In addition to classical adhesion structures like filopodia or focal adhesions, dendritic cells similar to macrophages and osteoclasts assemble highly dynamic F-actin structures called podosomes. They are involved in cellular processes such as extracellular matrix degradation, bone resorption by osteoclasts, and trans-cellular diapedesis of lymphocytes. Besides adhesion and migration, podosomes enable dendritic cells to degrade connective tissue by matrix metalloproteinases. SWAP-70 interacts with RhoGTPases and F-actin and regulates migration of dendritic cells. SWAP-70 deficient osteoclasts are impaired in F-actin-ring formation and bone resorption. In the present study, we demonstrate that SWAP-70 is not required for podosome formation and F-actin turnover in dendritic cells. Furthermore, we found that toll-like receptor 4 ligand induced podosome disassembly and podosome-mediated matrix degradation is not affected by SWAP-70 in dendritic cells. Thus, podosome formation and function in dendritic cells is independent of SWAP-70.

Le système ubiquitine-protéasome est le principal mécanisme par lequel les protéines intracellulaires sont dégradées. Le protéasome dit constitutif (PC) est donc essentiel à l’homéostasie mais aussi à la régulation de la majorité des processus cellulaires importants. La découverte d’un deuxième type de protéasome, appelé immunoprotéasome (IP), soulève toutefois de nouvelles questions. Pourquoi existe-t-il plus d’un type de protéasome ? L’IP a-t-il des rôles redondants ou complémentaires avec le PC ? L’IP étant présent principalement dans les cellules immunitaires ou stimulées par des cytokines, plusieurs groupes ont tenté de définir son rôle dans la réponse immunitaire. Or, l’implication de son homologue constitutif dans un éventail de processus non spécifiquement immunitaires nous laisse croire que l’IP pourrait lui aussi avoir un impact beaucoup plus large. L’objectif de cette thèse était donc de caractériser certains rôles cellulaires de l’IP dans les cellules dendritiques. Nous avons d’abord étudié l’impact global de l’IP sur la présentation antigénique de classe I. Ce faisant, nous avons pu déterminer ses deux contributions principales, soit l’augmentation drastique du nombre et de la diversité des peptides présentés sur les complexes majeurs d’histocompatibilité de classe I. Les différences de clivage entre le PC et l’IP pourraient expliquer en partie cette diversité du répertoire peptidique, notamment par l’affinité apparente de l’IP pour les régions protéiques non structurées. Dans un deuxième temps, nous avons dévoilé un nouveau rôle de l’IP sur un processus dépassant le cadre immunitaire : la transcription. Nous avons découvert que l’IP modifie l’abondance des ARNm en agissant principalement au niveau de leur synthèse. L’impact de l’IP sur le transcriptome est majeur et serait dû en partie à une dégradation différente de facteurs de transcription des familles IRF, STAT et NF-kB. Les cellules dendritiques IP-déficientes activent moins efficacement les lymphocytes T CD8+ et nous croyons que cette défaillance est causée (du moins en partie) par la perturbation transcriptomique provoquée par l’absence d’IP. Il importe donc de comprendre les différents rôles moléculaires de l’IP afin de mieux définir sa contribution globale au fonctionnement de la cellule et comprendre l’avantage évolutif, au niveau de l’organisme, procuré par une telle plasticité du système ubiquitine-protéasome. / The ubiquitin-proteasome system is the major mechanism by which intracellular proteins get degraded. Constitutive proteasomes (CPs) are thus essential for cellular homeostasis but also to regulate the majority of important cellular processes. However, the discovery of a second type of proteasome, named immunoproteasome (IP), raises new questions. Why are there more than one type of proteasome? Does the IP perform redundant or complementary roles with the CP? The IP is predominantly expressed in immune or cytokine-stimulated cells and several groups worked at defining its role during the immune response. Yet, the implication of its constitutive homolog in a variety of processes suggests that the IP may also have a much broader impact. The objective was to characterize cellular roles of the IP in dendritic cells. We first studied the global impact of the IP on class I antigen presentation. We discovered that the IP drastically increases the number and the diversity of peptide presented by class I major histocompatibility complexes. Cleavage differences between the CP and the IP are likely part of the explanation for this peptide repertoire diversity, notably due to IP’s apparent affinity for unstructured protein regions. Second, we discovered a new role for the IP in a process unrestricted to the immune system: transcription. We found that the IP affects transcript abundance mostly at the level of mRNA synthesis. The impact of IPs on the transcriptome is major and would be partly based on a different degradation of IRF, STAT and NF-kB transcription factor family members by the two types of proteasomes. IP-deficient dendritic cells are less potent activators of CD8+ T cells and we believe that this defect is at least partly caused by the transcriptome alterations induced by the absence of IPs. It is therefore important to understand the different molecular roles of the IP in order to better define its global contribution to cellular functions and to understand the evolutionary advantage, at the level of the organism, brought by such plasticity of the ubiquitin- proteasome system.

