Microfluidic devices are ideal for analysis of biological systems. The small dimensions result to controlled handling of the flow profile and the cells in suspension. Implementation of additional forces in the system, such as an electric field, promote further manipulation of the cells. In this dissertation, I show novel, unique microfluidic approaches for manipulation and analysis of mammalian cells by the aid of electrical methods or the architecture of the device. Specifically, for the first time, it is shown, that adoption of electrical methods, using surface electrodes, promotes cell concentration in a microchamber due to isoelectric focusing (IEF). In contrast to conventional IEF techniques for protein separation, a matrix is not required in our system, the presence of which would even block the movement of the bulky cells. Electric field is, also, used to breach the cell membrane and gain access to the cell interior by electroporation (irreversible and reversible). Irreversible electroporation is used in a unique, integrated microfluidic device for cell lysis and reagentless extraction of DNA. The genomic material is subsequently analyzed by on-chip PCR, demonstrating the possible elimination of the purification step. On the other hand, reversible electroporation is used for the delivery of exogenous molecules to cells. For the first time, the effect of shear stress on the electroporation efficiency of both attached and suspended cells is examined. On the second part of my dissertation, I explore the capabilities of the architecture of microfluidic devices for cell analysis. A simple, unique method for compartmentalization of a microchamber in an array of picochambers is presented. The main idea of the device lies on the fabrication of solid supports on the main layer of the device. These features may even hold a dual nature (e.g. for cell trapping, and chamber support), in which case, single cell analysis is possible (such as single cell PCR). On the final chapter of my dissertation, a computational analysis of the flow and concentration profiles of a device with hydrodynamic focusing is conducted. I anticipate, that all these novel techniques will be used on integrated microfluidic systems for cell analysis, towards point-of-care diagnostics. / Ph. D.

This work investigates the use of irreversible electroporation (IRE) for tissue engineering applications and as a cancer ablation therapy. IRE uses short, high-intensity electric pulses to create pores in a cell's membrane and disrupt its stability. At a certain energy level, damage to the cell becomes too great and it leads to cell death. The particular mechanisms that drive this response are still not completely understood. Thus, further characterization of this behavior for cell death induced by pulsed electric fields (PEFs) will advance the understanding of these types of therapies and encourage their use to treat unresectable tumors that can benefit from the non-thermal mechanism of action which spares critical blood vessels and nerves in the surrounding area. We evaluate the response to PEFs by different cell types through experimental testing combined with computer simulations of these treatments. We show that IRE can be used to kill a specific type of bacteria that produce cellulose which can be used as an implantable material to repair damaged tissues. By killing these bacteria at particular times and locations during their cellulose production, we can create conduits in the overall structure of this material for the transport of oxygen and nutrients to the cells within the area after implantation. The use of tissue models also plays a key role in the investigation of various cancer treatments by providing a controlled environment which can mimic the state of cells within a tumor. We use tumor models comprised of a mix of collagen and cancer cells to evaluate their response to IRE based on the parameters that induce cell death and the time it takes for this process to occur. The treatment of prostate and pancreatic cancer cells with standard monopolar (only positive polarity) IRE pulses resulted in different time points for a full lesion (area of cell death) to develop for each cell type. These results indicate the presence of secondary processes within a cell that induce further cell death in the border of the lesion and cause the lesion to increase in size several hours after treatment. The use of high-frequency irreversible electroporation (H-FIRE)--comprised of short bursts of high-intensity, bipolar (both positive and negative polarity) pulses--can selectively treat cancer cells while keeping healthy cells in the neighboring areas alive. We show that H-FIRE pulses can target tumor-initiating cells (TICs) and late-stage, malignant cancer cells over non-malignant cells using a mouse ovarian cancer model representative of different stages of disease progression. To further explore the mechanisms that drive this difference in response to IRE and H-FIRE, we used more complex tumor models. Spheroids are a type of 3D cell culture model characterized by the aggregation of one or more types of cells within a single compact structure; when embedded in collagen gels, these provide cell-to-cell