Current progress in the development of vaccines has decreased the incidence of fatal and non-fatal infections and increased longevity. However, new technologies need to be developed to combat an emerging generation of infectious diseases. DNA vaccination has been demonstrated to have great potential for use against a wide variety of diseases. Alone, this vaccine technology does not generate a significant immune response for vaccination, but combined with delivery by electroporation (EP), can enhance plasmid expression and immunity against the expressed antigen. Most EP systems, while effective, can be invasive and painful making them less desirable for use in vaccination. Our lab recently developed a non-invasive electrode known as the multi-electrode array (MEA), which lies flat on the surface of the skin without penetrating the tissue. This study evaluated the use of the MEA for the development of DNA vaccines. We assessed the appropriate delivery conditions for gene expression and the development of humoral immunity. We used both B. anthracis and HBV as infectious models for our experiments. Our results indicated that the MEA can enhance gene expression in a mouse model with minimal to no tissue damage. Optimal delivery conditions, based on generation of antibodies, were determined to be 125-175V/cm and 150ms with 200ug and a prime boost protocol administered on Day 0 and 14. Under these conditions, end-point titers of 20,000-25,000 were generated. Neutralizing antibodies were noted in 40-60% of animals. Additionally, we utilized a guinea pig model to assess the translation potential of this electrode. The plasmid encoding HBsAg, pHBsAg, was delivered intradermally with the MEA to guinea pig skin. The results show increased protein expression resulting from plasmid delivery using the MEA as compared to injection alone. Within 48 hours of treatment, there was an influx of cellular infiltrate in the experimental groups. Humoral responses were also increased significantly in both duration and intensity as compared to the injection only groups. Results from both experimental models demonstrate that protective levels of humoral immunity can be generated and that this electrode should translate well to the clinic.

Electroporation is the critical step in an electric field mediated drug or gene delivery protocol. Electroporation based protocols have been successfully demonstrated in cancer clinical trials, however, its impact in other applications is still under investigation. A significant roadblock to long term functioning of implantable biosensors in vivo is the tissue reaction in the form of fibrous encapsulation that results in reduced transport to the sensing element of the biosensor. In vivo gene electroporation has a great potential as a means to modify the transport properties of tissues in the proximity of the sensing element of implantable biosensors. This dissertation examines two postulated electroporation based strategies to modify tissue for enhanced performance of an implantable biosensor. In the first, the implantation protocol is modified to accommodate in vivo electroporation. In the second strategy, the the modification is applied post implantation. This post-implantation in vivo electroporation application requires that electric energy be delivered at the site of electroporation close to the biosensor while minimizing effects far from such site. A novel method, focusing electric fields, developed for this purpose is presented. A theoretical framework as well as in vitro and in vivo experiments are provided as the introduction to the method and in support of its potential as the basis of a viable technology.

The Signal Transducer and Activator of Transcription-3 (Stat3) is a transcription factor that is required for transformation by a number of oncogenes, while a constitutively active form of Stat3 alone is sufficient to induce neoplastic transformation. It was previously demonstrated that cell to cell adhesion causes a dramatic increase in the activity of Stat3 in both normal and tumour cells. This hinted for the first time at the possibility that the role of Stat3 may differ upon cellular confluence. To examine such a mechanism, it is important to evaluate the effect of Stat3 downregulation at different time-points relative to confluence. To examine this, two different approaches for Stat3 downregulation were used: (1) the introduction of high levels of peptidomimetics analogs, which block the Stat3-SH2 domain by using a technique of in situ electroporation. (2) Treatment with two platinum compounds that inhibit Stat3 binding to activated receptors and DNA. The results demonstrated that Stat3 downregulation in vSrc or TAg transformed mouse fibroblast cells or in breast carcinoma lines, induced apoptosis which was more pronounced post-confluence at the time of its peak activity. In contrast, in sparsely growing normal mouse fibroblasts, Stat3 inhibition induced merely a growth retardation. However, in densely growing normal fibroblasts, Stat3 inhibition induced apoptosis. At least in part, apoptosis induced by Stat3 inhibition was mediated by p53, as shown by the resistance to cell death by Stat3 downregulation in colon carcinoma cells, HCT116, where the p53 gene is ablated. Overall, our observations point to the possibility that constitutive activation of Stat3 may lead to tumourigenesis by downregulating wt-53 in cancers that do not have p53 mutations. As a result, targeting Stat3 in cancers with wt-p53 may be a promising therapeutic approach for restoring p53 function, thereby inducing p53-mediated apoptosis. Next, we examined the effect of constitutively activated Stat3 as an oncogene. Stat3C expression in rat F111 fibroblasts induced anchorage independence, but to a lower degree compared to other oncogenes, such as vSrc. Surprisingly Stat3C expression increased gap junction intercellular communication, despite the fact that other oncogenes such as vSrc or vRas effectively block gap junctions. / Thesis (Ph.D, Pathology & Molecular Medicine) -- Queen's University, 2009-04-26 01:09:21.654