This thesis describes the synthesis and characterization ofbranched semi-crystalline polymers. Included in this work isthe SEC characterization of a series of dendrimers. Thebranched semi-crystalline polymers were synthesized in order toinvestigate the concept of their use as powder coatings resins.This concept being that the use of branched semi-crystallinepolymers in a UV-cured powder coating system may offer a lowertemperature alternative thus allowing the use of heat sensitivesubstrates and the added benefit of a reduced viscositycompared to linear polymers. A series of branched poly(ε-caprolactone)’s (PCL)(degree of polymerization: 5-200) initiated from hydroxylfunctional initiators were synthesized. The final architectureswere controlled by the choice of initiator structure;specifically the dendritic initiators yielded starbranchedPCL’s while the linear initiator yielded comb-branchedPCL’s. The dendritic initiators utilized were: (1) a3rd-generation Boltorn H-30, commercially availablehyperbranched polyester with approximately 32 hydroxyl groups,(2) a 3rd-generation dendrimer with 24 hydroxyl groups, and (3)a 3rd-generation dendron with 8 hydroxyl groups. Linear PCL wassynthesized for comparison. All dendritic initiators are basedon 2,2- bis(methylol) propionic acid. The comb-branchedpolymers were initiated from a modified peroxide functionalpolyacrylate. The resins were end-capped withmethylmethacrylate in order to produce a cross-linkable system.The polymers and films were characterized using 1H NMR, 13CNMR, SEC, DMTA, DSC, FT-IR, FT-Raman, rheometry and a rheometercoupled to a UV-lamp to measure cure behavior. The star-branched PCL’s exhibited considerably lowerviscosities than their linear counterparts with the samemolecular weight for the molecular region investigated (2-550kg mol-1). It was also found that the zero shear viscosityincreased roughly exponentially with M. The PCL star-branched resins are semi-crystalline and theirmelting points (Tm) range from 34-50°C; films can beformed and cured below 80°C. The viscoelastic behaviourduring the cure showed that the time to reach the gel point, afew seconds, increased linearly with molecular weight. Thecrossover of G’and G’’was used as the gelpoint. Measurement of mechanical properties of films showedthat the low molecular weight polymers were amorphous whilethose with high molecular weight were crystalline after cure.The polymerization of 5,5-dimethyl-1,3-dioxane-2-one (NPC) fromoligo- and multifunctional initiators was evaluated utilizingcoordination and cationic polymerization. Two tin basedcatalysts, stannous(II) 2-ethylhexanoate and stannous(II)trifluoromethane sulfonate, were compared with fumaric acid.Fumaric acid under bulk conditions resulted in lowerpolydispersity and less chance of gelling. The synthesis ofstar-branched polymers was confirmed by SEC data. The starpolymers exhibited a Tg at 20-30°C and a Tm at about100°C. All semi-crystalline resins exhibited a fast decrease inviscosity at Tm. Blends of combbranched semi-crystalline resinsand amorphous resins exhibited a transition behavior inbetweenthat of pure semi-crystalline resins and that of amorphousresins. The SEC characterization of a series of dendrimers withdifferent cores and terminal groups showed that the core had animpact on the viscosimetric radius of the core while theterminal groups appeared to have no effect. Keywords:star-branched, semi-crystalline,comb-branched, ring-opening polymerization,poly(ε-caprolactone), dendritic, thermoset, lowtemperature curing, powder coating, UVcuring,poly(5,5-dimethyl-1,3-dioxane-2-one), size exclusionchromatography, rheology, dendritic aliphatic polyester / <p>NR 20140805</p>

Les vaccins à base de cellules dendritiques (DCs) constituent une avenue très populaire en immunothérapie du cancer. Alors que ces cellules peuvent présenter des peptides exogènes ajoutés au milieu, l’efficacité de chargement de ces peptides au le complexe majeur d'histocompatibilité (CMH) de classe II est limitée. En effet, la majorité des molécules du CMH II à la surface des DCs sont très stable et l’échange de peptide spontané est minime. Confinée aux vésicules endosomales, HLA-DM (DM) retire les peptides des molécules du CMH II en plus de leur accorder une conformation réceptive au chargement de peptides. Il est possible, cependant, de muter le signal de rétention de DM de façon à ce que la protéine s’accumule en surface. Nous avons émis l’hypothèse que ce