models of molecular flow through the cell membrane are developed. These simulations indicate that the cell’s surrounding environment during electroporation dramatically impacts the degree of molecular uptake. We use these models to predict how manipulating both the environment of cells during electroporation affects the induced membrane permeability. These experimental and computational results provide a foundation for future investigation and simulation of membrane electroporation.

Thesis (PhD)--Stellenbosch University, 2015. / ENGLISH ABSTRACT: In this work we investigate a new method to measure the electric field of ultrafast laser pulses by extending a known measurement technique, ptychography, in the spatial domain to the time domain which we call time domain ptychography. The technique requires the measurement of intensity spectra at different time delays of an unknown temporal object and a known probe pulse. We show for the first time by measurement and calculation that this technique can be applied with excellent results to recover both the amplitude and phase of a temporal object. This technique has several advantages, such as fast convergence, the resolution is limited by the usable measured spectral bandwidth and the recovered phase has no sign ambiguity. We then extend the technique to pulse characterization where the probe is derived form the temporal object by filtering meaning the probe pulse is also unknown, but the spectrum of the probe pulse must be the same as the spectrum of the temporal object before filtering. We modify the reconstruction algorithm, now called ptychographic iterative reconstruction algorithm for time domain pulses (PIRANA), in order to also reconstruct the probe and we show for the first time that temporal objects, a.k.a laser pulses, can be reconstructed with this new modality. / AFRIKAANSE OPSOMMING: In hierdie werk het ons ’n nuwe metode ondersoek om die elektriese veld van ’n ultravinnige laser puls te meet deur ’n bekende meettegniek wat gebruik word in die ruimtelike gebied, tigografie, aan te pas vir gebruik in die tyd gebied genaamd tyd gebied tigografie. Die tegniek vereis die meting van ’n reeks intensiteit spektra by verskillende tyd intervalle van ’n onbekende ‘tyd voorwerp’ en ’n bekende monster puls. Ons wys vir die eerste keer deur meting en numeriese berekening dat hierdie tegniek toegepas kan word met uitstekende resultate, om die amplitude en fase van ’n ‘tyd voorwerp’ te meet. Hierdie tegniek het verskeie voordele, die iteratiewe proses is vinnig, die resolusie van die tegniek word bepaal deur die spektrale bandwydte gemeet en die fase van die ‘tyd voorwerp’ word met die korrekte teken gerekonstrueer. Ons het hierdie tegniek uitgebrei na puls karakterisering waar die monster pulse afgelei word, deur ’n bekende filter te gebruik, van die onbekende ‘tyd voorwerp’ nl. die inset puls. Ons het die iteratiewe algoritme wat die ‘tyd voorwerp’ rekonstrueer aangepas om ook die monster puls te vind en ons wys dat ons hierdie metode suksesvol kan gebruik om laser pulse te karakteriseer