contact and cell-to-matrix adhesion by interactions of cells with the collagen fibers (closely mimicking the tumor microenvironment). The parameters for successful ablation with IRE and H-FIRE can be further optimized with the use of these models and the underlying mechanisms driving the response to PEFs at the cellular level can be revealed. / Ph. D. / This work investigates the use of irreversible electroporation (IRE) for tissue engineering applications and as a cancer ablation therapy. IRE uses short, high-intensity electric pulses to create pores in a cell’s membrane and disrupt its stability. At a certain energy level, damage to the cell becomes too great and it leads to cell death. The particular mechanisms that drive this response are still not completely understood. Thus, further characterization of this behavior for cell death induced by pulsed electric fields (PEFs) will advance the understanding of these types of therapies and encourage their use to treat unresectable tumors that can benefit from the non-thermal mechanism of action which spares critical blood vessels and nerves in the surrounding area. We evaluate the response to PEFs by different cell types through experimental testing combined with computer simulations of these treatments. We show that IRE can be used to kill a specific type of bacteria that produce cellulose which can be used as an implantable material to repair damaged tissues. By killing these bacteria at particular times and locations during their cellulose production, we can create conduits in the overall structure of this material for the transport of oxygen and nutrients to the cells within the area after implantation. The use of tissue models also plays a key role in the investigation of various cancer treatments by providing a controlled environment which can mimic the state of cells within a tumor. We use tumor models comprised of a mix of collagen and cancer cells to evaluate their response to IRE based on the parameters that induce cell death and the time it takes for this process to occur. The treatment of prostate and pancreatic cancer cells with standard monopolar (only positive polarity) IRE pulses resulted in different time points for a full lesion (area of cell death) to develop for each cell type. These results indicate the presence of secondary processes within a cell that induce further cell death in the border of the lesion and cause the lesion to increase in size several hours after treatment. The use of high-frequency irreversible electroporation (H-FIRE)—comprised of short bursts of high-intensity, bipolar (both positive and negative polarity) pulses—can selectively treat cancer cells while keeping healthy cells in the neighboring areas alive. We show that H-FIRE pulses can target tumor-initiating cells (TICs) and late-stage, malignant cancer cells over non-malignant cells using a mouse ovarian cancer model representative of different stages of disease progression. To further explore the mechanisms that drive this difference in response to IRE and H-FIRE, we used more complex tumor models. Spheroids are a type of 3D cell culture model characterized by the aggregation of one or more types of cells within a single compact structure; when embedded in collagen gels, these provide cell-to-cell contact and cell-to-matrix adhesion by interactions of cells with the collagen fibers (closely mimicking the tumor microenvironment). The parameters for successful ablation with IRE and H-FIRE can be further optimized with the use of these models and the underlying mechanisms driving the response to PEFs at the cellular level can be revealed.

Il est possible de perméabiliser la membrane plasmique des cellules par des agents chimiques (tels que les polyéthylènes glycols ou le diméthylsulfoxyde) ou par des agents physiques (tels que les ultrasons ou les impulsions électriques). Cette perméabilisation peut être réversible ou non, ce qui signifie qu’après la perméabilisation, la membrane retrouve son intégrité et ses propriétés d’hémi-perméabilité ou pas. Ces techniques peuvent être utilisées pour faire rentrer des médicaments ou des acides nucléiques dans les cellules ou pour générer des fusions cellulaires. Une approche récente, la dynamique moléculaire, utilise des simulations numériques pour prédire les effets des agents perméabilisants sur les membranes à l’échelle moléculaire, et permet d’apporter de nouvelles données pour comprendre les mécanismes moléculaires, encore peu connus à ce jour.Les impulsions dites « classiques » en électroperméabilisation, de l’ordre de la dizaine de millisecondes à la centaine de microsecondes et d’amplitude de champ de l’ordre de 100 kV/m, perméabilisent la membrane plasmique uniquement. Cependant, récemment, des impulsions plus courtes, dites impulsions nanoseconde (quelques nanosecondes) et de plus grande amplitude de champ (de l’ordre de 10 MV/m) ont été utilisées et permettent d’affecter également les membranes des organites cellulaires. Les travaux de cette thèse portent dans un premier temps sur les effets perméabilisants d’un agent chimique (le diméthylsulfoxyde, DMSO) en comparant les modèles prédictifs de la dynamique moléculaire avec des expériences in vitro sur des