<p>It is estimated that 1 in 500 Americans are inflicted with type I diabetes (T1D) with approximately 18,000 children and adolescents diagnosed each year. Islet/β cell replacement with long-lasting glucose-sensing and insulin-releasing functions has the potential to eliminate the need for insulin injections and minimize complications for individuals with T1D. However, limitations remain precluding it from widespread clinical use, including i) limited donor supply, ii) significant loss of functional islet mass upon transplantation, iv) limited functional longevity, and v) need for life-long systemic immunosuppression. To restore glucose-responsive insulin-release back to the patient’s body without the need for systemic immunosuppression, our approach involves a subcutaneous injection using a novel fibril-forming biologic, type I oligomeric collagen (Oligomer). Oligomer protects and in situ encapsulates replacement cells beneath the skin by transitioning from a liquid to a stable collagen-fibril scaffold, within seconds, just like those found in the body’s tissues. Preclinical validation studies in streptozotocin-induced diabetic mice show that replacement of islets at a dose of 500 or 800, results in a rapid (within 24 hours) reversal of hyperglycemia. All animals receiving syngeneic islets maintained euglycemia for beyond 90 days, while >80% of animals receiving allogeneic or xenogeneic (rat) islets remained euglycemia for at least 50 days. Histopathological analysis of Oligomer-islet implants showed normal morphology with no apparent evidence of a foreign body response and immune cell infiltrate. To our knowledge, this is the first report of an injectable subQ islet transplant strategy that yields rapid lowering and extended glycemic control without systemic immunosuppression.</p>

Student Number : 0213014A - MSc dissertation - School of Molecular and Cell Biology - Faculty of Science / Cytosolic glutathione transferases (GSTs) are obligate stable homo- and heterodimers comprising two GST subunits. Interactions across the subunit interface play an important role in stabilising the subunit tertiary structure and maintain the dimeric structure required for activity. The crystal structure of a rat Mu class GST consisting of two type one subunits (rGST M1-1) reveals a lock-and-key motif and a mixedcharge cluster at the subunit interface. Previous investigations revealed the lock-andkey motif was not essential for dimerisation. It was therefore postulated that the mixed-charge cluster at the dimer interface is primarily responsible for subunit association. Statistical analyses of individual rGST M1-1 chains did not predict the presence of any charge clusters. This suggests that the mixed-charge cluster forms only upon dimerisation and reinforces the probability that quaternary structure stabilisation is a major role of the mixed-charge cluster. Arginine 81 (Arg-81), a structurally conserved residue in the GST family involved in the mixed-charge cluster, was mutated to alanine. Phenylalanine 56 (Phe-56), the ‘key’ residue in the lock-and-key motif, was mutated to serine. These changes were engineered to disrupt the mixed-charge cluster and the lock-and-key motif situated at the dimer interface of rGST M1-1. Sizing by gel filtration chromatography of the mutant GST identified that these engineered amino acids resulted in a stable monomeric protein (F56S/R81A rGST M1). The F56S/R81A rGST M1 displayed almost no catalytic activity, suggesting perturbations of the active site or substrate binding sites. Structural investigations of the monomer by far- and near-UV circular dichroism revealed a similar secondary structural content to the wild-type. However, the tryptophan fluorescence properties suggested the tryptophans were situated in more hydrophilic environments than in the wild-type. ANS binding studies indicated a large increase in the accessible hydrophobic surface area of the monomer. Ureainduced equilibrium unfolding of F56S/R81A rGST M1 follows a cooperative twostate unfolding model. The unfolding data indicates decreased conformational stability and a large increase in the solvent exposed surface area of the monomer. In conclusion, the mixed-charge cluster at the dimer interface of rGST M1-1 is essential for monomeric association, which subsequently contributes to catalytic activity of the dimer and the stabilities of individual rGST M1-1 subunits.

