No description available.

No description available.

京都大学 / 新制・課程博士 / 博士(医科学) / 甲第25201号 / 医科博第157号 / 新制||医科||10(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 遊佐 宏介, 教授 近藤 玄, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

In order to achieve efficient therapeutic effect, it is important to understand the structure of biomaterials that are used in the therapeutic delivery system. This dissertation is dedicated towards understanding the hydrolysis pattern of plasmid DNA (pDNA) delivery vehicles comprised of poly(glycoamidoamine)s (PGAAs) under physiological conditions and effects of subtle changes in the chemical structure of the PGAAs on its biological performance. The unusual hydrolysis of the tartarate and galactarate based PGAAs was investigated by studying the hydrolysis of small model molecules which mimic the repeat unit of the respective polymers. In the case of galactarate and tartarate based molecules with terminal amines showed faster hydrolysis of the amide bonds. In addition for the tartarate based compounds, it was also found that it is necessary to have terminal amine functionality for the intramolecular hydrolysis to occur. The model compounds consists of two amide bonds and were designed symmetric, however amide bond on only one side of the tartarate moiety show underwent hydrolysis. Further studies show that one side of the amine assists the hydrolysis of the amide bond on the other side of the tartarate moiety. The degradation of poly(L-tartaramidopentaethylenetetramine) (<strong>T4</strong>) was also used to study the sustained release of pDNA from the layer-by-layer constructs of <strong>T4</strong>/pDNA. The thickness of the constructs was characterized by ellipsometry while the UV-visible spectroscopy was used to characterize the loading capacity of the constructs for pDNA. The indirect sustained release of pDNA under the physiological conditions with respect to time was characterized by the cellular uptake studies in HeLa cells. The increase in the uptake of the Cy5 labeled pDNA was seen at extended period of eleven days. The integrity of the sustained released pDNA for the transgene expression was characterized with an assay to see the expression of the green fluorescent protein (GFP) from the <strong>T4</strong>/GFP-pDNA layer-by-layer constructs. PGAAs show a very efficient delivery of the pDNA in a non-toxic manner. The chemical structure of the polymer can dictate the binding with pDNA and also the release of the pDNA form the polymer-pDNA complexes. In order to better understand the fundamentals of the nucleic acid delivery and to better design the nucleic acid delivery vehicles, subtle changes in the chemical structure of the PGAAs were designed and studied for the biological activity. The effect of charge type was investigated by designing and synthesizing guanidine based polymer series analogues to galactarate and tartarate based PGAAs (<strong>G1</strong> and <strong>T1</strong>) which incorporate secondary amines as the charge type on the polymer backbone. The guanidine based polymer series, poly(glycoamidoguanidine)s (PGAGs), show very non toxic behavior in HeLa cells at all the different polymer to pDNA ratio (<i>N/P</i> ratio) studied. Interestingly PGAGs are the only non-toxic guanidine containing polymers which are reported in the literature to the date. The cellular uptake of pDNA assisted from the PGAGs is a little higher than PGAAs compared although both the series of polymers show similar transgene expression. The transgene expression in case of PGAGs also imply the release of the polymer-pDNA complexes from the endosome. In another study of structure-bioactivity relationship based on the degree of polymerization (DP) of poly(galactaramidopentaethylenetetramine) (<strong>G4</strong>), it was found that the increase in the DP of <strong>G4</strong> increases the toxicity of the polymers in the HeLa cells. / Ph. D.

Text in Afrikaans / Hierdie studie ondersoek die moontlikheid dat aandagtekort versteuring met hiperaktiwiteit(ATHV/H) verband hou met probleme met voortgesette aandag, terwyl aandagtekort versteuring sander hiperaktiwiteit(ATHV/SO) verband hou met probleme met gefokusde aandag. Neurosielkundige toetse waarmee voortgesette- en gefokusde aandag gemeet word, word gebruik om drie groepe, naamlik ATHV/H(n=24), ATHV/SO(n=l5) en 'n kontrole groep(n=21), met mekaar te vergelyk. Resul tate dui daarop dat die groep ATHV/SO beduidend swakker presteer as die ander twee groepe op sommige van die toetse van gefokusde aandag, en ook neig om swakker te presteer op die toets van voortgesette aandag. Die groep ATHV/SO se probleme blyk gekoppel te wees aan 'n stadiger spoed van kognitiewe prosessering en 'n toestand van hipoopwekking. Die groep ATHV/H presteer nie beduidend swakker as die ander twee groepe op die toets van voortgesette aandag nie, maar presteer wel swakker as die kontrole groep op die Stroop Colour-Word Test waar inhibisie van ontoepaslike response vereis word. Die groep ATHV /H se probleme blyk verband te hou met frontale disfunksie wat manifesteer as 'n neighing tot impulsiwiteit en gebrekkige gedragsregulering. / This study examined the possibility that attention deficit disorder with hyperactivity(ADHD/H) is related to problems with sustained attention, while attention deficit disorder without hyperactivity (ADHD/WO) is related to problems with focussed attention. The performance of three groups: ADHD/H (n=24), ADHD/WO(n=15) and a control group(n=21) were compared. Results indicated that the ADHD/WO group performed significantly poorer than the other two groups on some of the tests of focussed attention and also tended to perform poorer on the test assessing sustained attention. This groups' symtoms appear to be related to a slowed speed of processing and a state of hypo-arousal. The ADHD/H group did not perform poorer than the other two groups on the test assessing sustained attention. The ADHD/H groups' symptoms appear to be related to frontal dysfunction resulting in impulsivity and poor behavioural control. / Psychology / M.A. (Sielkunde)