cellules. Le modèle numérique prédit trois régimes d’action en fonction de la concentration du DMSO. Utilisé à faible concentration, il y a déformation de la membrane plasmique. L’utilisation d’une concentration intermédiaire entraîne la formation de pores membranaires et les fortes concentrations de DMSO ont pour conséquence la destruction de la membrane. Les expériences in vitro faites sur des cellules ont confirmé ces résultats en suivant l’entrée de marqueurs de perméabilisation. Cette étude a été comparée avec la perméabilisation par un agent physique (les impulsions électriques). Dans un deuxième temps, ces travaux traitent du développement et de l’utilisation d’un nouveau dispositif d’exposition des cellules aux impulsions nanoseconde qui permet d’appliquer des champs électriques très élevés et d’observer par microscopie leurs au niveau cellulaire. Pour finir, ce dispositif a été utilisé avec des impulsions nanoseconde pour générer des pics calciques dans de cellules souches mésenchymateuses qui présentent des oscillations calciques spontanées liées à leur état de différenciation. Ces pics induits sont dus à la libération de calcium stocké dans les organites et/ou à la perméabilisation de la membrane plasmique permettant l’établissement d’un flux de calcium intramembranaire. Il est aussi possible d’utiliser des impulsions microseconde pour générer des pics calciques dans ces cellules. Dans ce cas, les pics calciques ne sont dus qu’à la perméabilisation de la membrane plasmique. En jouant sur l’amplitude des champs électriques appliqués et sur la présence ou l’absence de calcium externe, il est possible de manipuler les concentrations calciques cytosoliques en mobilisant le calcium interne ou externe. Une des particularités de ces nouveaux outils est de pouvoir être déclenchés et arrêtés instantanément, sans réminiscence, contrairement aux molécules chimiques permettant de produire des pics calciques. Ces outils pourraient donc permettre de mieux comprendre l’implication du calcium dans des mécanismes comme la différenciation, la migration ou la fécondation. / It is possible to permeabilize the cellular plasma membrane by using chemical agents (as polyethylen glycols or diméthylsulfoxyde) or physical agents (as ulstrasounds or electric pulses). This permeabilization can be reversible or not, meaning that after the permeabilization, the membrane recovers its integrity and its hemi-permeable properties. These techniques can be used for the uptake of medicines or nucleic acids or to generate cellular fusions. A recent approach, the molecular dynamics, uses numerical simulations to predict the effects of permeabilizing agents at the molecular scale, allowed generating of new data to understand the molecular mechanisms that are not completely known yet.The pulses so called “classical” in electropermeabilization, from the range of the ten of milliseconds to the hundred of microseconds and with a field amplitude in the range of 100 kV/m, can only permeabilize the plasma membrane. However, more recently, shorter pulses, so called nanopulses (few nanosecondes) and with an higher field amplitude (in the range of 10 MV/m) have been used and allow to affect also cellular organelles membranes.This thesis is, in a first time, about the permeabilizing effects of a chemical gent (the diméthylsulfoxyde, DMSO) by comparing predictive models from molecular dynamics with experiments in vitro on cells. The numerical model predicts three regimes of action depending on the DMSO concentration. Used at low concentration, there is a plasma membrane deformation. The use of an intermediate concentration lead to membrane pores formation and higher DMSO concentrations resulted in membrane destruction. The experiments done in vitro on cells confirmed these results using the following of permeabilization markers. This study has been compared to permeabilization due to a physical agent (electric pulses).Secondly, it is about the development and the use of a new cell exposure device for nanopulses that permit to apply very high electric fields and to observe induced cellular effects simultaneously by microscopy.To finish, this device has been used with nanopulses to generate calcium peaks in mesenchymal stem cells that are presenting spontaneous calcium oscillations in correlation to their differentiation state.. These induced peaks are due to the release of the calcium stored in organelles and/or to plasma membrane permeabilization leading to a intramembrane calcium flux establishment. It is also possible to use microsecond pulses to generate calcium peaks in these cells. In this case, the calcium peaks are due to the plasma membrane permeabilization . By changing the amplitude of the applied electric fields and the presence or the absence of external calcium, it is possible to manipulate cytosolic calcium concentrations by mobilizing internal or external calcium. One feature of these new tools is to be triggered and stopped instantly without reminiscence, unlike chemical molecules permitting the production of calcium peaks. These tools could therefore lead to a better understanding of the involvement of calcium in mechanisms such as differentiation, migration or fertilization.