Several approaches have been employed for directing the intracellular trafficking of DNA to the nucleus. Cationic polymers have been used to condense and deliver DNA and a few specific examples using chitosan as cationic polymer have been described. The concerted efforts in gene therapy to date have provided fruitful achievements toward a new era of curing human diseases. A number of obstacles, however, still must be surmounted for successful clinical applications. Therefore, chitosan-plasmid and quaternised chitosan-plasmid complexes (polyplexes) were investigated for their ability to transfect COS-1 cells and the results were compared with Transfectam/DNA lipoplexes for transfection efficiency. All of the chitoplexes utilised in this study proved to transfect COS-1 cells, however to a lesser extent than the Transfectam/DNA lipoplexes, which served as a positive control. Complexes formed with quaternised trimethyl and triethyl chitosan oligomers, specifically TMO L and TEO L, proved to be superior transfecting agents compared to other chitosans. The molecular mass of chitosan is considered to influence the stability of the chitosan/DNA polyplex, the efficiency of cell uptake and the dissociation of DNA from the complex after endocytosis. In literature it was shown that the toxicity of the chitosan1DNA polyplexes is relatively low compared to viral gene and lipid non-viral delivery vectors. This study showed that the percentage viable COS-1 cells when transfected with the chitosan polymers, oligomers, quaternised chitosan polymers and quaternised chitosan oligomers (chitoplexes) was higher than the percentage viable cells when transfected with lipoplexes prepared with Transfectam with the MTT assay. The Transfectam/DNA lipoplexes induced cell damage and a decreased viability of COS-1 cells were found. Chitosan/DNA and quaternised chitosan/DNA complexes did not affect the viability of the cell line. The degree of quaternisation of the polymers and oligomers and molecular size proved to be two important factors when considering effective non-viral gene delivery. It can be concluded that chitosan, especially quaternised oligomeric derivatives are polysaccharides that demonstrate much potential as a gene delivery system. The high solubility and low toxicity of chitosan allow its use in a wide variety of applications in the pharmaceutical industry and, as shown in this study, in gene delivery. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

A series of hybrid component based on reactive polyhedral oligomeric silsesquioxane(POSS) precusors and its reactive copolymers of PGMA were synthesized and utilized to nanobuild in epoxy. Reactive POSS and copolymer dispersed in homogenous in matrix, overcomed POSS-POSS interaction, which resulted in macroscale phase separation. The nanocomposites obtained were analyzed by Scanning electron microscopy, Transmission electron microscopy, X-ray scattering and dynamic mechanical. An analogue of POSS (denoted as POSSMOCA) was synthesized via addition reaction, which had reactive amino group bonding into epoxy network and improved the thermostability, because of the structural silicon, nitrogen and halogen. Epoxy/polyhedral oligomeric silsesquioxanes (POSS) hybrid composites were prepared from prereaction between trifunctional silanol POSS-OH and diglycidyl ether of bisphenol A (DGEBA) via silanol and the oxirane group. Reactive POSS-PGMA was polymerized via Reversible addition-fragmentation transfer polymerization. It was easy to tail the compatibility of the epoxide block copolymer with a step-growth polymerized matrix, to form nanostructure via reaction with PGMA segements. In the case of inert POSS-PMMA copolymers modified epoxy, topology of copolymer defined the final morphology and interaction between epoxy and them, because of directional hydrogen bonding and dilution effect. Tg of different epoxide conversion, obeyed of Gordon-Taylor equation and Kwei equation, k which reflected the interaction of modifier and DGEBA/MEDA and epoxy/amine oligomers, was consistent of the rheology and dynamic results.

Several approaches have been employed for directing the intracellular trafficking of DNA to the nucleus. Cationic polymers have been used to condense and deliver DNA and a few specific examples using chitosan as cationic polymer have been described. The concerted efforts in gene therapy to date have provided fruitful achievements toward a new era of curing human diseases. A number of obstacles, however, still must be surmounted for successful clinical applications. Therefore, chitosan-plasmid and quaternised chitosan-plasmid complexes (polyplexes) were investigated for their ability to transfect COS-1 cells and the results were compared with Transfectam/DNA lipoplexes for transfection efficiency. All of the chitoplexes utilised in this study proved to transfect COS-1 cells, however to a lesser extent than the Transfectam/DNA lipoplexes, which served as a positive control. Complexes formed with quaternised trimethyl and triethyl chitosan oligomers, specifically TMO L and TEO L, proved to be superior transfecting agents compared to other chitosans. The molecular mass of chitosan is considered to influence the stability of the chitosan/DNA polyplex, the efficiency of cell uptake and the dissociation of DNA from the complex after endocytosis. In literature it was shown that the toxicity of the chitosan1DNA polyplexes is relatively low compared to viral gene and lipid non-viral delivery vectors. This study showed that the percentage viable COS-1 cells when transfected with the chitosan polymers, oligomers, quaternised chitosan polymers and quaternised chitosan oligomers (chitoplexes) was higher than the percentage viable cells when transfected with lipoplexes prepared with Transfectam with the MTT assay. The Transfectam/DNA lipoplexes induced cell damage and a decreased viability of COS-1 cells were found. Chitosan/DNA and quaternised chitosan/DNA complexes did not affect the viability of the cell line. The degree of quaternisation of the polymers and oligomers and molecular size proved to be two important factors when considering effective non-viral gene delivery. It can be concluded that chitosan, especially quaternised oligomeric derivatives are polysaccharides that demonstrate much potential as a gene delivery system. The high solubility and low toxicity of chitosan allow its use in a wide variety of applications in the pharmaceutical industry and, as shown in this study, in gene delivery. / Thesis (Ph.D. (Pharmaceutics))--North-West University, Potchefstroom Campus, 2006.

