京都大学 / 0048 / 新制・課程博士 / 博士(薬学) / 甲第18551号 / 薬博第813号 / 新制||薬||238(附属図書館) / 31451 / 京都大学大学院薬学研究科医療薬科学専攻 / (主査)教授 橋田 充, 教授 髙倉 喜信, 教授 佐治 英郎 / 学位規則第4条第1項該当 / Doctor of Pharmaceutical Sciences / Kyoto University / DFAM

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Due to the safety concerns surrounding viral vectors, non-viral alternatives are desirable for fulfilling the aim of gene therapy. In this project gel mobility shift assays demonstrated how cross-linked PVA nanoparticles successfully form complexes with plasmid DNA and are of a size and charge that should, theoretically, permit endocytosis by eukaryotic cells. However, during in vitro transfection studies no reporter (GFP) gene expression was noted. The collective evidence from electroporation, fluorescent-DNA-tagging, Lipofectin® or calcium phosphate chimeric and chloroquine experiments suggest that a lack of cell uptake is responsible. Nevertheless, the same cross-linked PVA nanoparticles have been shown to exhibit much promise in the field of drug delivery during in vitro experiments, even when used to target the same cell types as those used during transfection studies. Nanagel®, a cross-linked PVA nanoparticle containing budesonide, achieved higher levels of drug delivery than a commercially available form of the same drug (Pulmicort®) after 1 or 24 hours drug exposure. Furthermore, by measuring superoxide production during a stimulated respiratory burst, the budesonide delivered to cells appears fully functional and significantly more effective than Pulmicort® in preventing the formation of reactive oxygen species, following a 24-hour pre-treatment period with the formulation. These findings have exciting possibilities for the use of hard-to-dissolve corticosteroids in the treatment of respiratory disease. / AGT Sciences Ltd

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No description available.

Herein, different polymer libraries were examined to determine the effect polymer structure has on intracellular events. The effect of different polyamine lengths in copolymers on cellular uptake, the effect of modifying end groups of trehalose-containing polymers on transfection efficiency, and the effect of different linker lengths between galactose and a hepatocyte-targeted polymer on transfection efficiency were studied. Furthermore, it was demonstrated that polymers with terbium chelated in their repeat units could potentially be used for Förster resonance energy transfer (FRET) studies to monitor pDNA release from the polymer. Much of the work in this dissertation focuses on elucidating the intracellular mechanisms of linear poly(ethylenimine) (PEI) and how it compares to poly(L-tartaramidopentaethylenetetramine) (T4) and poly(galactaramidopentaethylenetetramine) (G4), two poly(glycoamidoamine)s synthesized by our group. The long-term goal of this project is to develop structure-function relationships between polymers and pDNA delivery efficacy that will result in the rational design of safe, efficient vehicles for therapeutic nucleic acid delivery. Many polymers used as DNA delivery vehicles display high cytotoxicity. Often, the polymers with the highest transfection efficiency are the most toxic, as demonstrated herein by PEI and T4 with varying polymer lengths. Therefore, it was of interest to study how polymer structure influences mechanisms of cytotoxicity. To this end, studies on several mechanisms of cytotoxicity, including nuclear envelope permeabilization, were conducted. Longer polymers induced more cytotoxic responses than shorter ones, and it appears that hydroxyl groups in the repeat unit of polymers play a role in polyplex formation. This research has also led us to a potential link between transfection efficiency and cytotoxicity; the polymers with the highest transfection efficiency were also the most toxic, and were also able to induce the most nuclear envelope permeability. It is possible that these polymers' ability to permeabilize the nuclear envelope is what causes their high transfection efficiency and high toxicity. In addition, flow cytometry and confocal microscopy studies revealed that polymer structure plays a role in nuclear trafficking; poly(glycoamidoamine)s G4 and T4 more dependent on intracellular machinery than PEI. This research demonstrates the impact that changes in polymer structure have on intracellular mechanisms. / Ph. D.