This paper attempts to evaluate Training for Trainers (T4T) as an aid for developing healthy and sustained church planting movements (CPMs). The thesis is that Training for Trainers (T4T) can enable and sustain (by the Spirit’s power) healthy church planting movements because a discipleship process is built into the methodology that develops believers in their personal and communal growth and equips them to repeat the process with other individuals they reach. The very format of the T4T process provides a context for developing disciples inwardly and training disciples to minister outwardly. The T4T process continues over the course of months and years to systematically move believers through the essential stages of sustained church planting movements: Bridges in conversations with the lost from non-spiritual topics to the gospel in order to find those God is preparing (knowing whom to talk to and how to start) • Reproducible evangelism methods that are effective in the local context and can be learned by any new believer • Reproducible discipleship that addresses both short-term and long-term spiritual growth in a manner appropriate to the local worldview and able to be passed on by a new believer • Reproducible church models appropriate to the local context and able to be led and passed on by new believers • Leadership development and multiplication patterns that develop leaders rapidly in the context of ministry and enable the number of leaders to keep pace with the number of new churches. T4T moves each new generation of disciples (trainers) and churches through this process because it casts vision for and gives loving accountability for disciples to truly become trainers of others. It does this primarily through a three-thirds training process. T4T is training for trainers who will train trainers who will train trainers. T4T attempts to initiate movements of God in which at least four generations of new disciples and churches emerge. This paper evaluates T4T is based on case studies, survey instruments and biblical principles and then offers recommendations for CPM practitioners. / Christian Spirituality, Church History & Missiology / D. Th.(Missiology)

Le sujet de cette thèse consiste à formuler et caractériser de nouveaux matériaux hybrides inorganique/organique pour la formulation et la libération de principes actifs. Ces matériaux sont constitués soit par des hydroxydes doubles lamellaires (HDL) et des phospholipides (PL) soit par des hydroxydes doubles lamellaires et du chitosane. Les matériaux hybrides hydroxydes doubles lamellaires/phospholipides ont été obtenus par échange anionique entre les anions du feuillet d'HDL et les phospholipides chargés négativement sous forme de liposomes. Les techniques d'analyse utilisées (DRX, ATG, UV et MET) ont permis de déterminer l'influence de la nature des anions de compensation des feuillets d'HDL et des phospholipides sur le taux de intercalation de PL dans les HDL et le taux de chargement d'un principe actif modèle : l'estradiol. Nous avons effectué ensuite des cinétiques de libération des phospholipides et de l'estradiol intercalés en faisant varier le pH et la composition du milieu pour déterminer les propriétés de ce matériau en tant que système de libération sous forme d'implant afin de libérer le principe actif sur des temps très longs. Les matériaux hybrides hydroxydes doubles lamellaires/chitosane ont ensuite été synthétisés sous forme de billes par un procède sol-gel nous permettant d'optimiser la morphologie et la texture des matériaux par le contrôle de la viscosité et le choix du mode de séchage (étuve ou spray-drying). Le chitosane a été choisi pour ses propriétés muco-adhésives afin d'envisager une application du matériau sur les muqueuses. Les techniques d'analyse utilisées ont permis de confirmer la présence de HDL dans le chitosane (DRX), de déterminer la morphologie des billes (MET, MEB) ainsi que leur composition (ATG). Des cinétiques de libération au cours de temps et en fonction de pH ont été réalisées sur de billes chitosane/ hydroxydes doubles lamellaires chargées en principe actif (ibuprofène ou phospholipides charge en estradiol) afin de déterminer leur potentiel de libération du PA. Ces nouveaux hybrides inorganique/organique s'avèrent donc être des matériaux très intéressants en tant que systèmes de libération contrôlée de principes actifs dans le domaine pharmaceutique. / The aim of this work involves the preparation and the characterization of new inorganic/ organic hybrids materials as drug delivery systems. These materials are composed of layered double hydroxide (LDH) and phospholipids (PL) or layered double hydroxide and chitosan. The layered double hydroxide/phospholipids hybrids materials were obtained by anionic exchange between the anions from the HDL layers and the liposome composed of phospholipids with negative charges. Different analysis were used such as XRD, TGA, TEM and fluorescence for the characterization of these materials and the determination of parameters such as the nature of the LDH anions nature, the kind of phospholipids as well the ratio of used model drug (17ƒÒ-estradiol) allowing the optimal method. Release kinetic studies of the phospholipids and of the drug were carried out in different release media (pH, nature of ions) to evaluate the potential sustained release properties of these hybrid materials. Layered double hydroxide/chitosan beads were developed by a sol-gel process. Chitosan was chosen for its muco-adhesive properties permitting to broaden the possibility of pharmaceutical applications. This method allowed us to improve the morphology and the texture of the material by controlling the viscosity and the drying process. Materials were characterized by XRD, TGA, SEM and TEM to determine: the presence of LDH, the encapsulated LDH yield and the morphology of the beads. Releases kinetic of a model drug (ibuprofen) encapsulated in layered double hydroxide/chitosan hybrid materials allowed us to test its potential release properties. Such new inorganic/ organic hybrids materials proved to be interesting as sustained release systems in pharmaceutical domain.

