Dissertations / Theses on the topic 'Alginate capsule'

The work is devoted to the problem of three-dimensional reconstruction of alginate capsules and their subsequent analysis. The results of the software are shown and the main stages of its work are described. They include such image processing operations as filtering, segmentation, morphological operations, classification, construction of Hough space with the subsequent analysis stage.

Francisella tularensis is a Gram-negative, zoonotic bacterium that causes tularemia in animals and humans. The two subspecies tularensis (Type A) and holarctica (Type B) are considered Tier I Select Agents due to the bioweapon potential of these subspecies. Type A strains, considered the more virulent of the subspecies, are highly infective producing respiratory tularemia with inhalation of as few as 10 cells. Due to classification as a Select Agent, a vast amount of F. tularensis research has occurred in the last two decades after the September 11th terrorism attack and the use of Bacillus anthracis spores in a biological attack on the United States Postal Services in 2001. This research has uncovered many of the various virulence factors of F. tularensis including an intracellular nature, the unique lipopolysaccharide produced, and a genetic pathogenicity island. This dissertation aims to further characterize outer surface antigens of F. tularensis subspecies in regards to virulence, biofilm formation, and role in vaccine development. In addition, this dissertation will also investigate the use of a novel vaccine delivery vehicle, alginate microencapsulation, in increasing the efficacy of these mutant strains. F. novicida is a subspecies of F. tularensis and usually classified as being non-encapsulated. However, F. novicida has a similar capsule glycosylation locus as F. tularensis and could produce a similar capsule-like complex that has previously been described for the F. tularensis LVS strain. I was able to isolate and characterize this CLC of F. novicida, which contained a heterogenous mixture of proteins and possible glycosylated proteins. A mutant with a multi-gene interruption within the glycosylation locus (F. novicidaΔ1212-1218) produced significantly less carbohydrate than the parent strain, was attenuated in the mouse model, and was partially protective when used to immunize mice against a virulent challenge. Biofilms of F. novicida were also characterized in regards to biofilm formation in various growth media and biofilm formation of strains lacking the O-antigen of the lipopolysaccharide (LPS). In general, F. novicida produced the greatest amount of biofilm in a brain heart infusion (BHI) broth, compared to other media. Loss of the O-antigen led to increased biofilm production when grown in BHI and decreased or similar biofilm production as the wildtype when grown in other media. This highlights the need to carefully select the growth medium when assessing biofilm formation of Francisella strains in the future. A final study of this dissertation characterized the use of alginate microspheres as a vaccine vehicle for an attenuated F. tularensis type A O-antigen deficient strain. O-antigen deficient strains of F. tularensis are highly attenuated in vivo and would be a safe choice for a vaccine candidate. However, these strains produce less than ideal protection against virulent challenge when used to immunize mice, possibly due to a lack of persistence in the host. In an attempt to increase persistence, we encapsulated an O-antigen deficient strain within sodium alginate microspheres and used those microspheres to immunize mice. The immunized mice produced a higher level of antibody response than mice immunized with a non-encapsulated version. However, this immunization only partially protected mice from a virulent challenge and did not match the protection afforded by the former Live Vaccine Strain (LVS). In part the deficiency in protection appears to be due to a lack of a robust cellular immune response in mice immunized with the alginate microspheres. In summary, this dissertation focuses on the various extracellular polysaccharides of F. tularensis: the glycosylation of CLC, the O-antigen, and the biofilm. Each polysaccharide plays a role in the virulence and pathogenesis of F. tularensis. Glycosylation of the CLC and the O-antigen are important virulence factors in mammalian disease, and mutants lacking either (not type A strains) are attenuated in the mouse model. Both also appear to play a role in the formation of the F. tularensis biofilm in a manner dependent on the environment or culture medium used. Each of these extracellular polysaccharides contribute to the lifecycle of Francisella.
Ph.D.
Francisella tularensis is a highly infectious bacterial pathogen that can cause disease in a wide array of animals and in humans. F. tularensis is also considered a potential weapon of bioterrorism and the development of an effective vaccine is a critical area of research. One strategy of developing a tularemia vaccine includes mutating a strain of F. tularensis to reduce expression of extracellular components that include polysaccharides. Strains that cannot express these components are usually unable to produce clinical signs in the host and may provide protection against fully virulent F. tularensis strains. The work presented in this dissertation will focus on characterizing the polysaccharide extracellular components of F. tularensis and developing a novel vaccine vehicle to increase protection from strains that do not cause disease.