Le vieillissement de la peau est l'un des signes extérieurs du passage du temps. Avec l’âge, la peau devient plus sèche et se ride suite à la dégradation des macromolécules de la matrice extracellulaire par des enzymes cutanées telles que l’élastase, l’hyaluronidase et la collagénase. L’objectif de ce travail de thèse est de développer des tests enzymatiques miniaturisés en électrophorèse capillaire pour cribler des extraits de plantes et identifier de nouveaux bioactifs pour la cosmétique et le bienêtre de la peau. Ces essais ont été développés soit en dehors du capillaire (qui sert uniquement de milieu de séparation) ou dans le capillaire (qui sert alors de nanoréacteur enzymatique), puis optimisés pour permettre la détermination des constantes cinétiques (Km, Vmax et IC₅₀). La diffusion transversale des réactifs (TDLFP) a été appliquée pour mélanger les créneaux de réactifs injectés dans le capillaire. Des détecteurs tels que la fluorescence induite par laser ou la spectrométrie de masse à haute résolution ont été couplés à l’électrophorèse capillaire afin d’atteindre de fortes sensibilités de détection et la possibilité d’identifier les produits de la réaction enzymatique. Ces essais miniaturisés ont été appliqués à des algues extraites par électroporation ou à des plantes régionales extraites par des technologies vertes afin d’évaluer leur activité biologique vis-à-vis des enzymes de la peau. Les essais développés sont fiables, robustes et économes en réactifs. Enfin, l’utilisation d’une nouvelle technique d’analyse, la thermophorèse à micro-échelle, s’est montrée très utile et pleine d’espoir pour l’étude des interactions enzyme-effecteur. / Skin aging is one of the exterior/external signs of the passage of time. With age, the skin becomes drier and gets wrinkled due to the degradation of macromolecules of the extracellular matrix by skin enzymes such as elastase, hyaluronidase and collagenase. The aim of this thesis is to develop miniaturized enzymatic assays by capillary electrophoresis to screen plant extracts and identify new bioactives for cosmetics and skin wellbeing. These assays were developed either outside the capillary (which serves only as a separation tool) or in the capillary (which then serves as an enzymatic nanoreactor) then optimized to allow the determination of kinetic constants (Km, Vmax and IC₅₀). Tranvserse diffusion of laminar flow profiles (TDLFP) was applied to mix the reactants injected into the capillary. Detectors such as laser-induced fluorescence or high-resolution mass spectrometry have been coupled to capillary electrophoresis to achieve high sensitivities of detection and the possibility of identifying the products of the enzymatic reaction. These miniaturized assays were applied to algae extracted by electroporation or to regional plants extracted by green technologies in order to evaluate their biological activity towards skin enzymes. The assays developed are reliable, robust and economic in reactants consumption. Finally, the use of a new analytical technique, microscale thermophoresis, was shown to be very useful and hopeful for the study of enzyme-effector interactions.