Une nouvelle plateforme hydrogel uniquement formée par l’association de nanocapsules lipidiques (NCLs) a été développée en s’inspirant de précédents travaux utilisant une gemcitabine modifiée. Afin de limiter la toxicité de l’hydrogel, la gemcitabine a été remplacée par la cytidine, rendue amphiphile par une chaîne aliphatique (Cyt-C16). Placée à l’interface huile/eau des NCLs, la Cyt-C16 permet la formation d’un réseau tridimensionnel de NCLs à l’origine de la gélification. Un plan de mélange a permis d’optimiser les procédés de formulation de 4 tailles de NCLs modèles. Les propriétés viscoélastiques des hydrogels sont modulables. Plus les concentrations en NCLs et Cyt-C16 sont élevées, plus le gel est « rigide », indépendamment de la taille des NCLs qui doit être supérieure à 50 nm pour permettre la gélification. Les hydrogels sont injectables et permettent une libération prolongée de NCLs (de taille mono-disperse), sans toxicité supplémentaire in vitro, du fait de la présence de la Cyt-C16. De plus, uniquement solubilisée dans l’huile,la Cyt-C16 permet d’obtenir un organogel, dont les propriétés viscoélastiques sont renforcées en augmentant sa concentration. L’injection sous-cutanée (SC) in vivo des deux gels est bien tolérée et entraine une réaction inflammatoire locale comparable à celle provoquée par un excipient pharmaceutiquement acceptable. Ces deux formes pourront être utilisées pour libérer de façon prolongée différents actifs. Deux applications précliniques des hydrogels ont été explorées, l’une utilisant la voie SC pour cibler les ganglions lymphatiques, la seconde permettant un traitement local des suites opératoires d’une résection de glioblastome. / An innovative hydrogel platform obtained by the association of lipid nanocapsules (LNCs) was based on the previous work on modified gemcitabine. To limit the inherent toxicity of the hydrogel, gemcitabine was replaced by cytidine, then modified by an aliphatic chain (Cyt-C16). The hydrogel network was allowed by H-bond interactions between cytidine moieties exposed at the oil/water interfaces of LNCs. An experimental plan provided the formulation processes for 4 optimized sizes of model LNCs. The gelation was only possible for LNC sizes higher than 50 nm, and the hydrogel viscoelastic properties are versatile. The hydrogel is more “rigid” when LNC and Cyt-C16 concentrations increase, independently of the LNC size. The hydrogels are injectable and allow a sustained release of LNCs (withmonodisperse size), without additional in vitrocytotoxicity due to Cyt-C16. Moreover, when solubilized in oil, Cyt-C16 alone produced an organogel platform, whose viscoelastic properties are strengthened increasing its concentration. Both types of gels showed a good biocompatibility after an in vivo subcutaneous (SC) injection, with a local inflammatory response similar to that of induced by an approved excipient. These two forms could be used to sustain the release of various drugs, and two preclinical applications of hydrogels have been explored : one using the SC route to target lymph nodes, and the second for local treatment after glioblastoma resection.