Encapsulation of pancreatic islets in alginate microbeads and microcapsules show great promise for the treatment of Type 1 diabetes mellitus. Significant progress has been made in developing a biocompatible capsule that allows sufficient exchange of nutrients and products with the encapsulated cells, while at the same time maintaining a barrier to immune cells and preventing rejection of the transplanted cells. However, a truly biocompatible capsule has, as yet, not been developed, and implanted capsules often trigger low levels of inflammation leading to fibrosis, diminished insulin secretion, and sometimes death of the encapsulated cells. A lepirudin-based human whole blood model was used to demonstrate the inflammatory potential of a set of different alginate microcapsules and microbeads. This was performed to elucidate the effect of different capsule and bead parameters, such as the effect of a hollow versus solid inner core, polycation type, polycation concentration, alginate type, and capsule and bead diameter. Complement activation after incubation of capsules in whole blood was measured as sTCC generation. In addition, the secretion of chemokines, inflammatory cytokines, antiinflammatory cytokines, and growth factors was analyzed by ELISA and Bio-plex. Leukocyte activation as measured by CD11b expression was detected using flow cytometry. Finally, Confocal Laser Scanning Microscopy (CLSM) was used in order to screen for a set of plasma proteins and observe what proteins adsorbed to the capsule surface. TAM alginate microbeads did not trigger complement activation, secretion of cytokines, or up-regulation of CD11b expression, and thus appeared to have a minimal inflammatory potential. In addition, the protein adsorption assay showed no apparent protein surface deposition on the microbeads after 6 hours of incubation in plasma of the proteins screened for (complement protein C3, complement regulatory proteins factor H, factor I, C1 inhibitor, and vitronectin, as well as coagulation cascade proteins fibrinogen, plasminogen, and HMWK). Solid alginate APA microcapsules containing poly-L-lysine (PLL), on the other hand, showed an increase in complement component sTCC levels, in chemokine levels (IL-8, MCP-1, and MIP- 1α), in inflammatory cytokine levels (IL-6, IL-1β, and TNFα), in anti-inflammatory cytokine levels (IL-1RA and IL-10), and in growth factors levels (PDGF, HGF, and VEGF), as well as a decrease in cytokine IP-10 levels. In addition, the capsules also stimulated leukocyte activation by up-regulating the expression of CD11b. The solid APA micrcapsules showed heavy C3 adsorption, coupled with vitronectin and factor H surface deposition, indicating increased complement activity on these capsules. Hollow APA microcapsules with PLL triggered a rapid and strong sTCC response, as well as significantly increased secretion of the chemokine MCP-1. At the same time, a significant decrease in secretion of chemokines (IL-8 and MIP-1α) and inflammatory cytokines (IL-1β and TNFα), as well as a decrease in secretion of growth factor VEGF, and cytokine MIF, and an increase in cytokine IP-10 was observed. All these cytokine levels except the chemokine MCP- 1 and the complement complex sTCC suggested reduced inflammatory potential for hollow APA capsules. It was proposed that these capsules adsorbed the anaphylatoxins C3a and C5a, thus preventing the complement mediated activation of leukocytes. No increased surface adsorption of C3 was detected on hollow APA capsules compared to solid APA capsules. Conversely, the C3 adsorption was higher on solid APA capsules, thereby not reflecting the increased sTCC generation seen for hollow APA capsules. One explanation for this might be that the hollow capsules secreted some soluble molecule capable of triggering sTCC generation. No apparent change in inflammatory potential could be observed by exchanging the polycation PLL with PLO (poly-L-ornithine), except for abolishing the strong sTCC response observed for hollow APA capsules with PLL as well as lowering the MCP-1 response. It was suggested that this observation could be the result of PLO reducing the permeability of the capsules, thus preventing the diffusion of the hypothesized soluble trigger of sTCC. Increased sTCC was detected with increasing PLL concentration in High G alginate APA capsules. The same could not be observed for High M alginate capsules, however, the chemokine IL-8 and the inflammatory cytokines IL-1β and TNFα increased with increasing PLL concentration, suggesting increased inflammation with increasing PLL concentration. No change in inflammatory potential could be detected with varying alginate microbead diameter. Nor could any change in inflammatory potential be observed by the addition of HEPES in the gelling solution. TAM alginate microbeads appear to have the lowest inflammatory potential of the capsules tested, and are therefore the most suited for in vivo application from an inflammatory aspect, as demonstrated by the whole blood assay. A recent study in Type 1 diabetes patients however showed increased fibrosis when encapsulating human islet cells in barium alginate microbeads [61]. Further studies where incubation of TAM microbeads with isolated monocytes are co-cultured with fibroblasts could further elucidate the mechanisms of fibrosis on the microbeads. In addition, continued screening of protein adsorption on the bead surface should be performed.

