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.

In this study, we examined the effects of thyroid hormone on regulatory processes of cholesterol metabolism. Specifically, the pathways of cholesterol synthesis and cholesterol efflux were investigated. Hepatic HMG-CoA reductase (HMGR) is the rate-limiting enzyme in cholesterol synthesis. Hypothyroid rats exhibit decreased expression of this gene, which can be induced by subsequent treatment with thyroid hormone. The mechanism of this activation was previously unknown. Utilizing in vivo electroporation, we identified HMGR promoter elements necessary for the induction of HMGR by thyroid hormone. The -316/-321 element, the sterol response element, and nuclear factor-y site were all found to be necessary to induce HMGR promoter activity by thyroid hormone. We used electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) studies to demonstrate increased binding of upstream transcription factor-2 (USF-2) to the -316/-321 element in the HMGR promoter in response to thyroid hormone. Finally, co-electroporation of the wild-type HMGR plasmid with siRNA to USF-2, SREBP-2, or NF-Y nearly abolished the T3 induction as measured by promoter activity. Microarray and real-time PCR analysis demonstrated an induction of the apolipoproteins ApoA-I and ApoA-IV mRNA by T3. Serum levels of ApoA-I and ApoA-IV proteins were induced by T3. We collected serum from rats treated with or without T3 and used these sera in an in vitro macrophage efflux model. We found that T3 promoted cholesterol efflux via the ABCA1 cholesterol transporter and not via the ABCG1 transporter. We propose that the induction of serum ApoA-I and ApoA-IV by thyroid hormone promotes cholesterol efflux via the ABCA1 cholesterol transporter. Hepatic ABCG5 and ABCG8 are cholesterol transporters that promote biliary secretion of cholesterol. We utilized EMSAs to scan the shared ABCG5/G8 rat promoter for a thyroid hormone response element (TRE). We identified a TRß binding site at -392/-376 of the ABCG8 promoter. Collectively, these observations provide new insight into the cholesterol-lowering function of thyroid hormone.

Gene transfer plays an important role in bacterial evolution. Especially in an under explored species like Rhodococcus, a type of bacteria found in the soil. Rhodococcus has several applications in the pharmaceutical industry and in the production of antibiotics. Rhodococcus possess several unique sets of properties which makes it beneficial to have a reliable method of producing mutants of Rhodococcus. The goal of the experiment was to find an efficient way of forming Rhodococcus colonies with kanamycin resistant genes. The project began from an unexpected observation from an earlier experiment with Rhodococcus strain MTM3W5.2. where I attempted to transform this strain with a transposon via electro-transformation. The colonies that grew/ appeared transformants were screened to confirm the presence of kanamycin gene, however there was no amplified DNA seen on the PCR gel (i.e. absence of the kanamycin gene). The electro-transformant colonies were selected on LB plates containing different higher concentrations of kanamycin. Then the appeared transformants were again screened via disk diffusion assay and were classified into 3 different kanamycin resistant phenotypes. Majority of the “C” phenotypic colonies (i.e., high level resistance to kanamycin) appear to contain the kanamycin gene, but these colonies were less in numbers. This led us to try another method of gene transfer which is conjugation. Conjugation was carried on a double selection antibiotic plate containing both chloramphenicol (30 µg) and kanamycin (100 µg). The transconjugate colonies that appeared on the double selection plates were also screened by PCR, but none of the colonies had amplified DNA suggesting absence of the kanamycin gene. The colonies seen on the double selection plate were possibly due to spontaneous mutation or some type of unknown phenotypic variation. However, in the future, double selection plates with higher concentrations of antibiotics can possibly give us transconjugants with kanamycin genes.