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

Níveis sustentáveis de hormônio de crescimento humano (hGH) circulante e aumento de peso altamente significativo, avaliados também em comparação a repetidas injeções de hormônio, foram observados em trabalhos anteriores, baseados na eletrotransferência de DNA plasmidial no músculo de camundongos anões imunodeficientes (lit/scid). No presente trabalho, um modelo animal homólogo de terapia gênica para GH foi estudado mediante clonagem da sequência genômica do DNA de GH de camundongo (mGH-gDNA), a qual substituiu o hGH-gDNA no vetor que havia sido utilizado em camundongos anões imunodeficientes. O novo vetor, agora nomeado UBI-mGH-gDNA, foi utilizado em camundongos anões imunocompetentes (lit/lit). Foi primeiramente realizado um teste in vitro, transfectando-se células humanas HEK 293 com este plasmídeo e obtendo-se uma expressão de 3,0 &mu;g mGH/106 células/dia, contra 3,7 &mu;g mGH/106 células/dia, para o UBI-hGH-gDNA. Estes dois plasmídeos foram então injetados (50 &mu;g/animal) no músculo quadríceps de camundongos, seguido de eletroporação, realizando um ensaio de 94 dias. Enquanto após 15 dias, as inclinações das curvas de variação de peso relacionadas ao mGH, hGH e salina foram 0,130, 0,112 e 0,027 g/camundongo/dia, respectivamente, após 94 dias, as inclinações correspondentes foram 0,041, 0,028 e 0,033 g/camundongo/dia. As análises estatísticas mostraram que após 15 dias, as inclinações das duas curvas com o GH foram significativamente maiores que a inclinação do controle (P<0,001), enquanto que após 94 dias, somente a inclinação da curva do mGH foi maior que a do controle (P<0,005). A porcentagem de aumento de peso nos animais tratados com o gene do mGH, após 94 dias, foi de 34,3%, enquanto que o comprimento nariz-cauda e o comprimento do fêmur, dois parâmetros que medem diretamente o crescimento longitudinal, foram de 9,5% e 26%, respectivamente, quando comparados aos valores iniciais. A interrupção do crescimento progressivo do grupo tratado com hGH não foi inesperada, considerando a óbvia reação imunogênica dos animais imunocompetentes contra o GH humano e não contra o de camundongo (título do anticorpo anti-hGH 1:100 a 1:3200). A inclinação altamente positiva do grupo controle, já observada em camundongos lit/lit mas não em lit/scid, é provavelmente devida ao ganho de peso natural desta linhagem, não suportada, contudo, por um proporcional crescimento longitudinal. Níveis circulatórios de mGH da ordem de 4 ng/mL foram detectados após 15 dias para o grupo tratado com o mGH, enquanto o grupo controle apresentou níveis em torno de 0,7 ng mGH/mL (P<0,001). Níveis circulatórios de mIGF-I foram também determinados nos dias 15, 45 e 94 nos animais tratados com mGH, sempre mostrando valores 1,5 - 3,0 vezes maiores que o grupo controle, e valores 1,2-1,6 vezes maiores que o grupo tratado com hGH. Este modelo de tratamento homólogo pode ser considerado uma primeira abordagem e um importante suporte para futuros ensaios pré-clínicos baseados na administração de DNA plasmidial para o tratamento da deficiência de GH humano. / Sustained levels of circulating human growth hormone (hGH) and highly significant weight increases, also found comparable to repeated hormone injections, were observed in previous works, after electrotransfer of naked plasmid DNA into the muscle of immunodeficient dwarf mice (lit/scid). In the present work an homologous animal model for GH gene therapy was studied by cloning the genomic sequence of mouse GH-DNA (mGH-gDNA), which substituted hGH-gDNA in the plasmid that had been used in immunodeficient dwarf mice, now named UBI-mGH-gDNA and used in immunocompetent dwarf mice (lit/lit). An in vitro test was first carried out by transfecting HEK 293 human cells with this plasmid and obtaining an expression of 3.0 &mu;g mGH/106 cells/day, against 3.7 &mu;g /106 cells/day obtained with UBI-hGH-gDNA. The same two plasmids DNA (50 &mu;g/mouse) were then injected into the quadriceps muscle of lit/lit, followed by electroporation, carrying out a 94-day assay. While after 15 days the slopes of the weight variation curves related to mGH, hGH and saline were 0.130, 0.112 and 0.027 g/mouse/day respectively, after 94 days the corresponding slopes were 0.041, 0.028 and 0.033 g/mouse/day. Statistical tests showed that after 15 days the slopes of both GH curves were significantly higher than the control (P<0.001), while after 94 days only the slope of the mGH curve was significantly higher than the control (P<0.005). Weight increase for mGH-treated mice, after 94 days, was 34.3%, while nose-to-tail and femur length, both directly measuring longitudinal growth, increased 9.5% and 26.0%, respectively, when compared to the initial values. The progressive growth arrest of the hGH-treated mice was not unexpected, considering the obvious immunogenic reaction of the immunocompetent animals against human and not against mouse GH ( anti-hGH antibody title 1:100 to 1:3200). The highly positive slope of the control group, already observed in lit/lit but never in lit/scid, is probably due to the natural weight gain of this strain, not supported, however, by a proportional longitudinal growth. mGH circulating levels of the order of 4 ng/mL were detected after 15 days for mGH-treated mice, while the control presented levels around 0.7 ng mGH/mL (P<0.001). Mouse IGF-I serum levels were also determined on day 15, 45 and 94 in mGH-treated mice, always showing 1.5-3.0 fold higher values than the control and 1.2-1.6 fold higher values than hGH-treated mice. This homologous treatment model can be considered a first approach and an important support to the preclinical testing of naked DNA administration for the treatment of human GH deficiency.