Οι ολοένα αυξανόμενες ενεργειακές απαιτήσεις των σημερινών κοινωνιών και το γεγονός ότι οι ηλεκτρικές συσκευές και εξοπλισμός έχουν γίνει αναπόσπαστο κομμάτι της καθημερινότητάς μας, έχει οδηγήσει στην αναγωγή του ηλεκτρισμού σε κοινωνικό αγαθό. Παρόλα αυτά, η υπερβολική του χρήση έχει προκαλέσει έντονες ανησυχίες στο κοινό, όσον αφορά τα ηλεκτρομαγνητικά πεδία που εκπέμπονται λόγω του εναλλασσόμενου ρεύματος. Η παρούσα εργασία αποσκοπεί να ξεδιαλύνει το «θολό τοπίο» γύρω από τις επιδράσεις των χαμηλόσυχνων ηλεκτρομαγνητικών πεδίων (δηλαδή τα εκπεμπόμενα πεδία από τις εγκαταστάσεις ηλεκτρικής ενέργειας και τις ηλεκτρικές συσκευές) στην ανθρώπινη υγεία κι έτσι να συνεισφέρει ως ένα βαθμό στην άρση των ανησυχιών του κοινού και των επιπτώσεων που αυτές συνεπάγονται. Ειδικότερα, η εργασία μπορεί να συνοψιστεί ως εξής: Στο Κεφάλαιο 1 γίνεται μια θεωρητική αναφορά στις βασικές έννοιες της ηλεκτρομαγνητικής ακτινοβολίας. Προσδιορίζονται οι επιβλαβείς ακτινοβολίες για την υγεία καθώς επίσης και οι ιδιότητες των χαμηλόσυχνων ηλεκτρομαγνητικών πεδίων. Ακόμη, παρουσιάζονται οι κανονισμοί προστασίας και τα όρια έκθεσης σε ηλεκτρομαγνητικά πεδία, τόσο στην Ευρώπη, όσο και στην Ελλάδα. Στο Κεφάλαιο 2 παρουσιάζονται αναλυτικά οι διαφορετικές πηγές χαμηλόσυχνων ηλεκτρομαγνητικών πεδίων και γίνεται σύγκριση των πεδιακών εντάσεων στο περιβάλλον τους. Στο Κεφάλαιο 3 γίνεται λόγος για τη βιολογική επίδραση των ηλεκτρομαγνητικών πεδίων. Παρουσιάζονται οι επιδράσεις τους στην ανθρώπινη υγεία και οι δράσεις που λαμβάνουν χώρα τόσο για τη μελέτη αυτών των επιδράσεων, όσο και για την προστασία του κοινού. Στο Κεφάλαιο 4 γίνεται ανασκόπηση του προτύπου λήψης μετρήσεων που ακολουθήθηκε. Εξετάζεται το πρότυπο ΙΕC 61786 για μέτρηση χαμηλόσυχνων ηλεκτρομαγνητικών πεδίων και αναφέρονται κάποια χαρακτηριστικά του προτύπου EN 50413 για μέτρηση ηλεκτρομαγνητικών πεδίων όλων των συχνοτήτων. Στο Κεφάλαιο 5 περιγράφεται ο σκοπός και ο εξοπλισμός των μετρήσεων. Παρατίθενται τα αποτελέσματα των μετρήσεων συνοδευόμενα με διαγράμματα και σχόλια επί της μεταβολής της μαγνητικής επαγωγής στο περιβάλλον σχολικών συγκροτημάτων. Τέλος συνοψίζονται τα συμπεράσματα που εξάγονται με βάση όσα εξετάστηκαν στην παρούσα εργασία. Σύμφωνα με αυτά δεν πρέπει να υπάρχει κανένας λόγος ανησυχίας για την επίδραση των χαμηλόσυχνων ηλεκτρομαγνητικών πεδίων στην ανθρώπινη υγεία, καθώς τηρούνται όλοι οι κανονισμοί, οι οποίοι εμπεριέχουν μεγάλους συντελεστές ασφαλείας. / The ever-increasing energy demands of today's society and the fact that electrical appliances and equipment have become an integral part of our daily lives has led to the deduction of electricity into social good. However, its excessive use has caused great concern to the public, regarding to electromagnetic fields’ emissions because of the AC current. This essay aims to unravel the "misty area" around the effects of low frequency electromagnetic fields (i.e. fields emitted from power systems and electrical appliances) to human health and thus contribute to some extent to the disentanglement of public preoccupations and the consequences arising therefrom. In particular, this essay can be summarized as follows: In Chapter 1 there is a theoretical report in the basic concepts of electromagnetic radiation. Harmful radiation as well as the properties of low frequency electromagnetic fields is identified. Moreover, protection regulations and limits on exposure to electromagnetic fields are presented, both in Europe and in Greece. In Chapter 2 different sources of low frequency electromagnetic fields are presented and the fields’ intensities in their environment are compared. Chapter 3 talks about the biological effects of electromagnetic fields. It also presents the effects on human health and the actions that take place for the study of these effects, and for the public’s protection. Chapter 4 gives an overview of the pattern of taking measurements that was followed. The IEC 61786 standard for measuring low frequency electromagnetic fields is examined and some features of the standard EN 50413 for measuring electromagnetic fields of all frequencies are presented. Chapter 5 describes the measurements’ purpose and equipment. The measurements’ results are given, which are accompanied by charts and comments about the change of the magnetic induction around school buildings. Finally, the conclusions based on this essay’s data are summed up, according to which, there should be no reason for public concern about the effect of low frequency electromagnetic fields on human health, since compliance with all special regulations, which contain large safety factors, is being performed.

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The effects of short term exposure (5 minutes) to EEFs at relatively high dosages (10, 25, 39, kV/m) on the electrical activity in rat pineal glands was studied. Daytime and nighttime recordings were taken from an implanted microelectrode in the gland. The data show that (1) both the activity and frequency were enhanced when the animals were exposed to EEFs at 39 kV/m continuously and discontinuously; (2) the later condition yielded a sustained increase (36%) whereas the former a brief (10 sec) increase. This enhancement was statistically significant under both conditions (day and night). The effects observed were thought to be due to membrane alterations either in the pineal gland itself or in the neural inputs to the gland.

