Increasing consumer demand for new products with high nutritional qualities has spurred a search for new alternatives to food preservation. Pulsed electric field (PEF) is an emerging technology for non thermal food pasteurisation. Using this technology, enzymes, pathogenic and spoilage microorganisms can be inactivated without affecting the colour, flavour, and nutrients of the food. PEF treatment may be provided by applying pulsed electric field to a food product in a treatment zone between two electrodes at ambient , or slightly above ambient temperature. Exposure of microbial cells to the electric field induces a transmembrane potential in the cell membrane, which results in electroporation (the permeabilization of the membranes of cells and organelles) and/or electrofusion (the connection of two separate membranes into one) of the cells. An innovative pulsed electric field (PEF) unit was designed and constructed in the University of Auckland using modern IGBT technology. The system consists of main equipments, the high voltage pulse generator and the treatment chambers. The main focus of this work was to design an innovative PEF treatment chamber that provide uniform distribution of electric field, minimum increase in liquid temperature, minimum fouling of electrodes and an energy efficient system. Four multi pass treatment chambers were designed consisting of two stainless steel mesh electrodes in each chamber, with the treated fluid flowing through the openings of the mesh electrodes. The two electrodes are electrically isolated from each other by an insulator element designed to form a small orifice where most of the electric field is concentrated. Dielectric breakdown inside the chambers was prevented by removing the electrodes far from the narrow gap. The effect of the chambers different geometries on the PEF process in terms of electric parameters and microbial inactivation were investigated. Electric field intensity in the range of (17-43 kV/cm) was applied with square bipolar pulses of 1.7 µs duration. The effect of PEF treatment on the inactivation of gram-negative Escherichia coli ATCC 25922 suspended in simulated milk ultra-filtrate (SMUF) of 100%, 66.67% and 50% concentration was investigated. Treatments with the same electrical power input but higher electric field strengths provided larger degree of killing. The inactivation rate of E coli was significantly increased with increasing the electric field strength, treatment time and processing temperature. Morphological changes on E coli as a result of PEF treatment were studied under transmission electron microscopy (TEM). Significant morphological changes on E coli after PEF treatment were observed. The TEM studies suggested that the microbial inactivation was a consequence of electroporation and electrofusion mechanisms. Kinetic analysis of microbial inactivation due to PEF and thermal treatment of E coli suspended in SUMF were also studied. Comparison between measured (experimental) and predicted (theoretical) variation of E coli concentration with time following the PEF treatment was discussed, taking into consideration the recirculation mode of the PEF treatment. The treated liquid was circulated more than once through the treatment chamber to provide higher microbial inactivation. Arrhenius constants and activation energies of E coli inactivation using combined PEF and thermal treatment were calculated and generalized correlation for the inactivation rate constant as a function of electric field intensity and treatment temperature was developed. / Fonterra Research Institute (NZ) and the Foundation for Research Science and Technology (NZ)

<div>Pulsed electric fields (PEFs) can induce numerous biophysical phenomena, especially perturbation of the outer and inner membranes, that may be used for applications that include nonthermal pasteurization, enhanced permeabilization of tumors to improve the transport of chemotherapeutics for cancer therapy, and enhanced membrane permeabilization of individual cells to enhance RNA and DNA delivery for gene therapy. The applied electric field and pulse duration determine the density, size, and reversibility of the created membrane pores. PEFs with durations longer than the outer membrane’s charging time will induce pore formation with the potential for application in irreversible electroporation for cancer therapy and microorganism inactivation. Shorter duration PEFs, particularly on the nanosecond timescale (nsPEFs), induce a larger density of smaller membrane pores with the potential to permeabilize intracellular membranes, such as the mitochondria, to induce programmed cell death. Thus, the PEFs can effectively kill multiple types of cells, dependent upon the cells. This thesis assesses the ability of nsPEFs to kill different cell types, specifically microorganisms with and without antibiotics as well as varying the parameters to affect populations of immortalized leukemia cells (Jurkats).</div><div>Antibiotic resistance has been an acknowledged challenge since the initial development of penicillin; however, recent discoveries by the CDC and the WHO of microorganisms resistant to last line of defense drugs combined with predictions of potential infection cases reaching 50 million a year globally and the absence new drugs in the discovery pipeline highlight the need to develop novel ways to combat and overcome these resistance mechanisms. Repurposing drugs, exploring nature for new drugs, and developing enzymes to counter the resistance mechanisms may provide potential alternatives for addressing the scarcity of antibiotics effective against gram-negative infections. One may also leverage the abundance of drugs effective against gram-positive infections by using nsPEFs to make them effective against gram-negative infections, including bacterial species with multiple natural and acquired resistance mechanisms. Numerous drug and microbial combinations for different doses and pulse treatments were tested and presented here.</div><div>Low intensity PEFs may selectively target cell populations at different stages of the cell cycle (quiescence and mitosis) to modify cancer cell population dynamics. Experimental studies of cancer cell growth when exposed to a low number of nsPEFs, while varying pulse duration, field intensity and number of pulses reveals a threshold beyond which cell recovery is not possible, but also a point of diminishing returns if cell death is the intention. A theory comprised of coupled differential equations representing the proliferating and quiescent cells showed how changing PEF parameters altered the behavior of these cell populations after treatment. These results may provide important information on the impact of PEFs with sub-threshold intensities and durations on cell population growth and potential recurrence.</div>