Alginate is a popular candidate for encapsulation of cells due to the formation of gels with divalent ions under physiological conditions. Stable alginate gels can be formed by the selection of alginates with a high content of guluronic acid (G) and gelling in a mixture of calcium and barium. These alginate gels have been proposed as immune protective barriers for the transplantation of human pancreatic islets (insulin producing cells) for the treatment of type 1 diabetes where the alginate gel protects the transplant from the host immune system. Microencapsulation can thus provide a way to overcome the need for immunosuppressive drugs. Although showing promising results in animal models, there are potential limitations of the Ca2+/Ba2+-beads concerning growth of host cells on the surface of capsules in primate models. Development of coating strategies for alginate based capsules could thus be beneficial for reducing the attachment of host cells. Alginate microbeads/capsules were formed by electrostatic bead generator producing beads of 400µm. Afterward, the alginate beads were coated by fluoresceinamine labeled alginate that was visualized by confocal laser scanning microscopy (CLSM) and quantified by fluorescent spectroscopy. The binding of coating alginate to alginate-poly-L-lysine (PLL) capsules was also studied.In this project, in the optimalisation of coating of alginate beads some parameters were studies such as concentration of coating alginate, different gelling ions both for core and coating alginate, exposure time for gelling solution for fixation of coating layer and different washing solution.The long-term stability of coating layer of coated alginate beads was determined by measuring the fluorescent intensity of fluorescently labeled of coating alginate during a period of forty nine days. A stability study of alginate-alginate capsules revealed that Ca2+/Ba2+ alginate coated with high-G alginate and washed with saline and used Ca2+ and Ba2+ with ratio 50:1 for fixation of coating layer were more stable coating than other capsules. The alginate beads coated with high-M or epimerized alginate were produced. It shows higher intensity of coating layer in both capsules coated with high-M or epimerized alginate than alginate beads coated with high-G alginate. In continue of the study, the alginate-PLL capsules were coated with high-G, high-M, and epimerized and sulfated alginate. Alginate-PLL capsules coated with high-G, high-M and epimerized alginate shows no detective signal by confocal images and sulfated alginate coating shows some signal of coating. The stability of coating for alginate-PLL-alginate capsules and alginate beads coated with epimerized or high-M alginate revealed that both kind of coating have high-stability over one week screening.Three dimensional images of capsules, in confocal microscope, both epimerized and high-M coating alginates cover whole of capsules. However, in 3D images we have seen some fragment of coating gelling in the surround solution and attached to the capsules which can make disturbance in spectroscopy measurement. 3D images of alginate-PLL capsules coated with sulfated alginate show evenly distribution of coating.

Orientador: Ana Liz Garcia Alves
Resumo: As terapias regenerativas com a utilização de células tronco mesenquimais (CTMs) têm sido amplamente empregadas com a finalidade de modificar a progressão de enfermidades locomotoras em animais de grande porte. Estudos sobre o comportamento das células tronco, portanto, mostram-se de extrema importância para que, cada vez mais, elucidar sua ação, efeito e eficácia nos tratamentos propostos. A inserção das CTMs derivadas do tecido adiposo de muares em microcápsulas de hidrogel gera expectativas promissoras para a proteção da célula contra anticorpos do receptor, bem como processos inflamatórios exacerbados, distribuição de agentes terapêuticos e supressão de processos inflamatórios. O presente trabalho teve por objetivo verificar o comportamento das CTMs após o encapsulamento em hidrogel, quanto a sua viabilidade, migração, além da avaliação morfológica e imuno-histoquímica. Avaliação da morfologia da cápsula, dos poros, a rugosidade por microscopia eletrônica de varredura (MEV) e observação das células encapsuladas pela microscopia confocal de varredura a laser. A porcentagem de células viáveis manteve-se ao longo dos momentos em uma média de 93%, então o biomaterial permitiu a difusão de nutrientes e oxigênio adequadamente. A diminuição da quantidade de células no interior das cápsulas é justificada pela possível migração das mesmas através dos microporos das microcápsulas permitindo a aderência à placa de cultivo. Na avaliação morfológica foi possível identificar as células... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Regenerative therapies using mesenchymal stem cells (MSCs) have been widely widespread to treat locomotor diseases in large animals. Studies on the behavior of stem cells are extremely important to increase our knowledge regarding their action, effect and effectiveness in the proposed treatments. The insertion of muar adipose-derived MSCs into hydrogel microcapsules yields promising expectations for cell protection against immune response, as well as exacerbated inflammatory processes, delivery of therapeutic agents, and suppression of inflammatory processes. The present research aimed to verify the behavior of MSCs after hydrogel encapsulation, including cell viability, migration, morphological and immunohistochemical pattern. Evaluation of capsule morphology, pore size, roughness by scanning electron microscopy (SEM) and observation of encapsulated cells by confocal laser scanning microscopy. The percentage of viable cells remained throughout the moments at an average of 93%, so the biomaterial allowed the diffusion of nutrients and oxygen properly. A decreased amount of cells number inside the capsules is justified by the possible migration of them through the microcapsule micropores allowing adherence to the culture plate. The cells showed positive CD44 staining, absence in MHC II. The capsules were evaluated with SEM for their morphology, the area of circular and irregular pores and the size of the cells. It was possible to confirm the presence of stem cells in the micro... (Complete abstract click electronic access below)
Doutor