mutant de DM (DMY) sera aussi fonctionnel à la surface que dans la voie endosomale et qu’il favorisera le chargement de peptides exogènes aux DCs. Nous avons utilisé un vecteur adénoviral pour exprimer DMY dans des DCs et avons montrer que la molécule augmente le chargement de peptides. L’augmentation du chargement peptidique par DMY est autant qualitatif que quantitatif. DMY améliore la réponse T auxiliaire (Th) du coté Th1, ce qui favorise l’immunité anti-cancer. Du côté qualitatif, le chargement de peptides résulte en des complexes peptide-CMHII (pCMH) d’une conformation supérieure (conformère). Ce conformère (Type A) est le préféré pour la vaccination et DMY édite avec succès les complexes pCMH à la surface en éliminant ceux de type B, lesquels sont indésirables. La fonction de DM est régulée par HLA-DO (DO). Ce dernier inhibe l’habilité de DM à échanger le peptide CLIP (peptide dérivée de la chaîne invariante) en fonction du pH, donc dans les endosomes tardifs. Mes résultats indiquent que la surexpression de DO influence la présentation des superantigènes (SAgs) dépendants de la nature du peptide. Il est probable que DO améliore indirectement la liaison de ces SAgs au pCMH dû à l’accumulation de complexe CLIP-CMH, d’autant plus qu’il neutralise la polarisation Th2 normalement observée par CLIP. Ensemble, ces résultats indiquent que DMY est un outil intéressant pour renforcer le chargement de peptides exogènes sur les DCs et ainsi générer des vaccins efficaces. Un effet inattendu de DO sur la présentation de certains SAgs a aussi été observé. Davantage de recherche est nécessaire afin de résoudre comment DMY et DO influence la polarisation des lymphocytes T auxiliaires. Cela conduira à une meilleure compréhension de la présentation antigénique et de son étroite collaboration avec le système immunitaire. / Dendritic cell peptide-based vaccines are the most common immunotherapy approach in cancer therapy. While, in principle, dendritic cells (DCs) could be loaded efficiently by exogenously added tumor peptides, their loading efficacy is severely reduced due to low number of peptide-receptive MHC II on cell surface. Most surface MHC II molecules are either occupied by endogenous peptides or are inactive due to a conformation that is not receptive for free peptides. In MHC II antigen presentation pathway, HLA-DM (DM) in acidic endosomal vesicles removes the self-peptides and grants a peptide receptive conformation to MHC II. Mutating of an intracellular sorting motif in DM, renders its accumulation on cell surface. We hypothesized that the mutant DM (DMY) is functional on cell surface and can generate peptide receptive MHC II on surface of DCs for exogenous peptide loading. By using an adenoviral vector that expresses DMY, we found that DMY is functional on surface of DCs. DMY supplied peptide receptive MHC II on surface of DCs and improved exogenous peptide loading. The improvement of peptide loading by DMY is both quantitative and qualitative. DMY improves helper T cell (Th) response in Th1 direction that favors anti-cancer immunity. The qualitative improvement of peptide loading extends to loading of superior conformational isomer (conformer) of peptide-MHC complexes. This superior conformer (type A) is the favourite type for vaccination approaches and DMY successfully edits peptide-MHC conformers on cell surface level by eliminating undesirable one (type B). Function of DM is regulated by HLA-DO (DO) and it is well accepted that in acidic pH of late endosomes, DO inhibits function of DM by preventing removal of class II associated invariant chain peptide (CLIP) from peptide binding groove of MHC II. My results indicate that DO overexpression, changes binding of peptide-dependent superantigens to MHC II molecules. Superantigens (SAgs) are small microbial proteins that bind out side peptide binding groove of MHC II. DO probably enhances binding of peptide-dependent SAgs by forcing the accumulation of CLIP on the cell surface of antigen presenting cells. DO also neutralizes Th2 polarization by CLIP. Collectively, these results indicate that DMY is a valuable tool for improvement of exogenous peptide loading in DCs vaccines. An unnoticed effect of DO on SAgs binding was also recognized. Further investigations are needed to clarify the mechanisms by which, DMY and DO influence Th polarization. This would provide a better understanding of antigen presentation pathway and its interaction with immune system.

