Thesis (Ph.D.)--Hong Kong University of Science and Technology, 2007. / Includes bibliographical references (leaves 123-132). Also available in electronic version.

No description available.

Breast cancer (BC) is the second most common cause of cancer-related deaths for women in the United States, estimated to affect 1 in 8 women. Difficulties arise in BC treatment due to the hormone sensitivity and heterogeneity of the malignancies, and the poor prognosis after metastases. Due to the immense physical and psychological effects of conventional surgical methods, minimally invasive, non-thermal, focal electroporation-based ablation therapies are being investigated for the treatment of BC. Irreversible Electroporation (IRE) delivers a series of long, monopolar electrical pulses via electrodes inserted directly into the targeted tissue which disrupt cellular membranes by creating nano-scale pores, killing the cells via loss of homeostasis while promoting an immune response. However, IRE requires cardiac synchronization and a full-body paralytic to mitigate unwanted muscle contractions, which motivated the creation of second generation High-Frequency IRE or H-FIRE. H-FIRE delivers short, bipolar pulses to destroy cancer cells without muscle contractions and nerve excitation, and allows for more tunable treatment parameters. Throughout my thesis, I discuss investigations of H-FIRE for the treatment of triple-negative and hormone-sensitive BC cell lines and compare efficacy to IRE outcomes. To further establish the translation and understanding of H-FIRE for BC applications, my master's thesis focuses on: (1) determining the lethal electric field threshold of both cell lines in a 3D hydrogel matrix after H-FIRE and IRE; and (2) employ those values in a single bipolar probe numerical model to simulate in vivo treatments. The culmination of this thesis advances the use of H-FIRE in breast tissues, as well as demonstrates how in vitro data can be used to develop clinically relevant numerical models to better predict in vivo treatment outcome. / Master of Science / Breast cancer (BC) is one of the most deadly forms of cancer for women in the United States, affecting every 1 in 8 women. Difficulties arising in the treatment of BC include the hormone sensitivity of malignancies, metastatic tendencies, and the diversity of the tissue that characterizes the breast. Surgical options like mastectomy or lumpectomy are most often used when treating BC; however, these are incredibly taxing on the patient. This reason has sparked investigations of focused ablation modalities for the treatment of BC, specifically non-thermal mechanisms like electroporation-based therapies. Electroporation explains the phenomenon that cells subjected to a high enough electric field will result in increased membrane permeability, allowing for the entrance of therapeutic agents in reversible mechanisms, or cell death beyond an irreversible point. Irreversible Electroporation (IRE) has shown success for the treatment of prostate, liver, kidney, and pancreas. However, due to some drawbacks, second generation High-Frequency IRE (H-FIRE) is increasingly being investigated for certain cancer types and is the main focus of this thesis project. Within this thesis, I discuss investigations of H-FIRE with applications to treat malignant breast cell lines. Specifically, my thesis focuses on: (1) determining the point at which cancer cells damage is irreversible; and (2) incorporate those values into a numerical model used to simulate electroporation treatment if a tumor were embedded in a layer of fatty connective breast tissue. The culmination of this thesis enhances our understanding of H-FIRE in the breast, with the hopes of future transition of application into animal studies and ultimately the clinic.

No description available.

The invention of pronuclear microinjection initiated the field of transgenic research. Over 30 years later microinjection remains the most straight-forward and most commonly used transgene delivery option. In this work we address the current progress of microelectromechanical systems (MEMS) used as transgenic delivery mechanisms. The nanoinjector is a specially designed MEMS device which uses electrostatic charge to manipulate transgene molecules. The process of nanoinjection was designed as an alternative to microinjection which causes less damage to developing embryos, improves embryo survival, birth rates, and overall efficiency of injections. In vivo testing of nanoinjection demonstrates it is both safe and effective. Additionally nanoinjection has the potential to make transgenesis via yeast artificial chromosomes more practical as the nanoinjector may prevent shearing of the YAC molecules. A second nanoinjection protocol termed intracellular electroporetic nanoinjcetion (IEN) was designed to allow for cytoplasmic injections. Cytoplasmic injections are faster and easier than pronuclear injection and do not require the pronuclei to be visible; yet previous attempts to develop cytoplasmic injection have met with limited success. In IEN injections the nanoinjector is used to place transgenic molecules in the cytoplasm. The transgenes are then propelled through the cytoplasm and electroporated into the pronucleus using electrical pulses. Electroporation of whole embryos has not resulted in transgenic animals, but the MEMS device allows localized electroporation to occur within the cytoplasm, giving transgene access to the pronucleus before degradation can occur. In this report we describe the principles which allow for localized electroporation of the pronuclei including: the location of the pronuclei between 21-28 hours post-hCG treatment, modeling data predicting the voltages needed for localized electroporation of pronuclei, and data on the movement of transgenic DNA based on the voltages delivered by IEN. We further report results of an IEN versus microinjection comparative study in which IEN produced transgenic pups with viability, transgene integration, and expression rates statistically comparable to microinjection. The ability to perform injections without visualizing or puncturing the pronuclei will widely benefit transgenic research, and will be particularly advantageous for the production of transgenic animals with embryos exhibiting reduced pronuclear visibility.