Les ondes térahertz s’étendent de 0.1 à 10x1012 Hz, à la frontière entre les domaines de l’optique et des radiofréquences. Cette position intermédiaire originale en a longtemps rendu l’accès difficile : les technologies térahertz n’ont pris leur essor qu’au cours des années 90. Le domaine n’a pas encore atteint la maturité des domaines des microondes ou de l’infrarouge qui le jouxtent. Cependant, les motivations exploratoires sont fortes, de par la sensibilité spectroscopique du térahertz aux états moléculaires (rotationnels, vibrationnels..) et aux liaisons faibles établies dans et entre les molécules. Dans le cas des objets biologiques, le térahertz est particulièrement sensible à l’eau : sa quantité, son état physico-chimique et ses solutés.Nous avons mis en œuvre un montage d’imagerie en réflexion interne totale atténuée (ATR) pour pouvoir distinguer des cellules vivantes de leur milieu physiologique. Au cours de ce travail, le montage d’imagerie ATR a été caractérisé théoriquement, puis expérimentalement. La première démonstration de l’origine du contraste sur ces images térahertz a été réalisée. Il provient du contenu intracellulaire, plus spécifiquement des protéines et peptides dissouts dans le cytoplasme.Une analyse fine des mécanismes sous-jacents à la nature protéique du contraste térahertz a également été développée. Elle donne accès à des informations spectroscopiques inédites sur l’eau, les protéines dissoutes et la couche de solvatation les entourant.Mettant à profit cette compréhension de notre montage térahertz, nous l’avons proposé comme outil non invasif de suivi quantitatif de la perméabilisation de cellules en conditions physiologiques. Lors de la perméabilisation, augmentation des transferts moléculaires à travers la membrane, notre outil permet de quantifier le passage des peptides et protéines. La perméabilisation de cellules vivantes a une gamme d’application vaste, de l’entrée de fluorochromes pour l’imagerie ou de médicaments à la thérapie génique. Afin d’assurer ces passages à travers la membrane des cellules, il est nécessaire d’altérer ses propriétés, sans pour autant compromettre la viabilité cellulaire. L’étude de deux types de perméabilisation avec notre outil térahertz est proposée : la perméabilisation chimique et l’électroporation. Dans les deux cas, des mécanismes d’effet dose ont été caractérisés quantitativement. Notre outil térahertz a démontré de grands avantages devant les méthodes actuellement utilisées pour quantifier ces dynamiques de perméabilisation et en caractériser la réversibilité. / Lying between 0.1 to 10x1012 Hz, the terahertz radiation occupies a middle ground between microwaves and infrared light waves, sometimes named “the terahertz gap” for technologies relevant to generation and detection have only risen at the beginning of the 90’s and aren’t fully developed yet. Nevertheless, there are strong exploratory incentives because of terahertz spectroscopic sensitivity to molecular states (rotational, vibrational…) and weak bounds in and between molecules. In the case of biological object, terahertz waves are especially sensitive to water: its quantity, physico-chemical state and solutes. We implemented an Attenuated Total internal Reflection (ATR) imaging setup in order to distinguish live cells from their physiological bathing medium. Throughout this work, we characterized both experimentally and experimentally the ATR setup. The first demonstration of the contrast origin in the terahertz images obtained was done. It arises from the intracellular content, more specifically the proteins and peptides dissolved in the cytoplasm.A precise analysis of the underlying mechanism of this proteinaceous terahertz contrast has also been developed. It gives access to original spectroscopic information about water, dissolved proteins and the hydration shell around them.Taking advantage of our whole setup comprehension, we proposed it as a non-invasive tool for quantitative live-cell permeabilization assessment in physiological conditions. During permeabilization, aka increased molecular transfers through the cell membrane, our tool allows to quantify the transfer of peptides and proteins. Live-cell permeabilization has a large application range, from fluorochrome entry in imaging, to drugs or gene therapy. In order to ensure molecules crossing the cell membrane, it’s necessary to alter its properties without compromising cell viability.A study of two permeabilization methods is proposed: chemical permeabilization and electroporation. In both cases dose effect mechanisms were quantitatively characterized. Our terahertz tool demonstrated great advantages over classical permeabilization quantification methods and permeabilization reversibility assessment methods.

This study investigated the use of electroporation as an extraction method of lipids in oleaginous microorganisms. Electroporation is the process of placing a voltage gradient across a lipid membrane to create pores that vary in size and longevity with voltage magnitude and pulse duration. Once the voltage gradient is removed, the lipid membrane will seal the pore. The use of electroporation on oleaginous microorganisms to extract stored lipids could be a useful tool if the microorganism is allowed to regenerate and produce more lipids. Three high-lipid media were investigated: soybeans, Rhodococcus opacus (bacteria), and Rhodotorula glutinis (yeast). This study investigates varied voltage magnitude, pulse duration, quantity of pulses, and distance between electrodes. Electroporation proved to be moderately successful for lipid removal when using low voltages and long pulse durations. However, electroporation removed only a small percentage of the intracellular lipids.