This thesis investigates the transport and sorting of capsules (elastic membranes enclosing a liquid core) using viscous flow in complex vessel geometries. Of particular interest is passive sorting by deformability using only the fluid-structure interaction between the capsule, the viscous fluid and the geometry of the vessel. Millimetric alginate-ovalbumin capsules in the regime of negligible fluid inertia are used in this work. In order to characterise the elastic properties of the capsules, a novel numerical finite element model of the compression of a thick-shelled capsule between parallel plates is implemented. The constitutive model of the capsule membranes was determined by comparison to experimental data: a Yeoh constitutive model with the ratio of constants $C_1 = 1$, $C_2 = 0$ and $C_3 = 10$ describes the capsules used. Three geometries are investigated in this work. (i) A T-Junction bifurcation. Capsule deformation in the T-Junction bifurcation is characterised by the maximal length of the capsule $L_{max}$ and depends on the ratio of viscous to elastic forces, the capillary number $Ca$. The maximal length, $L_{max}$, is especially sensitive at distinguishing soft capsules by their deformability. The sensitivity of $L_{max}$ to capsule compliance and the large deformations that can be achieved makes the T-junction a promising geometry in which to measure elastic properties of the capsules. The rate of relaxation of the capsules after the bifurcation is independent of their deformation. (ii) A half-cylinder obstacle in a channel followed by a sudden expansion. We show that the half-cylinder obstacle causes capsule trajectories to vary depending on deformability. Capsules with a factor of three difference in deformability can be separated. A practical feature of the system is its relative insensitivity to the initial lateral position of the capsules in the channel. However, while the results are reproducible across different capsules, the variations in final position amount to 10 \% at fixed parameters. As these experiments were conducted with the same capsule under identical flow conditions, this is likely to represent the best case scenario. (iii) We adapt the pinched flow fractionation (PFF) geometry to the sorting of capsules. We show that the standard PFF device cannot be used to sort capsules. However, a novel mode of operation, termed the ``T-Junction'' mode, shows great promise for the sorting of capsules. The PFF device in the T-Junction mode separates capsules with a factor of 1.5 difference in deformability. This is twice as sensitive as the half-cylinder device, although larger variability was observed in the PFF device.

Actuellement les traitements thérapeutiques pour soigner la schizophrénie, par voie intraveineuse ou orale, ne sont qu’en partie efficaces et associés généralement à des effets extrapyramidaux souvent dangereux et très gênants pour les patients. Afin d’augmenter l’efficacité du traitement toute en neutralisant les effets indésirables, ce travail a eu comme objectif de concevoir des capsules composites (PLGA-PEG / alginate) destinées à être administrées par voie orale et capables de libérer localement, de façon spécifique et contrôlée, le neuroleptique halopéridol dans le cerveau. L’optimisation du protocole de synthèse a permis d’obtenir de façon reproductible des nanocapsules de PLGA poreuses monodisperses et peu agrégées, possédant un diamètre hydrodynamique moyen inférieur à 80 nm et une bonne stabilité en solution aqueuse. Une fois fonctionnalisées avec le Poly (éthylène glycol) diamine, des études in vitro ont montré la faible toxicité de ces nanoparticules furtives ainsi que leur capacité à encapsuler une quantité satisfaisante d’halopéridol et de libérer ce principe actif sur une durée d’un mois avec un faible effet « burst ». L’incorporation des nanoparticules pégylées dans des matrices préparées à haute concentration d’alginate et de 100 % CaCl2 a permis d’obtenir des billes nanocomposites possédants une meilleure stabilité à la sortie du milieu gastrique simulé et persistent environ 30 minutes en milieu intestinal simulé. Enfin des études in vivo préliminaires sur des souris adultes utilisant des nanoparticules injectées et des billes nanocomposites ingérées ont démontré l’efficacité de ces systèmes à délivrer l’halopéridol au cerveau
Currently therapeutic treatments for schizophrenia, intravenously or orally, are only partially effective and generally associated with extrapyramidal effects often dangerous and very troublesome for patients. In order, to increase the treatment efficiency by neutralizing any side effects the aim of this work was to design composite capsules (PLGA-PEG / alginate) intended to be administered by way oral and able to release locally, in a specific and controlled way, the neuroleptic “haloperidol” in the brain. The optimization of the protocol of synthesis allowed to obtain in a reproducible way of the nanocapsules of monodisperse and not very aggregate porous PLGA, having an average hydrodynamic diameter lower than 80 Nm and a good stability in aqueous solution. Once functionalized with Poly (ethylene glycol) diamine, in vitro studies showed the low toxicity of these furtive nanoparticles as well as their ability to encapsulate a satisfactory amount of haloperidol and release this active principle over a period of one month with a low burst effect. The incorporation of the PEGylated nanoparticles in matrices prepared with a high concentration of alginate and 100% CaCl2 made it possible to obtain nanocomposite beads having a better stability at the exit from the simulated gastric medium and persist approximately 30 minutes in simulated intestinal medium. Finally, preliminary in vivo studies on adult mice using injected nanoparticles and ingested nanocomposite balls showed the effectiveness of these systems to deliver haloperidol in the brain