The aim of this master thesis is to design a high voltage source for the generation of bipolar pulses, which are suitable for the process of reversible and irreversible electroporation. Further design is electronic control. The first part of the thesis analyzes the theoretical knowledge about the process of electroporation, the second part describes the principles of realization of such sources, the next part deals with the design of the source and its control. The penultimate part is a description of the user manual and the last part is dedicated to the measurement.

The aim of this master thesis is to design a power source for generating pulsed electric field for the needs of the technological process of electroporation of fruit musts and mashes. To design further the network of switched capacitors and inductors based on the required pulse and to design and implement the basic control unit together with the switching transistors. The thesis will include calculations and simulations used in the draft, also design solutions and measured values. This thesis is divided into several basic parts. In the first part there is discussed in detail the theoretical knowledge of electroporation and the desired characteristics of generated pulses are chosen according to this knowledge. Each part of electroporation workplace is described in the second part of this thesis, beginning from the source through the control system to the electrode chamber. For each of these parts are given different possible alternatives. In the next chapter is already proceeded to the design of the source. There are listed required parameters of the pulses and according to them calculations and the design are gradually carried out. Another chapter deals with the simulations, which are used to verify the calculated values and conditions in the electroporated sample. Last but one part discusses the mechanical design of the workplace. There are described all problems of the construction and commissioning of the product. The last section is dedicated to the workplace measurement and analysis of the measured results.

Les microalgues, de par leur diversité, peuvent offrir une multiplicité de molécules bio-sourcées pour des applications variées (alimentation, énergie, santé etc…). Cependant, la production de biodiesel à partir de microalgues, désignée comme la 3e génération de biocarburant, nécessite encore une optimisation lors de l’étape de culture de la biomasse ou lors de l’extraction de l’huile pour que le procédé soit énergétiquement viable. Parmi les voies d’amélioration, l’application de champs électriques pulsés (PEF) en prétraitement à la biomasse pourrait améliorer la rentabilité énergétique du procédé d’extraction de lipides. Ce procédé appliqué aux microalgues est étudié dans le contexte d’une collaboration entre le laboratoire SATIE de l’ENS Cachan Paris Saclay et le laboratoire LGPM de Paris Saclay.Un microsystème d’électroporation a été conçu afin d’étudier in situ l’impact des champs électriques pulsés sur les cellules de microalgue, Chlamydomonas reinhardtii chargées en lipides. Parmi les principaux résultats du projet, l’étude énergétique du procédé a montré que les impulsions de très courte durée (5 µs) sont les moins énergivores. Associées à un champ électrique de 4.5 kV/cm, ces impulsions entrainent une perméabilisation réversible (80 % de cellules atteintes) de quelques secondes tandis qu’un champ de 7 kV/cm entraine un effet irréversible. Après ce prétraitement, les algues sont ensuite mélangées à de l’hexane afin d’évaluer si les lipides sont extraits plus facilement de la cellule. / Microalgae offer a multiplicity of applications for the production of bio-sourced compounds such as proteins, pigments, sugars and oils. However, the energy spent for algae culture and lipid extraction hinder the energetic viability of the process for the production of biofuel derived from algae oils. Among possible improvements, pulsed electric fields (PEF) may be used as a pre-treatment to extract valuable compounds from microalgae and making the process less energy demanding.This project started with a collaboration between the team of bio-micro-systems Biomis, laboratory SATIE, with the team of bio-process engineering laboratory LGPM to study in situ the effects of PEF on microalgae.First, a energetic study is performed in a micro-system specially built for this project to characterize in situ, the effect of various treatment parameters (pulse duration / electric field) on Chlamydomonas reinhardtii cells with high lipid content.Among the outputs of this study, an energetic optimization of PEF conditions shows that a high level of permeability and low energy consumption are obtained when using short pulses of 5 µs. Associated with an electric field of 4.5 kV/cm, the pores are reversible (80% of the cells) during few seconds, and with a field of 7 kV/cm or higher, the permeabilization is irreversible. Afterwards, this PEFpre-treatment is associated with solvent mixing (hexane) to evaluate if lipid extraction is improved.