Seit 20 Jahren unterteilt man Monozyten in eine klassische CD14++/CD16-Population und eine proinflammatorische CD16+ Population. Letztere macht 10-20 % der peripheren Blutmonozyten aus und ist unter verschiedenen pathologischen Zuständen drastisch erhöht. Seit Kurzem wird die CD16+ Fraktion in zwei weitere Untergruppen aufgeteilt, die CD14++/CD16+ und die CD14+/CD16+ Monozyten. In der hier vorliegenden Arbeit wurde versucht, die relativ neue und wenig charakterisierte Subpopulation der CD14++/CD16+ Monozyten näher zu beschreiben. Es sollte die Frage beantwortet werden, ob diese sich von den übrigen Subpopulationen abzugrenzen lässt und damit als eigenständige Zellgruppe anzusehen ist. Die von gesunden Spendern gewonnen peripheren mononukleären Blutzellen (PBMCs) wurden mithilfe einer Magnetsäulenvorseparation von einem Großteil der T-, B- und NK-Zellen befreit. Die restlichen Zellen wurden mit Anti-CD14-FITC und Anti-CD16-PE markiert. In einem Hochgeschwindigkeitssortierer wurden diese, anhand ihres Fluoreszenzmusters, in die drei Subpopulationen CD14++/CD16- (Sub1), CD14++/CD16+ (Sub2) und CD14+/CD16+ (Sub3) aufgeteilt. Durch dieses Verfahren konnte ein hoher Reinheitsgrad erreicht werden. Die erhaltenen Subpopulationen wurden mit heterologen CD4+ T-Lymphozyten zusammen gebracht. Die Reaktion dieser T-Zellen auf die verschiedenen Subpopulationen wurde im Proliferations- und Sekretionsassay (IFN-γ, IL-4) studiert. Des Weiteren wurde die Zytokinsekretion der Monozytenarten nach definierter Stimulierung (LPS, Zymosan, aktivierte T-Zellen) analysiert. In einem zweiten Versuchskomplex wurden aus den jeweiligen Subpopulationen unreife dendritische Zellen (Sub-DCs) differenziert und diese auf bestimmte Oberflächenmarker bzw. deren Verhalten mit heterologen CD4+ T-Zellen im Proliferations-/Sekretionsassay (IFN-γ, IL-4) untersucht. Nach 7 Tagen Inkubation fanden sich im Sub2- und Sub3-Ansatz ca. 10 % mehr proliferierende T-Zellen als im Sub1-Ansatz. Dabei lag der Anteil der CD25+ T-Zellen in diesen beiden Versuchsansätzen ebenfalls um 10 % höher. Der Proliferationsassay über 14 Tage zeigte für die Sub3 ca. 10-15 % mehr proliferierende T-Zellen als für die anderen Populationen. Das Niveau der CD25-Expression der T-Zellen war hierbei in allen drei Ansätzen gleich. Im Sekretionsassay induzierten alle drei Subpopulationen eine Th1-Antwort, jedoch mit einem leicht höheren Anteil IFN-γ-positiver T-Zellen für die CD16+ Monozyten. Durch LPS-Gabe produzierten die CD14+/CD16+ Monozyten am meisten TNF-α, gefolgt von den CD14++/CD16+. Bei den CD14++/CD16- fanden sich die geringsten Mengen an TNF-α. Zusammen mit aktivierten T-Zellen demonstrierten die CD14++/CD16+ Monozyten die stärkste TNF-α-Sekretion unter allen anderen Subpopulationen. Für die beiden CD16+ Populationen wurden nach LPS-Stimulation höhere Werte für IL-1β bestimmt als für die klassischen Monozyten. Sowohl im LPS- als auch im Zymosanansatz lag die IL-6-Sekretion der CD14++/CD16- Subpopulation über der der anderen zwei Fraktionen. Unter allen drei Stimuli wurden die höchsten Werte für IL-8 bei den CD14++/CD16- Monozyten detektiert, gefolgt von den CD14++/CD16+. Am niedrigsten lag die IL-8-Produktion bei den CD14+/CD16+. Die IL-10-Sekretion im LPS- und im Zymosanansatz der CD14++/CD16- und CD14++/CD16+ Monozyten war gegenüber der CD14+/CD16+ Subpopulation erhöht. Nach Entwicklung der unreifen dendritischen Zellen aus den entsprechenden Monozytenarten weisen diese eine differenzierte Morphologie auf. Die DCs der CD14+/CD16+ Monozyten hatten eine stärkere HLA-DR-Expression in der mittleren Fluoreszenzintensität