Thesis (PhD) -- University of Western Sydney, 2007.
A thesis submitted to the University of Western Sydney, College of Health and Science, School of Natural Sciences, as a requirement for the degree of Doctor of Philosophy. Includes bibliography.

L'encapsulation des produits biologiques est particulièrement intéressante pour préserver leur intégrité et permettre leur relargage de façon contrôlée. L'alginate, polysaccharide anionique, est souvent utilisé pour la microencapsulation. Il présente des propriétés de gélification en présence de cations divalents (Ca++, Mg++). La microencapsulation de molécules biologiques et de cellules dans des billes d'alginate de calcium présente l'avantage d'être très douce et de maintenir ainsi l'activité de ces produits. Cependant pour limiter le relargage du matériel encapsulé, il est parfois nécessaire d'établir une membrane à la surface de la bille par formation d'un complexe polyélectrolyte avec un polymère polycationique. Le chitosane, polysaccharide cationique naturel, ou le diéthylaminoethyl-dextrane peuvent donc être utilisés pour l'élaboration d'une membrane autour de la bille d'alginate de calcium. Deux techniques de production de billes d'alginate ont été testées, la méthode à l'extrusion et celle à l'émulsion, qui ont permis d'obtenir des billes d'alginate de calcium de diamètre moyen différent soit respectivement de 1mm et 70m. Nous avons étudié les propriétés de rétention de ces billes recouvertes d'une membrane polyélectrolyte vis à vis de différentes molécules, des protéines (hémoglobine, albumine de sérum bovin), des dextranes de masses molaires différentes, le benzène tetracarboxylate. Les conditions de formation de la membrane, pH et concentration de la solution de polymère polycationique et la nature du polycation ont une influence sur les propriétés de rétention de ces billes recouvertes d'une membrane. La nature des produits encapsulés à également une grande influence sur leur rétention. Leur taux de libération varie en fonction de leur masse molaire, leur conformation tridimensionnelle ainsi que de leur composition chimique

Cette thèse a pour objectif d’étudier les propriétés physico-chimiques de capsules à cœur liquide à base d'hydrogel d'alginate et de contrôler leur perméabilité et propriétés mécaniques par ajout des biopolymères. Ces capsules sont préparées par un procédé de sphérification inverse par extrusion goutte à goutte d’une solution de chlorure de calcium dans un bain à base d’alginate. Dans un premier travail, l’influence des polymères utilisés pour contrôler la viscosité du cœur liquide lors de la préparation des capsules sur la perméabilité et la stabilité mécanique a été étudiée. Les propriétés mécaniques des capsules ont été corrélées avec les propriétés viscoélastiques d’hydrogels d’alginate caractérisés par rhéologie oscillatoire aux faibles amplitudes. Un second travail, a consisté à élaborer des capsules composites avec une membrane de caséinate de sodium/alginate qui présentent une meilleure stabilité et une libération pH-dépendante d’un colorant utilisé comme molécule modèle. Comme perspective a cette étude, des hydrogels sphériques à base d’alginate et de caséinate de sodium, avec différentes architecture ont été développés et leur efficacité a été testée sur trois colorants. Enfin, l’influence de l’incorporation de la gomme laque dans la membrane ou comme revêtement externe a permis de mettre en évidence une amélioration des propriétés barrières vis-à-vis de molécules de faible masse moléculaire (riboflavine dans ce cas). Les capsules à base d’alginate ont un large spectre d’utilisation allant de la cuisine moléculaire à la biotechnologie ce qui nécessite une meilleure compréhension et contrôle de leurs propriétés physicochimiques en fonction de l’application visée
The aim of this thesis is to study the physicochemical properties of alginate liquid-core capsules and to control their permeability and mechanical properties by biopolymers blending. These millimeter-scale size capsules are prepared by a reverse spherification process by dripping a solution of calcium chloride into an alginate gelling bath. In a first work, the influence of polymers used to control capsule liquid-core viscosity (thickening agent) during capsules preparation on permeability and mechanical stability of the alginate membrane was investigated. The mechanical properties of capsules were correlated with viscoelastic properties of plane alginate hydrogels characterized by small amplitude oscillatory shear rheology. In a second work, composite capsules with a membrane of sodium caseinate / alginate were developed and showed improved stability and pH-dependent release of a dye used as a model molecule. As a perspective, composite alginate/sodium caseinate microspheres with different architectures were developed and their effectiveness was tested against three anionic dyes. This type of system has applications in the removal of dyes from industrial wastewater by an adsorption mechanism. Finally, the influence of shellac incorporation in alginate membrane or as an external coating layer resulted in enhanced physicochemical properties and decreased membrane permeability against low molecular weight molecules (riboflavin in this case). Alginate capsules have a wide range of applications ranging from molecular gastronomy to biotechnology which requires a better understanding and control of their physicochemical properties according to the target application