Le travail de recherche mené au cours de cette thèse a été effectué dans le cadre d’une collaboration industrielle et a porté sur l’étude des caractéristiques physico-chimiques d’une matrice lipidique à libération prolongée. L’originalité de cette dernière tient à sa forme galénique, des microsphères calibrées d’environ 400 µm de diamètre, fabriquées par procédé de prilling puis conditionnées en sachets.Les caractérisations entreprises au cours de cette thèse ont été menées progressivement en partant de l’étude du comportement thermique individuel des constituants (excipients lipidiques et principe actif), puis de leurs mélanges pour définir les conditions d’existence et les caractéristiques structurales des phases solides qu’ils engendrent, notamment lors du prilling. Plusieurs diagrammes d’état partiels ont été construits pour permettre de cerner l’organisation de la matrice formant le produit fini et disposer de leviers fiables pour corriger d’éventuelles déviations du procédé de fabrication. Cette analyse était également indispensable pour décrire précisément le processus de libération du principe actif et en déterminer les paramètres cinétiques. / The research conducted in this thesis, during an industrial collaboration, is focused on the study of physicochemical characteristics of a sustained release lipid matrix. The originality of this matrix is its dosage form, microspheres of 400 microns in diameter, manufactured by a prilling process and packaged in stick-packs.The characterizations conducted during this thesis have been carried out gradually starting from the study of the thermal behavior of individual components (lipidic excipients and drug) and their mixtures to define the phase formation conditions and the structural characteristics of the solid phases generated, especially during the prilling process. Partial phase diagrams were constructed to understand the final product matrix organization and to identify actions to correct any deviations in the process. This analysis was also needed to accurately describe the drug release mechanism and to determine the kinetic parameters.

L’objectif général de cette thèse a été d’étudier le potentiel de billes à base de molécules d’α-cyclodextrine et d’huile de soja, pour l’administration orale de principes actifs peu solubles dans l’eau.Nous avons tout d’abord vérifié qu’il était possible d’encapsuler dans les billes des molécules actives (la progestérone et l’indométacine) autres que les rétinoïdes et le diazépam, avec une teneur élevée et un rendement de fabrication satisfaisant. L’étude du comportement des billes nues lyophilisées, en termes de stabilité et de libération dans des milieux digestifs simulés, nous a permis de proposer un mécanisme de libération de la molécule encapsulée qui se déroule en plusieurs étapes: i) hydratation des billes, ii) dissolution de la matrice hydrophile d’α-cyclodextrine, iii) libération de gouttelettes d’huile contenant le principe actif puis de la fraction dissoute dans l’huile par un phénomène de partage, iiii) fragmentation des billes fragilisées et libération totale de l’huile. La présence de sels biliaires dans le milieu, accélère à la fois la libération et la quantité dissoute, en fragilisant les billes et en réduisant la valeur du coefficient de partage du principe actif entre l’huile et le milieu digestif. Nous avons montré in vitro et in vivo qu’il est possible de moduler la libération d’un principe actif à partir d’une même formulation de départ, en jouant sur l’organisation du système (émulsion sèche, billes nues, billes coquées par un nouvel ajout d’α-cyclodextrine sur les billes nues). Les études in vivo chez le rat ont révélé que l’émulsion sèche se comporte comme une forme à libération immédiate, les billes coquées comme une forme à libération prolongée et les billes nues comme une forme à libération intermédiaire. Enfin, la libération du principe actif encapsulé peut également être modulée en modifiant le mode de séchage des billes. Comparativement à la lyophilisation, le séchage à l’étuve modifie les propriétés des billes en augmentant leur résistance dans les milieux digestifs simulés et prolonge la libération de la molécule encapsulée. / The general aim of this thesis was the study of the potential of beads, made of α-cyclodextrin and soybean oil, for the oral delivery of poorly water soluble drugs. We have first verified that it was possible to encapsulate in beads, active molecules (progesterone and indomethacin), other than retinoid and diazepam, with a high drug loading and a satisfying yied. The study of the behaviour of freeze-dried naked beads, in terms of stability and drug release in simulated gastro-intestinal fluids, allowed to propose a mechanism for the release of the encapsulated drug, involving several steps: i) hydration of the freeze-dried beads, ii) dissolution of α-CD hydrophilic matrix, iii) release of oily droplets containing the active drug and then of the fraction of drug dissolved in oil, following a partition phenomenon, iiii) fragmentation of the weakened beads and at last the total release of oil. The presence of bile salts in the medium accelerates both the release and the dissolved amount, by weakening the beads and reducing the partition coefficient value of the active molecule between oil and digestive medium.We have shown in vitro as well as in vivo that it is possible to modulate the release of a model drug from the same initial formulation, according to the degree of organization of the system (dry emulsion, naked beads, coated beads obtained by an additional amount of α-cyclodextrine to the preformed naked beads). In vivo studies in rats have highlighted that dry emulsion behaves as a fast release formulation, the coated beads as a sustained release formulation and the naked beads as an intermediate one. Finally, the release of the encapsulated drug can also be modulated by modifying the drying method of the beads. Compared to freeze-drying, oven-drying modifies the properties of the beads by increasing their resistance in simulated gastro-intestinal fluids and sustaining the release of the encapsulated drug.