CNT membrane platforms are biomimetic polymeric membranes imbedded with carbon nanotubes which show fast fluid flow, electric conductivity, and the ability to be grafted with chemistry. A novel micro-dialysis probe nicotine concentration sampling technique was proposed and proved in vitro, which could greatly improve the efficiency and accuracy of future animal transdermal studies. To enhance the scope of transdermal drug delivery which was limited to passive diffusion of small, potent lipophilic drugs, a wire mesh lateral electroporation design was also proposed which could periodically disrupt the skin barrier and enhance drug flux. It was shown that AMP binding aptamer at the tip of carbon nanotubes may act as gatekeepers and regulate ionic transport through CNT membrane. Multiple cycle gating of ionic transport upon AMP binding/unbinding which changes the aptamer conformation was displayed. This CNT membrane-aptamer system closely mimics how protein ion channels modulate ion flow by responding to stimuli, which may have significant impact on active membrane transport. Finally an enhanced electroosmosis concept by “ratchet” functionalization at both ends of carbon nanotubes in was discussed. Direct observation of water transport by electroosmosis was made possible through enhanced flow in vertically aligned high flux CNT membranes.

The inflammatory arthritis Ankylosing Spondylitis (AS) is linked to the human leucocyte antigen HLA-B27. HLA-B27 is thought to drive AS because it misfolds during assembly in the endoplasmic reticulum (ER), inducing ER cell stress. Modulating HLA-B27 folding in the ER is therefore a therapeutic target pathway. The recent discovery of polymorphisms in the ER-resident peptidase ERAP1 that can impact on HLA-B27 and AS, makes ERAP1 one such target. Exosomes are small, typically 50-200 nm sized particles, formed in the endosomal recycling pathway, which can be released into the extracellular environment. Exosomes have a wide range of biological activities depending on the cell type of origin, and on the delivered cargo, which can include bio-active proteins, lipids, mRNA and miRNA. There is interest in the use of exosomes as drug delivery agents. Here, exosomes were studied as a delivery agent to modulate ERAP1, as a potential therapeutic tool for the treatment of AS. Exosomes, isolated from cell lines including CEM and Jurkat (T cell lineage), Jesthom (B cell lineage), U937 (monocyte lineage) and the epithelial HeLa cell line, were characterized by nanoparticle tracking analysis, flow cytometry and immunoblotting using markers including CD9, CD63, CD81 and TSG101. Differential expression of these markers in the immune cell lines indicated the complexity of defining exosomes. EVs were then tested using cell penetrating peptides, electroporation, lipid transfection and sonication for their ability to load FITC-siRNA or FITC-antibody as cargo. Significantly, post-loading RNase A or trypsin incubation demonstrated that many techniques do not lead to efficient cargo loading of exosomes. Sonication proved the most effective technique, with up to 30% efficiency. Loading of exosomes with ERAP1-targetted siRNA did not however lead to notable ERAP1 inhibition. The data indicates that external loading of exosomes with cargo remains a significant challenge in developing exosomes as therapeutic tools.

Cette thèse présente des travaux concernant la modélisation mathématique de deux méthodes physiques existantes pour surmonter les barrières biologiques s’opposant à l’administration efficace de médicaments. Dans la première partie, plusieurs manières de modéliser l’électroporation sont exposées, aux échelles tissulaire et cellulaire. Des modèles phénoménologiques existants d’électroporation tissulaire sont présentés et comparés numériquement. Puis un modèle macroscopique d’électroporation est déduit d’un modèle d’électroporation cellulaire bien établi en utilisant des techniques d’homogénéisation. Dans la seconde partie, un nouveau modèle poroélastique est introduit pour décrire les écoulements dans un tissu biologique. Celui-ci prend en compte la dégradation tissulaire consécutive à un traitement par enzyme. Pour finir, un algorithme d’optimisation est proposé dans le but de déterminer un protocole optimal pour effectuer un traitement enzymatique. / This PhD thesis is devoted to the mathematical modeling and simulation of two existing physical methods to overcome the biological barriers to drug delivery. In the first part, several ways to model electroporation are considered, from the cell scale to the tissue scale. Existing phenomenological models of tissue electroporation are presented and numerically compared. Then a macroscopic model of electroporation is derived from a well-established model of cell elecroporation using homogenization techniques. In the second part, a new poroelastic model for the flows in biological tissues is presented to account for tissue degradation after an enzymatic treatment. To finish, an optimization algorithm is suggested in attempt to determine an optimal protocol when considering enzyme based therapies.