Ce travail de thèse concerne l'étude des propriétés physico-chimiques de capsules millimétriques à cœur liquide possédant une membrane fine d'hydrogel d'alginate. Nous avons dans un premier temps caractérisé le transport de molécules à travers la coque d'hydrogel et estimé le rayon de coupure de cette membrane semi-perméable. De plus, il est possible de limiter la fuite de petits solutés hydrophiles en ajoutant une couche hydrophobe entre le cœur aqueux et la membrane d'alginate. Nous démontrons alors que le temps caractéristique de fuite est déterminé par la géométrie du système (épaisseur de la couche hydrophobe et rayon de la capsule) ainsi que la solubilité et le coefficient de diffusion du soluté dans la phase huileuse. D'autre part, le gonflement d'une capsule soumise à une différence de pression osmotique nous a permis d'identifier les régimes viscoélastique et viscoplastique de la membrane sous étirement. Nous en déduisons un module élastique de l'hydrogel, qui diminue au-delà du seuil de plasticité. A contrario, lors de la compression de capsules, l'hydrogel est concentré par expulsion irréversible de l'eau, ce qui entraîne une rigidification de sa structure. Enfin, nous nous sommes intéressés à l'écoulement d'une suspension concentrée de capsules à coeur huileux dans l'eau. Il existe une friction entre les capsules qu'il est possible de contrôler par ajout de tensioactif. Nous avons donc étudié son rôle dans l'écoulement de capsules dans une constriction de type sablier. Ainsi, nous démontrons que le flux est constant et qu'il ne dépend pas de la friction, mais qu'il est fixé par la taille de l'étranglement. En effet, l'écoulement est uniquement déterminé par l'hydrodynamique en phase fluide dans le col du sablier.

Renal failure uremia occurs when the kidneys fail to function properly. Despite being the main treatment, dialysis and other therapeutic approaches are not only associated with numerous long-term adverse complications often leading to morbidity and mortality events, but are also not affordable. Orally administrating Alginate-Poly-L-Lysine-Alginate microcapsules entrapping live yeast cells to treat renal failure uremia has not yet been investigated. In this thesis, the growth and microencapsulation of yeast were optimized. The efficacy of these microcapsules in removing unwanted electrolytes was tested in vitro in simulated gastro-intestinal media, in vitro in a column bioreactor and in vivo in an uremic rat model. Results showed that these novel microcapsules can not only maintain morphological stability and membrane integrity under gastro-intestinal environments and mechanical stresses, but also, preserve the viability of yeast. These microcapsules were successful in reducing urea concentrations while not harming the human GI tract's microbial flora.