Les protéines membranaires représentent environ 2/3 des cibles thérapeutiques. Le développement de nouveaux médicaments est toutefois limité par l'absence de données structurales pour de nombreuses protéines. Les protéines membranaires s'avèrent en effet difficiles à manipuler et à maintenir en solution ce qui complique leur étude structurale. Les protéines sont en général solubilisées grâce à des surfactants comme les détergents, les amphipols, les hémifluorés et les peptergents. Il est aussi possible de les étudier dans des conditions plus physiologiques en les insérant dans des membranes lipidiques telles que des liposomes, des bicelles, ou des nanodisques.Les nanodisques sont des particules protéolipidiques autoassemblées, composées de protéines d'assemblages et de lipides, qui constituent un système de membranes modèles très prometteur permettant de solubiliser des protéines membranaires dans un milieu dépourvu de détergent. D'autres avantages sont aussi la variabilité de la constitution en lipides et l'accessibilité des deux côtés de la membrane.Dans le cadre de ma thèse, j'ai mis au point l'insertion de plusieurs protéines membranaires en nanodisques afin de permettre leur caractérisation fonctionnelle, biophysique et structurale. Nous avons en particulier étudié le transporteur ABC BmrA impliqué dans la résistance aux antibiotiques et cherché à identifier les changements conformationnels de la protéine en nanodisques par microscopie électronique. Les interactions de la protéine YedZ, un homologue de NADPH oxydases, avec ses partenaires solubles potentiels ont été étudiés par différentes méthodes telles que le pontage chimique, la résonance plasmonique de surface et la spectrométrie de masse native. En parallèle, le mécanisme d'assemblage des nanodisques a été investigué. Une interaction entre les protéines d'assemblages et des cations divalents a été mise en évidence. Cette interaction a un effet sur l'oligomérisation de la protéine d'assemblage mais également sur l'homogénéité des nanodisques. Ces observations nous ont permis d'améliorer les conditions de préparation des nanodisques, condition déterminante pour le succès de nombreuses approches structurales. Nous avons pu en particulier explorer la possibilité de cristalliser ces particules en vue d'études cristallographiques. / Membrane proteins represent around 2/3 of therapeutic targets. However, the development of new drugs is hampered by the lack of structural data for many proteins. Membrane proteins are indeed difficult to handle and to maintain stable in solution, which complicates their study by structural methods. Proteins are usually stabilized by surfactants like detergents, amphipols, hemifluorinated compounds and peptergents. It is also possible to study those proteins in an environment mimicking their native conditions by incorporating them in lipid membranes such as liposomes, bicelles or nanodiscs.Nanodiscs are self-assembled proteolipidic particles, composed of a scaffold protein and lipids. This technology is a top-notch model membrane system, which provides a detergent free environment to study membrane proteins in solution. Further advantages are the possibility to vary the lipid composition and the accessibility of the incorporated protein from both sides of the membrane.During my PhD project, I have achieved the insertion of several membrane proteins into nanodiscs for functional, biophysical and structural characterizations. In particular, we have studied Bmra, an ABC transporter involved in multidrug resistance and tried to identify the conformational changes of the protein in nanodiscs by electron microscopy. The interaction of YedZ, a NADPH oxidase homologue, with potential soluble partners has been studied by various methods such as cross-linking, surface plasmon resonance and native mass spectrometry. In parallel, the mechanism of nanodiscs assembly has been investigated. An interaction between the scaffold protein and divalent cations has been revealed. This interaction influences the oligomerization of the scaffold protein but also the nanodiscs homogeneity. Those observations allowed us to improve the preparation of the nanodiscs, which was an essential step torward the success of many structural approaches. In particular, we were able to explore their accessibility to protein crystallography.