L’intégration croissante des procédés innovants comme les ultrasons, les champs magnétiques, et les champs électriques pulsés a pour but d’améliorer et de stabiliser le déroulement des procédés de fermentation. Le champ électrique pulsé (CEP) est un procédé athermique généralement utilisé pour l’inactivation des pathogènes (Barbosa-Cánovas et al., 2001) ainsi que pour l’extraction des composés intracellulaires d’intérêt (El Zakhem et al., 2006a; Vorobiev & Lebovka 2006). Dans ce travail de thèse, nous proposons d’évaluer l’activité microbienne des cellules soumises à un traitement électrique modéré. Un intérêt particulier est apporté à des aspects fondamentaux comme la croissance et le métabolisme des cellules. Sur le plan technologique, le but fondamentale est de mettre en place et optimiser des protocoles de stimulation de microorganismes pour intensifier les bioprocédés. La fermentation de microorganismes stimulés par CEP a montré des cinétiques plus rapides que les levures non traitées. La stimulation de l’activité fermentaire s’est révélée grâce au suivi de la masse du milieu, les solutés solubles, l’absorbance, les sucres... L’optimisation des protocoles de stimulation a permis de réveler deux comportements logarithmique et saturé. Il a été montré une dépendance importante de l’énergie spécifique sur certains aspects physiologiques notamment la taille et le nombre de colonies. / The continually increasing integration of innovative technologies such as ultrasound, magnetic fields, and pulsed electric fields aims to improve and stabilize the course of fermentation processes. The pulsed electric field (PEF) is an athermal process generally used for pathogen inactivation (Barbosa-Canovas et al., 2001) and for the extraction of intracellular compounds of interest (El Zakhem et al., 2006a; Vorobiev & Lebovka 2006). In this thesis, we propose to evaluate the microbial activity of cells subjected to a moderate electric treatment. Special consideration is given to key aspects such as growth and cell metabolism. Technologically, the fundamental purpose is to implement and optimize microorganisms’ stimulation protocols to intensify their bioprocesses. The positive impact of PEF pre-treatment on yeast cells was shown by their faster fermentation kinetics compared to the control. This was proven by monitoring the weight of the ferment, the soluble solutes, the UV absorbance, and sugar consumption profiles. Two behaviors of electrostimulation, “logarithmic” and “saturated”, were revealed by optimization of the stimulation protocols. Finally, a relationship between the growth rate, the size of the colonies and the applied specific energy is deduced.

DNA polymerases are a family of molecular machines involved in high-fidelity DNA replication and repair, of which DNA polymerase I (Pol) is one the best-characterized members. Pol is a strand-displacing polymerase responsible for Okazaki fragment synthesis and base-excision repair in bacteria; it consists of three protein domains, which harbour its 5’-3' polymerase, 3’-5’ exonuclease and 5’ endonuclease activities. In the first part of the thesis, we use a combination of single-molecule Förster resonance energy transfer (smFRET) and rigid-body docking to probe the structure of Pol bound to its gapped-DNA substrate. We show that the DNA substrate is highly bent in the complex, and that the downstream portion of the DNA is partly unwound. Using all-atom molecular dynamics (MD) simulations, we identify residues in the polymerase important for strand displacement and for downstream DNA binding. Moreover, we use coarse-grained simulations to investigate the dynamics of the gapped-DNA substrate alone, allowing us to propose a model for specific recognition and binding of gapped DNA by Pol. In the second part of the thesis, we focus on the catalytically important conformational change in Pol that involves the closing of the ‘fingers’ subdomain of the protein around an incoming nucleotide. We make use of the energy decomposition method (EDM) to predict the stability-determining residues for the closed and open conformations of Pol, and test their relevance by site-directed mutagenesis. We apply the unnatural amino acid approach and a single-molecule FRET assay of Pol fingers-closing, to show that substitutions in the stability-determining residues significantly affect the conformational equilibrium of Pol. In the final part of the thesis, we attempt to study Pol in its native environment of the living cell. We make use of the recently developed method of internalization by electroporation, and optimize it for organically labelled proteins. We demonstrate the internalization and single-molecule tracking of Pol, and provide preliminary data of intra-molecular FRET in Pol, both at the single-cell and single-molecule levels. Finally, by measuring smFRET within an internalized gapped-DNA construct, we observe DNA binding and bending by endogenous Pol, confirming the physiological relevance of our in vitro Pol-DNA structure.