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Irreversible electroporation has recently emerged as an effective focal ablation technique. When performed clinically, the procedure involves placing electrodes into, or around, a target tissue and applying a series of short, but intense, pulsed electric fields. Oftentimes, patient specific treatment plans are employed to guide procedures by merging medical imaging with algorithms for determining the electric field distribution in the tissue. The electric field dictates treatment outcomes by increasing a cell's transmembrane potential to levels where it becomes energetically favorable for the membrane to shift to a state of enhanced permeability. If the membrane remains permeabilized long enough to disrupt homeostasis, cells eventually die. By utilizing this phenomenon, irreversible electroporation has had success in killing cancer cells and treating localized tumors. Additionally, if the pulse parameters are chosen to limit Joule heating, irreversible electroporation can be performed safely on surgically inoperable tumors located next to major blood vessels and nerves. As with all technologies, there is room for improvement. One drawback associated with therapeutic irreversible electroporation is that patients must be temporarily paralyzed and maintained under general anesthesia to prevent intense muscle contractions occurring in response to pulsing. The muscle contractions may be painful and can dislodge the electrodes. To overcome this limitation, we have developed a system capable of achieving non-thermal irreversible electroporation without causing muscle contractions. This progress is the main focus of this dissertation. We describe the theoretical basis for how this new system utilizes alterations in pulse polarity and duration to induce electroporation with little associated excitation of muscle and nerves. Additionally, the system is shown to have the theoretical potential to improve lesion predictability, especially in regions containing multiple tissue types. We perform experiments on three-dimensional in vitro tumor constructs and in vivo on healthy rat brain tissue and implanted tumors in mice. The tumor constructs offer a new way to rapidly characterize the cellular response and optimize pulse parameters, and the tests conducted on live tissue confirm the ability of this new ablation system to be used without general anesthesia and a neuromuscular blockade. Situations can arise in which it is challenging to design an electroporation protocol that simultaneously covers the targeted tissue with a sufficient electric field and avoids unwanted thermal effects. For instance, thermal damage can occur unintentionally if the applied voltage or number of pulses are raised to ablate a large volume in a single treatment. Additionally, the new system for inducing ablation without muscle contractions actually requires an elevated electric field. To ensure that these procedures can continue to be performed safely next to major blood vessels and nerves, we have developed new electrode devices that absorb heat out of the tissue during treatment. These devices incorporate phase change materials that, in the past, have been reserved for industrial applications. We describe an experimentally validated numerical model of tissue electroporation with phase change electrodes that illustrates their ability to reduce the probability for thermal damage. Additionally, a parametric study is conducted on various electrode properties to narrow in on the ideal design. / Ph. D.

Electroporation affects the dielectric properties of cells. Dielectric measurement techniques can provide a label-free and non-invasive modality to study this phenomenon. In this thesis we introduce a dielectrophoresis (DEP) based technique to study changes in the cytoplasm conductivity of single Chinese hamster ovary (CHO) cells immediately after electroporation. Using a microfluidic chip, we study changes in the DEP response of single CHO cells a few seconds after electroporation. First, in order to quantify our DEP measurement results and relate them to the cells internal conductivity, we introduce a dielectric model for CHO cells. This is achieved by measuring the DEP response of many individual cells in the β-dispersion frequency region and curve fitting to the measured data. Second, we present quantitative results for changes in the cytoplasm conductivity of single cells subjected to pulsed electric fields with various intensities. We observe that when electroporation is performed in media with lower ionic concentration than cells cytoplasm, their internal conductivity decreases after electroporation depending on the intensity of applied pulses. We also observe that with reversible electroporation there is a limit on the decrease in the cells’ internal conductivity. We hypothesize the reason is the presence of large and relatively immobile negative ions inside the cell which attract mobile positive ions (mainly sodium and potassium) to maintain cell electrical neutrality. We monitor the temporal response of cells after electroporation to measure the time constant of changes due to ion transport and observe this ranges from seconds to tens of seconds depending on the applied pulse intensity. This result can be used to infer information about the density and resealing time of very small pores (not measurable with conventional marker molecules). Lastly, we measure the electroporation of cells in media with different conductivities. Our results show that electroporation in very low conductivity media requires stronger pulses to achieve a similar poration extent as in high conductivity media. The outcome of this thesis can be used to improve our understanding of the dynamics of electroporation as well as its modelling in order to make more accurate predictions or optimize the process for specific applications. / February 2017