als die anderen beiden Sub-DC-Populationen, wobei sich keine Unterschiede in der HLA-DRExpressionsintensität zwischen Sub1- und Sub2-DCs feststellen ließen. Der Anteil der CD11c positiven Zellen war bei den Sub1-DCs deutlich größer als bei den Sub2-DCs. Dagegen exprimierten die Sub3-DCs praktisch kein CD11c. Im Proliferationsassay über 7 Tage ergaben sich keine signifikanten Differenzen zwischen den Subpopulationen. Allerdings zeigte sich eine Tendenz zu geringeren Werten im Anteil proliferierender bzw. CD25-positiver T-Lymphozyten für den Sub2-DC-Ansatz. Nach 14 Tagen im Assay war der Anteil der proliferierenden T-Zellen bei den Sub1-DCs um 10 % höher als bei den Sub2-DCs. Es fanden sich ca. 10 % mehr CD25+ T-Zellen im Sub1-DC-Ansatz als in den anderen beiden Ansätzen. Der Sekretionsassay erbrachte für alle Sub-DCs eine Th1-Induktion der T-Zellen. Dabei ließen sich keine Abweichungen im Anteil IFN-γ-positiver T-Zellen zwischen den einzelnen Sub-DC-Kulturen nachweisen. Die gewonnen Ergebnisse dieser Arbeit verdeutlichen auf der einen Seite das proinflammatorische Potential (z.B. TNF-α-Sekretion, erhöhte Proliferation), auf der anderen Seite die antiinflammatorische Komponente (IL-10-Sekretion) der CD14++/CD16+ Monozyten. Eine Rolle in der Regulation von entzündlichen und infektiologischen Erkrankungen erscheint für diese Subpopulation denkbar. Die Subpopulation 2 kann dabei als eigenständige Monozytenfraktion betrachtet werden. Die funktionellen Unterschiede zwischen den analysierten Monozytensubpopulationen zeigten sich auch nach deren Differenzierung zu unreifen dendritischen Zellen. In Anbetracht des erhöhten Anteils der CD16+ Monozyten im Rahmen diverser autoimmuner Krankheiten und der immer klarer werdenden Unterteilung in eine CD14++/CD16+ und eine CD14+/CD16+ Subpopulation, sollten weitere Untersuchungen zur klinischen Relevanz dieser Monozytengruppen durchgeführt werden. Die in der vorliegenden Arbeit erzielten Ergebnisse könnten bei der Zuordnung und Interpretation in diese zukünftigen klinischen Befunde behilflich sein. / For more than 20 years monocytes were subdivided in the classical CD14++/CD16- population and the proinflammatory CD16+ populations. The latter one includes about 10-20 % of the peripheral blood monocytes and is dramatically expanded under different pathological circumstances. Recently, the CD16+ fraction was further subdivided into two subpopulations: The CD14++/CD16+ and CD14+/CD16+ monocytes. In the present paper, the subpopulation of CD14++/CD16+ monocytes was characterized in order to answer the question if this subset can be distinguished as an independent cell population. T cells, B cells and NK cells were depleted from peripheral mononuclear blood cells (PBMCs) from healthy donors using immunomagnetic cell separation. Subsequently, the pre-separated cells were stained with anti-CD14-FITC and anti-CD16-PE and separated by fluorescence activated cell sorting (FACS) in three subpopulations: The CD14++/CD16- (Sub1), the CD14++/CD16+ (Sub2) and the CD14+/CD16+ (Sub3). The achieved purity was sufficient for the subsequent experiments. The obtained subpopulations were co-cultured together with heterologous CD4+ T lymphocytes. The reaction of these T cells to the different subpopulations was studied in the proliferation and secretion assay (IFN-γ, IL-4). Furthermore, the cytokine secretion of the monocyte subsets after defined stimulation (LPS, Zymosan, activated T cells) was analysed. In a second set of experiments, immature dendritic cells were differentiated from the monocyte subpopulations (Sub-DCs) and phenotypically and