<p>One in eight woman develop breast cancer in the United States of America and is the most common type of cancer in the world. Breast cancer has the highest rate of death compared to any other form of cancer. Triple Negative Breast Cancer (TNBC) is the most lethal type of breast cancer, which is the most fatal of all breast cancer types. TNBC is onerous to treat since it lacks all the three most commonly targeted hormones and receptors. Current patients afflicted with TNBC are treated with platinum core chemotherapeutics, namely Cisplatin. Despite the anticancer effects shown by Cisplatin, TNBC attenuates its effect and develops a resistance eventually, which results in reoccurrence of TNBC after few years. Hence there is a demand for effective and alternative ways to treat TNBC. To inhibit the TNBC cell proliferation, blocking the key glycolytic enzyme Lactase Dehydrogenase B (LDHB) is studied and validated. Galloflavin (GF), a proven LDHB inhibitor is utilized in this series of studies and analysis. In addition, Electrochemotherapy, which involves the application of electrical pulses (EP) were utilized to enhance the uptake of GF. The combination of Electrochemotherapy (ECT) with LDHB is a novel way to treat TNBC to produce an alternative to traditional chemotherapy. EP+GF will be subjected onto TNBC cells at various concentrations and pulse parameters. The purpose of this study is to test the effect of alternative chemotherapeutic drug delivery methods for TNBC patients for decrease in mortality rate and improve quality of life. Results indicate TNBC cell viability is the least for EP+GF treatments and the maximum Reactive Oxygen Species (ROS) levels and a maximum decrease in Glucose and Lactate uptake for EP+GF treatments relative to control. Immunoblotting studies indicate the inhibition of LDHB is the most on EP+GF treatments, indicating that this could be a novel modality to treat TNBC.<br></p>

This thesis investigates the applicability of novel approaches designed to study the molecular mechanisms required for the initiation of organogenesis within the early endoderm. The endoderm is the germ layer that gives rise to the gut-tube and associated organs including the thyroid, lung, liver and pancreas. Our laboratory focuses on understanding the molecular mechanisms governing the developmental transition from endoderm to liver and pancreas. Several signaling pathways including Wnt, Retinoic Acid (RA), Bone Morphogenetic Protein (BMP) and Transforming Growth Factor-β (TGFβ) have been implicated in the emergence of the liver bud from the endoderm in the mouse or other vertebrate species. However, neither the exact signals nor the precise roles during budding process have been identified, due to the complexity of specifically altering these essential pathways using traditional genetic approaches during the earliest stages of endoderm organogenesis. These traditional techniques include transgenic, knockout or conditional knockouts strategies. To overcome the difficulties of genetic accessibility, our laboratory has optimized two complementary approaches, electroporation and addition of activators or inhibitors directly to the culture media, to study the earliest stages of organ formation using an ex vivo culture system (whole embryo culture), that allow us for normal embryonic development for up to two days. This ex-vivo technique also provides the opportunity to access and manipulate the endoderm, specifically the liver and pancreas precursor cells, prior to organ specification. Because the endoderm undergoes normal liver and pancreas specification in our ex vivo system by 24 hours after culture begin, we reason that it is possible to manipulate gene expression at the onset of culture. We then determine the effects of this manipulation on liver or pancreas development by molecular and morphological analysis after culture. The first approach we developed is the use of directional electroporation of nucleic acids to manipulate a specific region of the endoderm, particularly on liver and pancreas developmental processes. The second method is global inhibition or activation using inhibitors or growth factors activators, focusing on the TGFβ signaling pathway. These techniques will be performed prior to, or concurrent with, liver and pancreas specification, followed by embryo culture until after the onset of organogenesis. The combination of these techniques constitutes a practical approach to stage-manage the endoderm in a temporally and spatially distinct manner. In addition, it will allow us to alter specific signaling pathways without the labor-intensive generation of genetically modified animals. Indeed, establishment of these methodologies may provide a robust tool for rapid screening of candidate genes and signaling molecules underlying organogenesis in any endodermally derived organ in mouse embryos.