Ce travail scientifique a abordé la problématique du vieillissement des îlots pancréatiques et l’effet de la senescence endothéliale et des microparticules (MPs) sur la fonction des îlots. Nous avons exploré l’impact du vieillissement du pancréas sur la morphologie, le devenir et la fonction de l’îlot pancréatique par analyse comparative entre pancréas de rats jeunes et d’âge moyen et le rôle des MPs endothéliales pro-sénescentes sur la fonction des îlots et leur sénescence prématurée. Nos résultats in vivo montrent que le pancréas est un organe précocement sensible au stress oxydant s’accumulant avec l’âge. Il conduit à la surexpression des marqueurs procoagulants et de senescence sans apparition d’apoptose. In vitro, les MPs de cellules endothéliales sénescentes ont un effet pro-sénescent sur les îlots pancréatiques isolés de rats jeunes avec une activité SA-β-galactosidase caractéristique, la surexpression des marqueurs p53, p21 et p16 et la réduction de la capacité de la sécrétion d’insuline en réponse au glucose. L’ensemble de nos résultats in vivo et in vitro désigne la contribution de la sénescence endothéliale comme une cause probable à la dysfonction de greffon. / This scientific work has tackled the question of the pancreatic islets aging and the effect of endothelial senescence and microparticles (MPs) on islet function. We investigated the impact of aging on pancreas morphology, fate and on the function of the pancreatic islet by comparative analysis between pancreas in young and middle-aged rats, as well as the role of pro-senescent endothelial MPs on islet function and their premature senescence. Our in vivo data show that the pancreas is an early sensitive organ to oxidative stress accumulating with age and leading to overexpression of the procoagulant and senescence markers without appearance of apoptosis. In vitro, MPs of senescent endothelial cells have a pro-senescent effect on pancreatic islets isolated from young rats with characteristic SA-β-galactosidase activity, overexpression of p53, p21 and p16 markers and reducing the ability of insulin secretion in response to glucose. Altogether, our in vivo and in vitro data indicate the contribution of endothelial senescence as a possible contributor to graft dysfunction.

L'objectif de cette thèse est de préparer et de caractériser les particules cationiquesbiodégradables à base de cyclodextrine. Ces microparticules doivent également presenter unestructure et morphologie éponge et multifonctionnelle. L'encapsulation simultanée deplusieurs actifs cosmétiques (bien etre) et thérapeutiques (anti-inflammatoire) dans cesmicroparticules peut ouvrir de nouvelles applications une fois appliquées sur un textilespécifiquement sélectionnés pour leur fonctionnalisation. Pour atteindre cet objectif, desmicroparticules à base de polyméthacrylate cationique ont été préparés en utilisant le procédéémulsion double et diffusion de solvant suivi de l'évaporation du solvant. Des étudessystématiques ont été effectuées pour l'optimisation des paramètres du procédé ce qui apermis de contrôler la taille des particules et leur stabilité colloïdale. Une fois la taille désiréedes particules de type éponge a été obtenu, la surface d'un textile model de polyamide a étéfonctionnalisé via l'adsorption de ces particules tout en consacrent une attention particulièreaux propriétés électrocinétiques du textile avant et après fonctionnalisation en fonction d'ungrand nombre de paramètres physico-chimiques tels que; le pH, la salinité, la quantité initialede particules présente lors de l'adsorption. Enfin, l'encapsulation de la vitamine E, LIB(Lauryl Isoquinolinium Bromide), IMC (indomethacin) ainsi que l'encapsulation decomplexes de vitamine E-HPBCD, LIB-HPBCD et IMC-HPBCD dans microparticules a étéeffectuée séparément. Ces particules ont été caractérisées et la pénétration cutanée en utilisantune peau humaine a été examinée / The objective of this thesis was to prepare and characterize cyclodextrin based biodegradable,cationic and sponge like multifunctional microparticles that not only can be used for thedelivery of cosmetic and therapeutic agent (anti-inflammatory) but also can be potentiallyapplied onto specifically selected textile for their functionalization. For achieving these goals,polymethylmethacrylate based microparticles were prepared using double emulsion-diffusionsolvent evaporation technique. Systematic studies were performed for optimization of processcontrol parameters. Once desired size of sponge like particles was obtained from thesystematic study, the polyamide textile surface was functionalized with these particles whilededicating special attention to electrokinetic properties of textile as a function of variousparameters such as; particles amount present during adsorption process. Finally, encapsulationof pure vitamin E, Lauryl Isoquinolinium Bromide (LIB), Indomethacin (IMC) as well asencapsulation of complexes of vit E-Hydroxypropyl-beta-cyclodextrin (HPBCD), LIBHPBCDand IMC-HPBCD into microparticles was done separately. These particles werecharacterized for skin penetration and encapsulation efficiency

<p>Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of <i>Salmonella enterica</i> serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic <i>Escherichia coli</i>.</p><p>The immune response elicited using secreted proteins from <i>S. enterica</i> serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant <i>Salmonella</i> atypical fimbriae (SafB/D) complexes, a conserved structure within <i>Salmonella</i> species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. </p><p>Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. </p><p>Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.</p>