Nous avons étudié l’effet d’une stimulation ultrasonore sur les propriétés mécaniques et le relargage de billes et capsules d’alginate. Les capsules sont fabriquées avec le même d’extrusion que les billes, mais selon un nouveau procédé mis au point. Elles sont constituées d’un fortement visqueux entouré d’une mince membrane hyperélastique. En utilisant une technique ultrasonore de pulse-écho, nous avons d’abord montré que le gel d’alginate est quasiment incompressible et puis il a ainsi un coefficient de Poisson très proche de 0. 5. Le module de cisaillement des billes et capsules d’alginate a été mesuré par compression. Nous avons montré qu’elles retrouvent leurs propriétés mécaniques après la sonication. Cependant, le sonication peut conduire à la rupture des capsules pour des intensités élevées et/ou longues durée d’exposition à cause de la fatigue de la membrane. Un tel phénomène n’a pas été observé sur les billes. Le relargage est étudié en sonifiant des billes et capsules chargées mises en suspension. Le relargage qui résulte de la sonication des capsules est proportionnel à la durée de sonication et à l’amplitude de l’onde de pression. Une interprétation physique possible est que l’écoulement de streaming acoustique induit par l’onde ultrasonore augmente la convection à proximité de la membrane de capsule et donc le relargage. Le relargage pour les billes d’alginate augmente également avec les paramètres ultrasonores, mais dans une moindre mesure. Nous avons finalement quantifié le relargage passive après une sonication de faible intensité : il est en moyenne identique à celui mesuré sur les billes et capsules non-sonifiées. Globalement, le gel d’alginate retrouve donc ses propriétés physiques et mécaniques après la sonication. Si la sonication conduit à une augmentation de la porosité, l’augmentation est temporaire et réversible à la fin de stimulation ultrasonore
We have investigated the effect of ultrasonic stimulation on the mechanical properties and release of alginate beads and capsules. Capsules are fabricated with the same technique of extrusion as the beads, but using a new process that has been developed. They are made of a highly-viscous liquid core enclosed in a thin hyperelastic alginate membrane. Using an ultrasonic pulse-echo technique, we have first shown that the alginate gel is quasi-incompressible and thus has a Poisson ratio very close to 0. 5. The shear modulus of the alginate beads and capsules has then been measured by compression. We have shown that they recover their mechanical properties after sonication. Still, at high intensities and/or long exposure times, sonication can lead to the rupture of the capsule membrane due to fatigue. No similar phenomenon has been observed for the beads. Release is studied by sonicating loaded beads and capsules suspended in an aqueous solution. The mass release that results from the sonication of capsules is found to be proportional to the sonication duration time and pressure wave amplitude. A possible physical interpretation is that the acoustic streaming flow induced by the ultrasonic wave enhances convection in the vicinity of the capsule membrane and thus mass release. The release from alginate beads is also increased by the ultrasonic parameters, but to a smaller extent. We have finally quantified the passive release subsequent to low-intensity sonication: it is on average identical to the one measured on non-sonicated beads and capsules. Overall the alginate gel therefore recovers its physical and mechanical properties after sonication. If sonication leads to an increase in porosity, the increase is temporary and reversible at the end of the ultrasonic stimulation

L’intérêt pour de nouveaux modèles de foie, plus proches physiologiquement du foie in vivo, est élevé, en particulier en provenance de l’industrie pharmaceutique. En effet, les systèmes standards, tels que la culture en 2D ne sont pas très prédictifs pour certaines études et de meilleurs modèles sont nécessaires, à la fois dans le cadre des études ADME/Tox pour le développement de médicaments ou pour modéliser les nombreuses maladies hépatiques. Afin de reproduire le foie humain, un modèle doit imiter sa structure de façon plus proche que les systèmes en 2D et refléter sa composition cellulaire. Pour produire ce modèle, nous avons utilisé une technologie de micro-encapsulation basée sur la co-extrusion dans l’air d’un jet biphasique, composé d’une phase externe de solution d’alginate et d’une phase interne contenant les cellules. Ce jet est ensuite fragmenté puis l’alginate est réticulé produisant des micro-capsules cœur/coque. La coque poreuse d’alginate protège les cellules du stress mécanique tout en permettant le passage de l’oxygène et des nutriments.Les cellules s’auto-assemblent dans ces capsules en sphéroïdes hépatiques pouvant être cultivés pendant un mois en gardant une bonne fonctionnalité et pouvant être utilisés dans le cadre de criblage à haut-débit. Cette thèse a porté sur l’utilisation de cette technologie pour mettre au point un modèle 3D de foie humain contenant des hépatocytes primaires humains, des cellules de Kupffer, et des cellules endothéliales sinusoïdales. Dans un premier temps, les conditions de culture de ce modèle ont dû être optimisées, notamment le ratio entre les différents types cellulaires et le milieu de culture adapté à ceux-ci. Puis, une fois ces conditions établies, le modèle a été caractérisé, structurellement par microscopie,ainsi que fonctionnellement, par l’étude de l’expression génique de plusieurs protéines hépatiques importantes, telles que les cytochromes P450 ou des récepteurs nucléaires. Des études d’activité enzymatique, de sécrétion d’albumine et d’urée ont également été menées. Ces capsules nous permettent d’obtenir un modèle en 3D, plus proche de la structure du foie humain, et capable de reproduire les interactions complexes entre les différents types cellulaires
Interest for new and more physiologically relevant liver models is high, particularly from pharmaceutical companies. Standard systems, like 2D culture, are indeed not enough predictive and better models are needed, either for drug candidates screening in ADME/Tox studies or for hepatic diseases modelling. To be closer to the human liver, a new model needs toreplicate liver structure and cellular composition better than the 2D. To this end, we used a micro-encapsulation technology, developed by the laboratory and based on the co-extrusion of a two-phases jet, composed of an alginate external phase and a cell-containing internal phase. This jet is then fragmented into micro-droplets and the alginate reticulated to form core-shell microcapsules. The porous alginate shell protects the cells from shear stress while letting oxygen and nutrients pass, and by preventing cell adhesion, enables the cells to self-assemble into hepatic spheroids which can bekept alive during one month, retain good functionality and can be used for high-throughput screening. This thesis focusedon using this technology to develop a next 3D liver model containing human primary hepatocytes, Kupffer cells and liver sinusoidal endothelial cells. Firstly, culture conditions for this model had to be optimized, particularly the ratio between these different cell types and the culture medium, which had to be suitable for these cell types. Then, once the culture conditions had been established, the model was characterized, structurally by immunofluorescence staining, and functionally by studying gene expression of important liver proteins, like cytochromes P450 or nuclear receptors. Enzymaticactivity, albumin and urea secretion were also studied. These capsules allow us to obtain a model able to replicate the complex interactions between these cell types and structurally closer to the human liver