With the current state of climate change and world peak oil on the horizon, it is important to focus our research efforts on alternative sources of energy. Ethanol obtained from the digestion of biomass (bioethanol) and oil harvesting from algae (biodiesel) are two promising fields of study for transportation fuel production. However, in their current state of development, neither option is capable of reasonably replacing the transportation fuel demand for this country. The land demand needed is too large for either process to become a viable option, albeit the land demand for biodiesel is considerably smaller than that of bioethanol. Therefore, when moving forward with alternative transportation fuel, harvesting oil from algae is a more promising option. Therefore, I investigated oil producing green algae to better understand algal growth, the algal cytoskeleton, and tried to establish a methodology to genetically manipulate algae. I developed a microgrowth assay in order to investigate algal growth and proliferation, while at the same time using considerably less material and space. This assay can directly monitor algal growth in response to media contents, and overcomes many of the limitations of existing microassays due to its use of solid media agar and fluorescent imaging. I also investigated algal genetic manipulation with the intention of creating a standard operating procedure, which could lead to further investigation of how to increase lipid output and increase lipid harvesting cycles through studying lipid production and cell division. Electroporation and PEG mediated transformation were the two chief methods investigated for nuclear transformation. Lastly, I performed an algal kinesin phylogenetic study to characterize the currently available algal kinesin superfamily, providing insight to proteins that are important for cell division as well as other functions within this superfamily. Kinesins 5, Kinesin 7s Class II and Class V, and Kinesin 14 Class I were identified to be important for algal cell division, while Kinesin 8, 12, 11, and some orphan kinesins will require further investigation due to their unknown plant function. Overall, this research provides a foundation for future algal studies required for optimal oil production necessary for a more sustainable future.

Níveis sustentáveis de hormônio de crescimento humano (hGH) circulante e aumento de peso altamente significativo, avaliados também em comparação a repetidas injeções de hormônio, foram observados em trabalhos anteriores, baseados na eletrotransferência de DNA plasmidial no músculo de camundongos anões imunodeficientes (lit/scid). No presente trabalho, um modelo animal homólogo de terapia gênica para GH foi estudado mediante clonagem da sequência genômica do DNA de GH de camundongo (mGH-gDNA), a qual substituiu o hGH-gDNA no vetor que havia sido utilizado em camundongos anões imunodeficientes. O novo vetor, agora nomeado UBI-mGH-gDNA, foi utilizado em camundongos anões imunocompetentes (lit/lit). Foi primeiramente realizado um teste in vitro, transfectando-se células humanas HEK 293 com este plasmídeo e obtendo-se uma expressão de 3,0 &mu;g mGH/106 células/dia, contra 3,7 &mu;g mGH/106 células/dia, para o UBI-hGH-gDNA. Estes dois plasmídeos foram então injetados (50 &mu;g/animal) no músculo quadríceps de camundongos, seguido de eletroporação, realizando um ensaio de 94 dias. Enquanto após 15 dias, as inclinações das curvas de variação de peso relacionadas ao mGH, hGH e salina foram 0,130, 0,112 e 0,027 g/camundongo/dia, respectivamente, após 94 dias, as inclinações correspondentes foram 0,041, 0,028 e 0,033 g/camundongo/dia. As análises estatísticas mostraram que após 15 dias, as inclinações das duas curvas com o GH foram significativamente maiores que a inclinação do controle (P<0,001), enquanto que após 94 dias, somente a inclinação da curva do mGH foi maior que a do controle (P<0,005). A porcentagem de aumento de peso nos animais tratados com o gene do mGH, após 94 dias, foi de 34,3%, enquanto que