<p>Most pathogens enter the body via mucosal surfaces. In contrast to parenterally administered vaccination, mucosal vaccination has the advantage of eliciting both a systemic and a local mucosal immune response. An oral biodegradable adjuvant with these features would have great potential. </p><p>This thesis has focused on the development of a new oral vaccine against diphtheria. Biodegradable polyacryl starch microparticles were used as a mucosal adjuvant. Diphtheria toxin or cross-reacting material of diphtheria toxin (CRM197) was covalently conjugated to the microparticles and fed to mice by oral gavage. Formaldehyde treatment was also studied as a means of either detoxifying (diphtheria toxin) or stabilising (CRM197) these formulations. All formulations given to mice orally or parenterally, but not intranasally, induced a strong systemic immune response and diphtheria toxin neutralising antibodies. Only formulations administered orally induced a mucosal IgA response as well. </p><p>The non-toxic recombinant protein CRM197 proved to be a promising antigen candidate in an oral diphtheria vaccine when conjugated to the microparticles. Mild treatment of CRM197 with formaldehyde before conjugation to the starch microparticles potentiated the immunogenicity of the formulation. However, no immune response was detected in healthy volunteers after administration of this vaccine in a phase I trial. The possible reasons for the difference in response between mouse and man are discussed.</p><p>The use of cDNA expression macro array technology was also evaluated as a tool in vaccine-related research. Tetanus toxoid and aluminium phosphate were used as model parenteral antigen and adjuvant. It was concluded that the antigen modulates the molecular mechanisms of the aluminium phosphate adjuvant to a greater extent than previously recognised.</p>

<p>Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response.</p><p>When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. </p><p>Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. </p><p>Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.</p>

Polyacryl starch microparticles have been developed as a new mucosal vaccine adjuvant intended for use in oral vaccination. The main objectives of this thesis were to evaluate the efficacy of these polyacryl starch microparticles and to study their uptake through mucosal tissues. Secreted or surface components of Salmonella enterica serovar Enteritidis were used in free form or were conjugated to or mixed with the microparticles in vaccination studies in mice in order to find components suitable for use in a future combination vaccine against enteric bacteria such as enterotoxigenic Escherichia coli. The immune response elicited using secreted proteins from S. enterica serovar Enteritidis was shown to be mainly directed against flagella-related antigens and partly by LPS. Flagellin was purified and used in C3H/HeJ mice that do not respond to LPS. Strong immune responses were observed even when the flagellin was given orally alone. Recombinant Salmonella atypical fimbriae (SafB/D) complexes, a conserved structure within Salmonella species, were also studied and shown to be immunogenic after administration both subcutaneously and nasally, but not orally. Oral challenge using live bacteria, showed that mice orally immunised with the secreted antigens, resulted in a lower degree of infection than that seen in non-vaccinated mice. Similarly, mice that had been immunised with purified free flagellin had a lower degree of infection than untreated mice. However, with mice, immunised with SafB/D complexes plus rCTB, only the subcutaneous route resulted in a lower degree of infection than seen in untreated mice. The polyacryl starch microparticles were effective as an adjuvant with secreted proteins, but did not potentiate the immune response in the study using flagellin. Confocal laser-scanning and transmission electron microscopy demonstrated that the microparticles were taken up by pig respiratory nasal mucosa mounted in horizontal Ussing chambers. Although anticytokeratin 18 stained mucus-producing cells, M cells were not seen in the studied area. Changing the route of administration of the microparticles conjugated with serum albumin can cause differences in the IgG-subclass ratios. The mucosal immune response measured as specific s-IgA levels, was induced by oral but not parenteral immunisation.

Most pathogens enter the body via mucosal surfaces. In contrast to parenterally administered vaccination, mucosal vaccination has the advantage of eliciting both a systemic and a local mucosal immune response. An oral biodegradable adjuvant with these features would have great potential. This thesis has focused on the development of a new oral vaccine against diphtheria. Biodegradable polyacryl starch microparticles were used as a mucosal adjuvant. Diphtheria toxin or cross-reacting material of diphtheria toxin (CRM197) was covalently conjugated to the microparticles and fed to mice by oral gavage. Formaldehyde treatment was also studied as a means of either detoxifying (diphtheria toxin) or stabilising (CRM197) these formulations. All formulations given to mice orally or parenterally, but not intranasally, induced a strong systemic immune response and diphtheria toxin neutralising antibodies. Only formulations administered orally induced a mucosal IgA response as well. The non-toxic recombinant protein CRM197 proved to be a promising antigen candidate in an oral diphtheria vaccine when conjugated to the microparticles. Mild treatment of CRM197 with formaldehyde before conjugation to the starch microparticles potentiated the immunogenicity of the formulation. However, no immune response was detected in healthy volunteers after administration of this vaccine in a phase I trial. The possible reasons for the difference in response between mouse and man are discussed. The use of cDNA expression macro array technology was also evaluated as a tool in vaccine-related research. Tetanus toxoid and aluminium phosphate were used as model parenteral antigen and adjuvant. It was concluded that the antigen modulates the molecular mechanisms of the aluminium phosphate adjuvant to a greater extent than previously recognised.