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The heterogeneity of pancreatic cancer renders many available general therapies ineffective holding the five-year survival rate close to 10% for decades. Surgical resection eligibility, resistance to chemotherapy and limited efficacy of immunotherapy emphasize the dire need for diverse and innovative treatments to combat this challenging disease. This study evaluates co-therapy strategies that combine non-thermal, minimally invasive ablation technology and targeted drug delivery to enhance treatment efficacy. Our research begins by uncovering the multifaceted potential of Irreversible Electroporation (IRE), a cutting-edge non-thermal tumor ablation technique. This study demonstrates IRE-mediated ability to trigger programmed necrotic cell death, induce cell cycle arrest, and modulate immune cell populations within the tumor microenvironment. This transformation from a pro-tumor state to a proinflammatory milieu, enriched with cytotoxic T lymphocytes and neutrophils. IRE-induced proinflammation in the tumor site renders immunologically "cold" tumor into immunologically "hot" tumor and holds significant promise of improving treatment efficacy. Notably, IRE-treated mice exhibited an extended period of progression-free survival, implying clinical potential. The transient nature of these effects suggests potential mechanisms of tumor recurrence highlighting the need for further studies to maximize the efficacy of IRE. Our mechanistic studies evaluated the IFN-STAT1-PD-L1 feedback loop as a possible reason for pancreatic tumor recurrence. Our data also suggest a stronger IFN-PD-L1 feedback loop compared to mammary, osteosarcoma and glioblastoma tumors rendering pancreatic cancer immunologically "cold". This study also investigates the use of histotripsy (a non-thermal, noninvasive, nonionizing ultrasound-guided ablation modality) to treat pancreatic cancer utilizing a novel immunocompromised swine model. We successfully generated human orthotopic pancreatic tumors in the immune deficient pigs, which allowed for consequent investigation of clinical challenges presented by histotripsy. While rigorous clinical studies are indispensable for validation, the promise of histotripsy offers new hope for patients. In parallel, we used our immunocompromised swine model of orthotopic pancreatic cancer to investigate the SonoTran® system, which employs ultrasound-activated oscillating particles to enhance drug delivery within hard-to-reach tumors. Our study demonstrates that SonoTran® significantly enhances the intratumoral penetrance of therapeutic agents, including commonly used chemotherapy drugs like paclitaxel and gemcitabine. Additionally, SonoTran® improved delivery of the anti-epidermal growth factor (EGFR) monoclonal antibody, cetuximab- which is frequently used in cancer immunotherapy. Together, our findings address challenges in the delivery of a range of therapeutics while simultaneously exposing challenges like off-target damage. In conclusion, this study presents a multifaceted approach to confront the complex characteristics of pancreatic cancer. Given the variations in patient response and the complexity of the disease, it is clear that a singular solution is unlikely. Our research, which combines IRE, histotripsy, and SonoTran®, to interrogate a promising array of tools to tackle different challenges to provide tailored treatments. In the ever-evolving landscape of pancreatic cancer therapy, this research opens new avenues to investigate deeper into molecular mechanisms, co-therapy treatment options, future preclinical and clinical studies which eventually encourage the potential for improved patient outcomes. / Doctor of Philosophy / Pancreatic cancer is a formidable disease, known for its late-stage diagnosis and limited treatment options with a poor 5-year survival rate of ~10%. However, a promising frontier in the battle against this lethal disease has emerged through combining mechanical, cell based and immunotherapies to attack the cancer from multiple angles at once. In my PhD research, I explored novel approaches to transform the landscape of pancreatic cancer treatment. We began by investigating Irreversible Electroporation (IRE), a non-thermal method to ablate tumors. Beyond its known function of reducing tumor size, IRE initiated programmed necrotic cell death, halted tumor cell division, and triggered changes in the immune landscape within the tumor. In response to IRE treatment, the immune environment shifted from pro-tumor to proinflammatory state, showing potential for clinical use. Mice treated with IRE experienced extended cancer progression-free survival temporarily, followed by eventual relapse. During relapse, we found that immune cells reverted back to their original, pre- IRE treated state. This observation logically implies combining IRE and immune checkpoint inhibitors aimed towards maintaining the IRE-altered immunological environment. Next, we developed and used novel pig models that closely resemble human pancreatic cancer patients to test histotripsy, a first phase toward making histotripsy as a non-invasive treatment approach for pancreatic cancer. Use of orthotopic tumor in a large animal model and clinical device allowed us to expose some challenges of ultrasound guidance of histotripsy. Notably, the treatment results in partial ablation and a reduction in stroma materials, which play a role in the tumor's resistance to commonly used