functionally characterized according to the expression of cell surface markers and the response to heterologous CD4+ T cells in the proliferation/secretion assay (IFN-γ, IL-4). After 7 days of incubation, the Sub2 and Sub3 of the monocytes induced approximately 10 % higher proliferation rates of T cells than the Sub1. The resulting frequency of CD25+ T cells in these two co-culture settings was also 10 % higher. The proliferation analysis after 14 days again showed for the Sub3 ca. 10-15 % more proliferating T cells than for the other populations. At this, the frequency of CD25 expression was equal in all co-cultures. In the secretion assay all three subpopulations induced a Th1 response, but the range of IFN-γ-positive T cells was somewhat higher for the CD16+ monocytes. Under LPS stimulation the CD14+/CD16+ monocytes produced the highest amounts of TNF-α followed by the CD14++/CD16+. The CD14++/CD16- showed the lowest amounts of TNF-α. In co-culture with activated T cells the CD14++/CD16+ monocytes demonstrated the strongest TNF-α secretion from all subpopulations. After LPS stimulation, a higher level of IL-1β was measured for both CD16+ populations than for the classical monocytes. In the LPS and the Zymosan stimulated cultures, the IL-6 secretion of the CD14++/CD16- subpopulation was higher than in the two other fractions. Under all three stimuli the highest levels of IL-8 were detected for the CD14++/CD16- monocytes followed by the CD14++/CD16+. The lowest IL-8 production was found by the CD14+/CD16+. The IL-10 secretion of the CD14++/CD16- and CD14++/CD16+ monocytes was increased compared to the CD14+/CD16+ subpopulation after LPS and Zymosan stimulation. The in vitro generated immature dendritic cells from the different monocyte subsets showed a differentiated morphology. The DCs of the CD14+/CD16+ monocytes had the strongest HLA-DR expression compared to the other two Sub-DC populations. No differences in the HLA-DR intensity were found between the Sub1- and Sub2-DCs. The rate of CD11c-positive cells was significantly higher in the Sub1-DCs than in the Sub2-DCs. However, the Sub3-DCs expressed no CD11c altogether. The proliferation assay over 7 days showed no significant differences between the subpopulations. Nevertheless, a tendency for lower levels of proliferating or CD25-positive T lymphocytes was seen in T cells co-cultured with the Sub2-DC. After 14 days, the ratio of proliferating T cells was 10 % higher with the Sub1-DCs than with the Sub2-DCs. The Sub1-DC co-culture yielded ca. 10 % more CD25+ T cells than the other two. The secretion assay revealed for all Sub-DCs a Th1 response of the T cells, with no differences in the amount of IFN-γ-positive T cells between the Sub-DC cultures. The results illustrate on one hand the proinflammatory potential (e.g. TNF-α secretion, higher proliferation), on the other hand the antiinflammatory effect (IL-10 secretion) of CD14++/CD16+ monocytes. A role in the regulation of inflammatory and infectious diseases seems to be possible for this subpopulation. The subpopulation 2 can be regarded as an independent fraction of monocytes. The functional differences between the analyzed monocyte subpopulations are further underscored following differentiation into immature dendritic cells. Considering the increased proportion of CD16+ monocytes in various autoimmune diseases and their clear subdivision in a CD14++/CD16+ and a CD14+/CD16+ subpopulation, new investigations about the clinical relevance are warranted. The findings obtained in the work presented could be in the basis for these future clinical studies.