Polyacryl starch microparticles have been developed as an oral vaccine adjuvant capable of inducing strong local and systemic immune responses in mice. In this thesis, the starch microparticles were studied in order to increase basic understanding of their function. In particular, the thesis addressed aspects of the uptake of the particles and their presentation to the immune system after different routes of administration, in correlation with the differentiation of the induced immune response. When using human serum albumin as a model antigen conjugated to the microparticles, it was found that the route of administration and the use of different combinations of routes, parenteral or oral, affect the profile (Th1/Th2 balance) of the induced immune response. It was also found that oral boosters are needed for the development of a local s-IgA response. Ligated mouse intestinal loops in combination with confocal laser-scanning microscopy demonstrated that the uptake of the particles by the intestinal mucosa takes place over the follicle-associated epithelium (FAE) that covers the Peyer’s patches. The particles are also taken up in the villus epithelium when conjugated with rCTB, a ligand to the GM1 receptor. This qualitative difference in uptake did not affect the induced immune response. Thus, the addition of rCTB to the microparticles did not improve or influence the profile of the immune response. Chronic stress, known to alter the barrier function of the FAE, increased the cellular response but did not affect the humoral immune response. Despite positive results in rodents, the particles were not able to boost a humoral immune response in man when tested with diphtheria toxin-cross reacting material (CRM197). Possible methods of improving the adjuvant effect in man are discussed.

The derivation of embryonic stem cells (ESCs) has created an invaluable resource for scientific study and discovery. Further improvement in differentiation protocols is necessary to generate the large number of cells needed for clinical relevance. The goal of this work was to develop a method to incorporate biomaterial microparticles (MPs) within stem cell aggregates and to evaluate their use for local control of the cellular microenvironment for directed differentiation. The effects of unloaded MPs on ESC differentiation were first determined by controlled incorporation of poly(lactic-co-glycolic acid) (PLGA), agarose and gelatin MPs. Embryoid body (EB) formation, cell viability, and gross morphology were not affected by the presence of the MPs. Further analysis of gene expression and patterns of phenotypic marker expression revealed alterations in the differentiation profile in response to material incorporation. The ability of MPs to direct ESC differentiation was investigated by incorporation of growth factor loaded MPs within EBs. MPs were loaded with bone morphogenetic protein-4 (BMP-4). BMP-4 loaded MPs incorporated within EBs induced mesoderm gene expression while inhibiting expression of an ectoderm marker compared to untreated EBs. Finally, magnetic MPs (magMPs) were incorporated within EBs to induce magnetic sensitivity. The responsiveness of EBs to applied magnetic fields was controlled by the number of magMPs incorporated within the aggregates. Magnetic guidance was then used to control the precise location of single EBs or populations of EBs for bioreactor culture and for construction of heterogeneous cell constructs. Overall, the results indicated that PSC differentiation within spheroids is sensitive to various types of biomaterials. Incorporation of MPs within EBs can be used to direct ESC differentiation by control of the cellular environment from microscale interactions, by delivery of soluble factors, to macroscale interactions, by control of EB position in static and suspension cultures.

Poly(3-hydroxybuttersäure-co-3-hydroxyvaleriansäure), PHBV, ist ein Copolyester, der auf biologischem Weg durch Bakterien herstellbar und ein steifes sowie relativ festes Polymer ist. Seine Biokompatibilität und biologische Abbaubarkeit weckt das Interesse für diverse Anwendungen in Pharmazie und Medizin. PHBV reagiert mit Abbau empfindlich auf zugleich thermische und mechanische Belastungen, was ein Problem für die Verarbeitung darstellt. Produkte aus PHBV aus einmal geschmolzenem und verarbeitetem Pulver sind hochkristallin. Daher ist das Material spröde. Ferner wirkt sich die hohe Kristallinität sowie eine große Änderung der Dichte beim Abkühlen der Schmelze nachteilig auf die Spinnbarkeit des Materials aus. Nach dem Passieren der Spinndüse ziehen sich die Spinnfäden zusammen, was die Gefahr eines Fadenrisses beim Spinnen erhöht. Aufgrund der relativ hohen Kristallinität des Materials und einer verzögerten Kristallisationskinetik bei gesponnenen Polymerfäden kommt es zur Nachkristallisation in einem erheblichen Ausmaß, die Fäden verkleben nach dem Aufwickeln auf den Galetten und reißen beim Abwickeln. Zur Behebung der Nachteile wurden Verarbeitungsbedingungen vor allem bei Schmelzspinnprozessen mit der Kolbenspinnanlage und bei Mischungsprozessen optimiert. Die Polymermischungen und ?verbundstoffe enthalten kugelförmige Mikropartikel verschiedener Morphologie, die zuvor synthetisiert und charakterisiert wurden. Vor allem mit Vinylgruppen modifizierte Silikat-Submikropartikel mindern die Sprödigkeit von PHBV.