treatments. While rigorous clinical studies are needed for validation, this approach offers hope in the quest for innovative pancreatic cancer treatment. Another promising approach we investigated involves SonoTran® particles, ultrasound-activated oscillating particles that can increase drug absorption in a targeted fashion. Our study demonstrated increased concentrations of commonly used therapeutic agents within tumors through SonoTran®-facilitated delivery, providing an effective means to overcome drug delivery issues within pancreatic tumors. There is no one size fits all treatment to address the complexity of pancreatic cancer. The future of treatment lies in the integration of IRE, histotripsy and SonoTran® into clinical practice. In summary, this PhD research identified promising novel technologies and combinations of treatments for pancreatic cancer, reaffirming the importance of exploring innovative solutions to combat pancreatic cancer. The dynamic nature of the pancreatic tumor microenvironment underscores the importance of further research to extend the positive impacts of these treatments and improve tumor debulking.

The most common and aggressive malignant brain tumor, glioblastoma (GBM), demonstrates on average a 5-year survival rate of only 6.8%. Difficulties arising in the treatment of GBM include the inability of large molecular agents to permeate through the blood-brain barrier (BBB); migration of highly invasive GBM cells beyond the solid tumor margin; and gross, macroscopic intratumor heterogeneity. These characteristics complicate treatment of GBM with standard of care, resulting in abysmal prognosis. Electroporation-based therapies have emerged as attractive alternates to standard of care, demonstrating favorable outcomes in a variety of tumors. Notably, irreversible electroporation (IRE) has been used for BBB disruption and nonthermal ablation of intracranial tumor tissues. Despite promising results, IRE can cause unintended muscle contractions and is susceptible to electrical heterogeneities. Second generation High-frequency IRE (H-FIRE) utilizes bursts of bipolar pulsed electric fields on the order of the cell charging time constant (~1 μs) to ablate tissue while reducing nerve excitation, muscle contraction, and is far less prone to differences in electrical heterogeneities. Throughout my dissertation, I discuss investigations of H-FIRE for the treatment of malignant gliomas and other intracranial disorders. To advance the versatility, usability, and understanding of H-FIRE for intracranial applications, my PhD thesis focuses on: (1) characterizing H-FIRE-mediated BBB disruption effects in an in vivo healthy rodent model; (2) the creation of a novel, real-time impedance spectroscopy technique (Fourier Analysis SpecTroscopy, FAST) using waveforms compatible with existing H-FIRE pulse generators; (3) development of FAST as an in situ technique to monitor ablation growth and to determine patient-specific ablation endpoints; (4) conducting a preliminary efficacy study of H-FIRE ablation in an orthotopic F98 rodent glioma model; and (5) establishing the feasibility of MRI-guided H-FIRE for the ablation malignant gliomas in a spontaneous canine glioma model. The culmination of this thesis advances our understanding of H-FIRE in intracranial tissues, as well as develops a novel, intraoperative impedance spectroscopy technique towards determining patient-specific ablation endpoints for intracranial H-FIRE procedures. / Doctor of Philosophy / The most aggressive malignant brain tumor, glioblastoma (GBM), demonstrates on average a 5-year survival rate of only 6.8%. Difficulties arising in the treatment of GBM include the inability of chemotherapy agents to diffuse into brain tumor tissue as these molecular are unable to pass the so-called blood-brain barrier (BBB). This tumor tissue also presents with cells with the propensity to invade healthy tissue, to the point where diagnostic scans are unable to capture this migration. These characteristics complicate treatment of GBM with standard of care, resulting in abysmal prognosis. Electroporation-based therapies have emerged as attractive alternates to standard of care, demonstrating favorable outcomes in a variety of tumors. For instance, irreversible electroporation (IRE) has been used to successfully treat tumors in the prostate, liver, kidney, and pancreas. Second generation High-frequency IRE (H-FIRE) may possess even greater antitumor qualities and this is the focus of my dissertation. Throughout my dissertation, I discuss investigations of H-FIRE with applications to treat malignant gliomas and other intracranial disorders. My PhD thesis focuses on: (1) characterizing H-FIRE effects for enhanced drug delivery to the brain; (2) the creation of a new, real-time electrical impedance spectroscopy technique (Fourier Analysis SpecTroscopy, FAST) using waveforms compatible with existing H-FIRE pulse generators; (3) development of FAST as a technique to determine H-FIRE treatment endpoints; (4) conducting a preliminary efficacy study of H-FIRE to ablate rodent glioma tumors; and (5) establishing the feasibility of MRI-guided H-FIRE for the ablation malignant gliomas in a spontaneous canine glioma model. The culmination of this thesis advances our understanding of H-FIRE in intracranial tissues, as well as develops a new impedance spectroscopy technique to be used in determining patient-specific ablation endpoints for intracranial H-FIRE procedures.