Graduation date: 1993

Vaccination is one of the major achievements of modern medicine. As a result of vaccination, diseases such as polio and measles have been controlled and small pox has been eliminated. However, despite these successes there are still many diseases of microbial origin that cause tremendous suffering because there are no vaccines or the vaccines available are inadequate. The development of DNA based vaccines and immunostimulatory CpG oligonucleotides (ODNs) as adjuvants offer new possibilities for developing new vaccines. The objectives of this research were to improve the delivery of polynucleotides and oligonucleotides to enhance their potency and to evaluate the feasibility of non-invasive methods for the delivery of vaccines through the skin in order to improve the safety and the ease of administration of human and veterinary vaccines. The results demonstrated that topical administration of plasmids in a lipid-based delivery system (biphasic lipid vesicles [Biphasix]) resulted in gene expression in the draining lymph nodes, as well as induction of antigen specific immune responses in mice. The use of electroporation significantly enhanced both gene expression and immune responses to DNA vaccines in pigs. Prior treatment with electroporation enhanced immune responses to both protein and DNA vaccines indicating that both gene expression and tissue damage are important mechanisms that electroporation uses to enhance immune responses. In addition, the formulation of CpG ODNs in biphasic lipid vesicles (BiphasixTM) called Vaccine-Targeting Adjuvant (VTA) enhanced immune response to protein antigens following systemic and mucosal administration.

Vaccination is one of the major achievements of modern medicine. As a result of vaccination, diseases such as polio and measles have been controlled and small pox has been eliminated. However, despite these successes there are still many diseases of microbial origin that cause tremendous suffering because there are no vaccines or the vaccines available are inadequate. The development of DNA based vaccines and immunostimulatory CpG oligonucleotides (ODNs) as adjuvants offer new possibilities for developing new vaccines. The objectives of this research were to improve the delivery of polynucleotides and oligonucleotides to enhance their potency and to evaluate the feasibility of non-invasive methods for the delivery of vaccines through the skin in order to improve the safety and the ease of administration of human and veterinary vaccines. The results demonstrated that topical administration of plasmids in a lipid-based delivery system (biphasic lipid vesicles [Biphasix]) resulted in gene expression in the draining lymph nodes, as well as induction of antigen specific immune responses in mice. The use of electroporation significantly enhanced both gene expression and immune responses to DNA vaccines in pigs. Prior treatment with electroporation enhanced immune responses to both protein and DNA vaccines indicating that both gene expression and tissue damage are important mechanisms that electroporation uses to enhance immune responses. In addition, the formulation of CpG ODNs in biphasic lipid vesicles (BiphasixTM) called Vaccine-Targeting Adjuvant (VTA) enhanced immune response to protein antigens following systemic and mucosal administration.

Nanosecond pulse electroporation of biological cells is gaining significant interest due to its ability to influence intracellular structures. In nanosecond pulse electroporation of biological cells nanosecond duration pulses with high frequency spectral content are applied to the cell. In this research we show that accurate modeling of the nanosecond pulse electroporation process requires considering the effect of the membrane dielectric relaxation on the electric potential across the membrane. We describe the dielectric relaxation of the membrane as dispersion in the time-domain and incorporate it into the nonlinear asymptotic model of electroporation. Our nonlinear dispersive model of a biological cell is solved using finite element method in 3-D space enabling arbitrary cell structures and internal organelles to be modeled. The simulation results demonstrate two essential differences between dispersive and non-dispersive membrane models: the process of electroporation occurs faster when the membrane dispersion is considered, and the minimum required electric field to electroporate the cell is significantly reduced for the dispersive model.

Nanosecond pulse electroporation of biological cells is gaining significant interest due to its ability to influence intracellular structures. In nanosecond pulse electroporation of biological cells nanosecond duration pulses with high frequency spectral content are applied to the cell. In this research we show that accurate modeling of the nanosecond pulse electroporation process requires considering the effect of the membrane dielectric relaxation on the electric potential across the membrane. We describe the dielectric relaxation of the membrane as dispersion in the time-domain and incorporate it into the nonlinear asymptotic model of electroporation. Our nonlinear dispersive model of a biological cell is solved using finite element method in 3-D space enabling arbitrary cell structures and internal organelles to be modeled. The simulation results demonstrate two essential differences between dispersive and non-dispersive membrane models: the process of electroporation occurs faster when the membrane dispersion is considered, and the minimum required electric field to electroporate the cell is significantly reduced for the dispersive model.