碩士
東吳大學
微生物學系
100
Waste water generated in the daily life will into the sewage treatment plant for processing, and brominated organic pollutants contained in wastewater, such as dibromo-diphenyl ether, decabromodiphenyl ether, tetrabromobisphenol A, etc., will accumulate in the sludge of sewage treatment plants the sludge resources to produce risk. The previous reserch shows the strain has the ability to degrade or enzyme can remove a variety of organic pollutants. But add to the degradation ability strains, due to environmental conditions may lead to added strain can not be effective to achieve the removal of organic pollutants. In this study, strain laboratory by Erhjen river isolated PBDE degradation ability of strain, Bacillus sp. (W1) and Pseudomonas sp. (W4), and lignin enzymes through solid culture extraction of the enzymes were extracted in the matrix of the enzyme and the cultivation of White rot fungi. In this study, alginate micro-embedding technique to observe the strain has the ability to decompose and extracellular lignin degradative enzymes containing bromine organic pollutant removal ability after embedding and embedded. In the batch experiments, we found that the dibromo ether after processing seven days to remove the order of the W1 strain (100%)> W4, strains (92%)> lignin degradative enzymes (83%). DecaBDE removal experimental results, the strain of the added W1, W4 strains and enzymes DecaBDE can not reach the effect of biodegradation. Tetrabromobisphenol A removal capacity of the display order for enzymes of lignin (90%)> W1, strains (22%)> W4, strains (19%). In the degradation of tetrabromobisphenol A and dibromo-diphenyl ether experimental results show that by embedding the enzyme capsule removal efficiency and embedding and micro-embedding of the removal efficiency was no significant difference. Speculated that the reasons may be after the alginate entrapped porosity greater than the lignin extracellular enzymes size, so the enzymes are free to move inside and outside the capsule, with or without the use of embedded technology does not affect the enzyme on the pollutant removal efficiency. In the column system experiment results show that the continuous pollutants from entering the column in response to contaminants adsorbed on the column of the sludge after its removal reaction added to the strain or enzyme. Finally, out of the water of pollutants are reduced to very low concentrations or not detected. Column system in practical application to remove the bromine-containing contaminants with good removal efficiency.

Encapsulation is a novel technique that involves the entrapment of materials such as cells, enzymes or chemicals within a semi-permeable matrix and is being explored as a drug delivery system. This project investigated the encapsulation of Streptomyces nodosus in alginate to assess whether this organism can produce the antifungal drug amphotericin B from within the matrix. New methods were developed to immobilise S. nodosus mycelia and spores in alginate capsules, assess bacterial viability and detect ng mL–1 quantities of amphotericin B in culture fluids. When capsules were cultured and cell proliferation was encouraged, organisms formed protrusions on the surface of the capsules. Differentiated branched hyphae that never progressed to sporogenic hyphae were observed on the surface of these structures. Viability was maintained for up to 30 days and low levels of amphotericin B were produced. The emergence of a co-existing free-dwelling population was also observed. Culturing immobilised organisms using conditioned media from an amphotericin deficient S. nodosus strain, augmented the development of the free-dwelling population resulting in the detection of amphotericin B in the culture fluid and full differentiation to sporogenic hyphae. This is the first report of sporulation of S. nodosus in liquid environments and demonstrates that immobilised S. nodosus can produce antibiotics. The sporulation of free-dwelling organisms was also induced using conditioned media and manipulation of quorum size, indicating a solid surface is not required for sporulation. Conditioned media from other Streptomyces spp. induced variable responses including sporulation, pigment formation and antibiotic production, possibly demonstrating communication between species and/or alteration in nutritional status. This new model for the life cycle of S. nodosus will permit the study of developmental pathways, antibiotic production, microbial community structure and inter-species and intra-species signalling.
Doctor of Philosophy (PhD)