Microfluidics has revolutionized how molecular biology studies are conducted. It permits profiling of genomic and epigenomic features for a wide range of applications. Microfluidics has been proven to be highly complementary to NGS technology with its unique capabilities for handling small volumes of samples and providing platforms for automation, integration, and multiplexing. In this thesis, we focus on three projects (diffusion-based PCR, MID-RRBS, and SurfaceChIP-seq), which improved the sensitivities of conventional assays by coupling with microfluidic technology. MID-RRBS and SurfaceChIP-seq projects were designed to profiling genome-wide DNA methylation and histone modifications, respectively. These assays dramatically improved the sensitivities of conventional approaches over 1000 times without compromising genomic coverages. We applied these assays to examine the neuronal/glial nuclei isolated from mouse brain tissues. We successfully identified the distinctive epigenomic signatures from neurons and glia. Another focus of this thesis is applying electrical field to investigate the intracellular contents. We report two projects, drug delivery to encapsulated bacteria and mRNA extraction under ultra-high electrical field intensity. We envision rapid growth in these directions, driven by the needs for testing scarce primary cells samples from patients in the context of precision medicine. / Ph. D.

Microfluidics, the manipulation of fluids at nanoliter scale, has emerged to offer an ideal platform for biological analysis of a low number of cells. The technological advances in microfluidics have allowed both forming of valves, mixers and pumps and integrating of optic and electronic components into microfluidic devices to construct complete and functional systems. In this dissertation, I present novel microfluidic techniques and their applications in cellular probes delivery, cell separation and epigenetic study. In the first part of the dissertation, electroporation is implemented on microfluidic platform to generate uniform delivery of "exposed" nanoparticle or protein into cells. In contrast to endocytosis, electroporation is a physical method to breach cell membrane and does not involve vesicle encapsulation of delivered probes, which means these probes have exposed surface in the cytosol. Such trait enables the use of delivered nanoparticle and protein for intracellular targeting of native biomolecules. Laser-induced fluorescent microscopy was used for single particle illuminating to track single molecules in cells. Microfluidic device provide integrated platform for conducting electroporation, cell culture and imaging. In the second part, microfluidic immunomagnetic cell separation is introduced. I showed two new approaches to enhance immunomagnetic cell separation based on (1) uniquely microfabricated paramagnetic patterns inside separation channels; and (2) using combination of nonmagnetic beads and magnetic beads for selection of tumor initiating cells based on two markers of opposite preference in one step. Enhancement in cell isolation (high capture efficiency or high selection purity) is experimentally observed and the former is explained by computational model. In the final part of the dissertation, microfluidic device incorporating valves and mixers for sensitive study of chromosome conformation is presented. This device has small reaction chamber minimizing sample requirement, and allows multiple steps of biological analysis in a single chip avoiding sample loss during sample transfer. Several orders of magnitude improved detection sensitivity is achieved with our microfluidics based method. I envision all novel techniques discussed in this dissertation have great potential in application of disease prognosis, diagnosis and treatment. / Ph. D.