Le néocortex déclenche des activités paroxystiques suite aux lésions cérébrales traumatiques. Avec des blessures cérébrales pénétrantes, la déafférentation s'accompagne d'une longue période silencieuse du cortex atteint, et d'une augmentation des périodes d'hyperpolarisation du néocortex entourant la lésion. L'activité synchrone du réseau neuronal après une période de latence conduirait à l'épileptogenèse. Des tentatives de modifier la nature des crises ont été effectuées à l'aide de modèles animaux. L'une des plus prometteuse étant la chimiogenèse par l'injection des vecteurs viraux afin d'anéantir la réponse de certains récepteurs couplés aux protéines G aux ligands habituels, alors qu'ils réagissent aux médicaments désirés, comme N-oxyde de clozapine. Ces récepteurs appelés « DREADDs » exclusivement activés par ces médicaments ont été étudiés pour réduire le nombre et la sévérité des crises. Selon les résultats non-publiés de notre laboratoire, l'utilisation d'un DREADD excitateur près de «undercut» serait antiépileptogène. Nous pensons qu'une excitation ciblée pourrait optimiser l'effet antiépileptogène. L'excitation est directement liée aux neurones transduits. Nous postulons qu'une transduction optimale des neurones pourrait être atteinte par un dosage optimisé du virus injecté, tant au titre viral qu'au volume injecté. Pour prouver notre hypothèse nous avons utilisé trois différentes titrations de AAV2/8 et injecté différents volumes de ces titrations aux souris. Le volume de transfection corticale et le nombre des neurones transduits ont été quantifiés. Avec la titration E11gc/ml aucune transfection n'a été observée. Avec la titration E12gc/ml une corrélation quasi-linéaire a été observée entre le volume viral injecté, et le volume cortical transfecté, ainsi que le nombre de neurones transduits. Avec la titration E13gc/ml, une meilleure corrélation a été observée à la transduction neuronale qu'à la transfection corticale, par rapport au volume viral injecté. En conclusion, la titration E12gc/ml paraît être un meilleur choix pour nos futures études, la fiabilité de la titration E13gc/ml n'ayant pas été démontrée. / The neocortex is the origin of paroxysmal activities that occur after traumatic brain injuries. In penetrating brain injuries, deafferentation causes long silent periods in affected cortex and increased hyperpolarization period in neocortical tissue around the injury. The synchronous neural network activity after a latent period may lead to epileptogenesis. Some attempts to alter seizures were done using animal models. One of the most promising involves chemogenetic tools via AAV viral vector injection to make some G protein-coupled receptors unresponsive to their natural ligands, and activated by the desired drug, such as clozapine-N-oxide. These designer receptors exclusively activated by designer drugs (DREADDs) have been studied in reducing the numbers and severity of seizures. According to unpublished works in our lab, using excitatory DREADDs in the vicinity of undercut was antiepileptogenic. We believe there could be an optimal level of excitation for yielding an optimal antiepileptogenic response. This excitation is in direct relation with neurons transfected. We hypothesize that the optimal neuronal transduction might be achieved with optimal dosage of virus delivered, in terms of viral titration and the volume of virus injected. To test this, we used three different titrations of AAV2/8 and we injected different volumes of these titrations in adult mice. Cortical transfection volume and number of neuronal transductions were estimated. With E11gc/ml titration, no transfection was visible. With E12gc/ml titration, an almost linear correlation was observed between the volume of virus injected and the number of neurons transduced and the cortical volume of transfection. With E13gc/ml titration the correlation between the injected AAV volume and the number of neuronal transductions was still good but there was a poor correlation between AAV volume and transfection volume. We concluded that E12gc/ml titration was a more reliable option for our further studies. The reliability of E13gc/ml titration needs to be proven.

Recently, laser light sources of different regimes have emerged as an essential tool in the biophotonics research area. Classic applications include, for example: manipulating single cells and their subcellular organelles, sorting cells in microfluidic channels and the cytoplasmic delivery of both genetic and non-genetic matter of varying sizes into mammalian cells. In this thesis several new findings specifically in the optical cell sorting as well as in the photo-transfection study fields are presented. In my optical cell sorting and guiding investigations, a new technique for enhancing the dielectric contrast of mammalian cells, which is a result of cells naturally engulfing polymer microspheres from their environment, is introduced. I explore how these intracellular dielectric tags influence the scattering and gradient forces upon these cells from an externally applied optical field. I show that intracellular polymer microspheres can serve as highly directional optical scatterers and that the scattering force can enable sorting through axial guiding onto laminin coated glass coverslips upon which the selected cells adhere. Following this, I report on transient photo-transfection of mammalian cells including neuroblastomas (rat/mouse and human), embryonic kidney, Chinese hamster ovary as well as pluripotent stem cells using a tightly focused titanium sapphire femtosecond pulsed laser beam spot. These investigations permitted advanced biological studies in femtosecond laser transfection: firstly, the influence of cell passage number on the transfection efficiency; secondly, the possibility to enhance the transfection efficiency via whole culture treatments of cells thereby, synchronizing them at the mitotic (M phase) as well as the synthesis phases (S phase) of the cell cycle; thirdly, this methodology can activate the up-regulation of the protective heat shock protein 70 (hsp70). Finally, I show that this novel technology can also be used to transfect mouse embryonic stem (mES) cell colonies and the ability of differentiating these cells into the extraembryonic endoderm.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.