Magister Scientiae - MSc / Malaria remains the most important parasitic disease worldwide. It is estimated that over 500 million infections and more that 2.7 million deaths arising from malaria occur each year. Most (90%) of the infections occur in Africa with the most affected groups being children of less than five years of age and women. this dire situation is exacerbated by the emrggence of drug resistant strains of Plasmodium falciparum. The work reported in this thesis focuses on improving the purification of PfHPRT by investigating the characteristics of anion exchange DE-52 chromatography (the first stage of purification), developing an HPLC gel filtration method for examining the quaternary structure of the protein and possible end stage purification, and initialcrystalization trials. a homology model of the open, unligaded PfHPRT is constructed using the atoomic structures of human, T.ccruz and STryphimurium HPRT as templates. / South Africa

Surfactants from the 'Triton' range, manufactured by Rohm and Haas, Germany, were used to study the adsorptive behaviour of non-ionic surfactants (of the alkyl polyoxyethylene type) from aqueous solution onto mineral oxide surfaces. The oligomeric distributions of the surfactants were characterised using the HPLC technique. Two gradients were used: a normal phase gradient was used to study the surfactants from non-aqueous solution; an unusual gradient, which could not be definitively categorised as either normal or reversed phase and which was developed at Brunel, was used to analyse surfactants directly from aqueous solution. Quartz was used as a model mineral oxide surface. The quartz surface was characterised using a range of techniques: scanning electron microscopy (SEM), X-ray photoelectron spectroscopy, X-ray fluorescence -analysis, Fourier transform-infra red spectroscopy and BET analysis. It was found that washing the quartz with concentrated HCI removed any calcium ions present on the surface and also removed 02- ions. Calcining the sample removed carbonaceous materials from the surface and also caused a decrease in the surface area. The quartz was shown to be non-porous by SEM and BET analysis. The adsorption experiments for this study were carried out using a simple tumbling method for which known ratios of surfactant in aqueous solution and quartz silica were mixed together for a known length of time. The amounts of surfactant present were measured using ultra-violet analysis and the HPLC techniques mentioned above. It was found that the smallest oligomers were adsorbed the most. An addition of salt to the system caused an overall increase in adsorption of the bulk surfactant, and increase in temperature caused an initial decrease in adsorbed amounts before the plateau of the isotherm and a final increase in bulk adsorption at the plateau of the isotherm. The oligomeric adsorption generally appeared to mirror the behaviour of the bulk surfactant. Atomic force microscopy (AFM), dynamic light and neutron scattering studies were used to analyse the character of the adsorbed surfactant layer. It was shown that the layer reached a finite thickness that corresponded to a bilayer of adsorbed surfactant. According to AFM data, this value of thickness was not consistent over the whole of the quartz surface.

付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 新制・課程博士 / 博士(工学) / 甲第23227号 / 工博第4871号 / 新制||工||1760(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 田中 一生, 教授 秋吉 一成, 教授 古賀 毅 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

The use of autografts is the gold standard for peripheral nerve regeneration (PNR) while biomedical engineering made some contributions to improve PNR. A next generation of nerve guidance conduits (NGC) is required to transmit topographical and biochemical signals towards severed nerves. In this thesis, the gelatin hydrolyzate Collagel® (COL) and anhydride-containing cross-linkers (oPNMA, oPDMA) were used to fabricate crosslinked hydrogels (cGEL) for PNR. At first, established cGEL formulations were adjusted towards an injection-molding tool with static mixer. Therefore, the gelation kinetic was modified by variation of the gelation base. Hence, high reactive oPNMA was available for fabrication of robust cGEL based NGC. Secondly, novel cGEL and molding technique were adapted towards the fabrication of cGEL-based filler for polymer-derived braided NGC. Shear-thinning filler was developed that allowed direct application inside the conduit lumen with minimal mechanical stiffness but sufficient scaffolding properties. Besides pristine filler, chemically modified filler was designed with a small mimetic of the nerve growth factor, LM11A-31, that was grafted to oPNMA. In a rat sciatic nerve model, the performance of this derivatized filler was comparable to the control and underlined the potential of chemical cues in PNR. A number of small diamines were further integrated into oPNMA and oPDMA to modify cGEL bulk. In addition to chemical feasibility, the cytocompatibility and cellular response were tested on L929 mouse fibroblasts and human adipose-derived stem cells. The functionalization showed an impact on the cell behavior with differences in cell proliferation, migration and spreading. Finally, modified oPNMA-derived hydrogels were tested on neonatale Schwann cells. The cell viability and extension was maintained in all hydrogels while the impact of LM11A-31 was not as pronounced. This thesis emphasizes the potential of cGEL hydrogels in nerve implants as fillers or conduits and, thus, is a promising building block for a new generation of NGC.