Electric fields, which are generated by the movement of charged ions across membranes, are found in all biological systems and can influence cellular components ranging from amino acids to biological macromolecules. Physiological field strengths range from 1 – 200 mV/mm, and these electric fields are especially elevated at sites of cellular growth during development and regeneration. It has previously been demonstrated that elevated electric fields induce alignment of astrocyte processes in vitro, enhancing the rate of neurite outgrowth. It is believed that electric fields of varying physiological strength affect other astrocytic responses associated with regeneration. To characterize the duration over which these changes emerge, cultured rat astrocytes were exposed to different direct-current electric field strengths. The resulting cellular behaviors were recorded every three minutes with an inverted microscope equipped with DIC optics and a stage incubator. Electric fields were found to induce astrocyte responses similar to those observed during periods of neurodevelopment and regeneration. Changes in astrocyte movement, proliferation, & morphology emerged within the first hour and persisted through the course of the electric field application, leading mammalian astrocytes to revert to an earlier maturation state resembling those seen in amphibian astrocytes associated with central nervous system regeneration. Collectively, these results suggest that applied electric fields lead to astrocyte dedifferentiation, with certain electric field strengths eliciting and enhancing specific cell responses.

Le procédé industriel d’extraction d'inuline consiste en une diffusion de longue durée et à température élevée. Ce procédé induit l’extraction des sucres mais également des impuretés. Les étapes de purification de jus deviennent alors plus complexes. Pour pallier à ces problèmes, il est essentiel de réduire la température de diffusion pour limiter l’extraction des impuretés. Récemment, la technique de champs électriques pulsés (CEP) a démonté ses avantages dans le domaine sucrier et également dans d’autres applications agro-alimentaires. L’objectif de ce travail de thèse est de d’optimiser et valider la faisabilité de cette techniques dans le cas de la chicorée. Une purification par filtration dynamique est envisagée et proposée pour remplacer la purification classique. Les résultats de l’extraction montrent qu’il est possible d’intensifier l’extraction de l’inuline par application de CEP. Le prétraitement des cossettes de chicorées à 600 V/cm et 50 ms, réduit de 20°C la température de diffusion (60°C au lieu de 80°C actuellement). Une économie d’énergie importante est réalisée (gain 90 kJ/kg chicory juice). La combinaison innovante d’un prétraitement électrique et un traitement thermique (chauffage ohmique) provoque une perméabilisation importante des cellules et permet de réaliser des diffusions « à froid ». Les analyses qualitatives de jus de diffusion classique et jus CEP montrent que la pureté des extraits CEP est plus élevée (87.2% vs 86.7%). Les essais de filtration sur membrane montrent que les performances de la filtration (pureté, flux, colmatage,…) dépendent énormément de l’origine de l’extrait. Le jus CEP contient moins d’impureté que le jus thermique, est donc plus facile à filtré. Cependant, col matage de membrane restent dans les deux cas important. Un module de filtration dynamique avec disque rotatif a été utilisé pour améliorer les performances de la filtration. Ce module a permit de réduire le colmatage des membranes, améliorer la pureté des extraits et atteindre des facteurs de réduction volumique important (10). / Industrial inulin extraction requires high temperature and long duration to ensure inulin productivity. In addition complex steps are necessary to purify extracted chicory juice and to obtain inulin with desirable purity. In order to avoid the extracting of inulin at high temperature, to obtain chicory juice with better quality and to purify chicory juice with more simple operation, pulsed electric fields (PEF) and membrane filtration were applied to this study : PEF was used to intensify inulin extraction from chicory roots. By pre-treatment of chicory slices at 600 V/cm, diffusion temperature at pilot scale can be decreased to 60oC from about 80oC (industrial extraction condition) to obtain comparable juice inulin concentration. Moreover, this moderate temperature diffusion brought out interesting energy savings compared to that of conventional diffusion. For the goal of extracting inulin at “cold” condition (for example 30oC) PEF induced combined electroporation/ohmic heating pretreatment was applied for chicory tissue denaturation. Better solute extraction was observed after effective damage of chicory tissue diffusivity.Juice extracted from PEF assisted diffusion (PEF juice) was confirmed had higher inulin purity and less impurity in comparison to juice from conventional thermal diffusion process. The juice was purified by membrane filtration in order to investigate a simple chicory juice purification method. Since it contains less impurity, dead-end filtration performance of PEF juice was significantly improved, with less membrane fouling and higher inulin purity in filtrate. However, permeate flux of membrane filtration was seriously restricted by the membrane fouling. In order to improve filtration flux, rotating disk module was introduced in this study for clarification and purification of chicory juice. Elevated shear rate of rotating disk (1500–2000 rpm) could effectively control or even eliminate membrane fouling and lead to satisfying carbohydrate transmission (98%) and desirable permeate flux even at high volume reduction ratio (10). In view of energy saving, optimization of RDM assisted chicory juice filtration was carried out and yielded optimal operation conditions to maximize average flux and minimize specific energy consumption.