o comprimento nariz-cauda e o comprimento do fêmur, dois parâmetros que medem diretamente o crescimento longitudinal, foram de 9,5% e 26%, respectivamente, quando comparados aos valores iniciais. A interrupção do crescimento progressivo do grupo tratado com hGH não foi inesperada, considerando a óbvia reação imunogênica dos animais imunocompetentes contra o GH humano e não contra o de camundongo (título do anticorpo anti-hGH 1:100 a 1:3200). A inclinação altamente positiva do grupo controle, já observada em camundongos lit/lit mas não em lit/scid, é provavelmente devida ao ganho de peso natural desta linhagem, não suportada, contudo, por um proporcional crescimento longitudinal. Níveis circulatórios de mGH da ordem de 4 ng/mL foram detectados após 15 dias para o grupo tratado com o mGH, enquanto o grupo controle apresentou níveis em torno de 0,7 ng mGH/mL (P<0,001). Níveis circulatórios de mIGF-I foram também determinados nos dias 15, 45 e 94 nos animais tratados com mGH, sempre mostrando valores 1,5 - 3,0 vezes maiores que o grupo controle, e valores 1,2-1,6 vezes maiores que o grupo tratado com hGH. Este modelo de tratamento homólogo pode ser considerado uma primeira abordagem e um importante suporte para futuros ensaios pré-clínicos baseados na administração de DNA plasmidial para o tratamento da deficiência de GH humano. / Sustained levels of circulating human growth hormone (hGH) and highly significant weight increases, also found comparable to repeated hormone injections, were observed in previous works, after electrotransfer of naked plasmid DNA into the muscle of immunodeficient dwarf mice (lit/scid). In the present work an homologous animal model for GH gene therapy was studied by cloning the genomic sequence of mouse GH-DNA (mGH-gDNA), which substituted hGH-gDNA in the plasmid that had been used in immunodeficient dwarf mice, now named UBI-mGH-gDNA and used in immunocompetent dwarf mice (lit/lit). An in vitro test was first carried out by transfecting HEK 293 human cells with this plasmid and obtaining an expression of 3.0 &mu;g mGH/106 cells/day, against 3.7 &mu;g /106 cells/day obtained with UBI-hGH-gDNA. The same two plasmids DNA (50 &mu;g/mouse) were then injected into the quadriceps muscle of lit/lit, followed by electroporation, carrying out a 94-day assay. While after 15 days the slopes of the weight variation curves related to mGH, hGH and saline were 0.130, 0.112 and 0.027 g/mouse/day respectively, after 94 days the corresponding slopes were 0.041, 0.028 and 0.033 g/mouse/day. Statistical tests showed that after 15 days the slopes of both GH curves were significantly higher than the control (P<0.001), while after 94 days only the slope of the mGH curve was significantly higher than the control (P<0.005). Weight increase for mGH-treated mice, after 94 days, was 34.3%, while nose-to-tail and femur length, both directly measuring longitudinal growth, increased 9.5% and 26.0%, respectively, when compared to the initial values. The progressive growth arrest of the hGH-treated mice was not unexpected, considering the obvious immunogenic reaction of the immunocompetent animals against human and not against mouse GH ( anti-hGH antibody title 1:100 to 1:3200). The highly positive slope of the control group, already observed in lit/lit but never in lit/scid, is probably due to the natural weight gain of this strain, not supported, however, by a proportional longitudinal growth. mGH circulating levels of the order of 4 ng/mL were detected after 15 days for mGH-treated mice, while the control presented levels around 0.7 ng mGH/mL (P<0.001). Mouse IGF-I serum levels were also determined on day 15, 45 and 94 in mGH-treated mice, always showing 1.5-3.0 fold higher values than the control and 1.2-1.6 fold higher values than hGH-treated mice. This homologous treatment model can be considered a first approach and an important support to the preclinical testing of naked DNA administration for the treatment of human GH deficiency.

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.