Dendritische Zellen (DCs) sind befähigt, als potenteste Antigen-präsentierende Zelle anti¬genspezifische Immunantworten zu initiieren und zu regulieren. Mittels in vivo Antigen¬beladung von spezifischen DC-Subpopulationen mit rekombinanten Antigen-gekoppelten Antikörpern gegen C-Typ-Lektinrezeptoren konnte im Mausmodell erfolgreich adaptive zelluläre und humorale Immunantworten hervorgerufen werden. Im Menschen kann dieser Ansatz therapeutisch noch nicht zum Einsatz kommen, da DC-Subpopulationen im humanen Gewebe momentan nicht ausreichend charakterisiert sind. Im Rahmen dieser Arbeit wurden humane Gewebe auf das Vorhandensein der im peripheren Blut beschriebenen DC-Subpopulationen (mDC-1 DCs: CD11c+BDCA1+, mDC-2 DCs: CD11clowBDCA3+ und pDCs: CD11c-CD123+BDCA2+) und die Expression von C-Typ-Lektinrezeptoren (DC-SIGN, MMR, Langerin, DCIR und DEC205) mittels Immun¬fluoreszenz untersucht. Anhand der vorge¬gebenen Marker im peripheren Blut konnten alle drei DC-Subpopulationen in der Milz, Thymus und Tonsillen detektiert werden. Zusätzlich konnte hier erstmalig eine vierte CD11c-CD123-BDCA2+ pDC-2 DC-Subpopulation in der Milz beschrieben werden, deren Funktion derzeit noch näher untersucht wird. Im Thymus konnte CD26 nach FACS-Analysen von Gordon Heidkamp als spezifischer Marker für mDC-2 DCs identifiziert und dies in der vorliegenden Arbeit auch durch Immun¬fluores¬zenzaufnahmen von Gewebeschnitten verifi¬ziert werden. CD26 stellt damit erstmalig einen Marker dar, der erfolgreich als alternativer Marker für BDCA3, welcher unspezifisch an Thrombomodulin bindet, zur Identifikation von mDC-2 DCs in der Immunfluoreszenz von Thymusproben eingesetzt werden könnte. Die getesteten Anti¬körper XCR-1 (monoklonal) und Clec9a (polyklonal) hingegen erschienen in der Immun¬fluoreszenz sowie in FACS-Analysen (Gordon Heidkamp) nicht geeignet. Weiterhin wurde die Expression ausgewählter C-Typ-Lektinrezeptoren (MMR, Langerin, DCIR, DC-SIGN und DEC205) im vorhandenen Gewebe näher betrachtet. Nach Auswertung der Immun¬fluoreszenzen konnte eine weit verbreitete Expression der untersuchten C-Typ-Lektin¬rezeptoren in humaner Milz und Thymus gefunden werden. Einzig hDCIR war auf vereinzelten Zellen exprimiert, und Langerin im Thymus nicht detektierbar. Um nicht verfügbare monoklonale Antikörper gegen C-Typ-Lektinrezeptoren zu produzieren und später Antigene an diese koppeln zu können, sollten lösliche Proteine einiger C-Typ-Lektinrezeptoren (humanes und murines Clec9a, Dectin-1 und -2, Langerin) produziert werden. Dabei gelang es bereits, lösliche Proteine der C-Typ-Lektinrezeptoren von humanem und murinem Dectin-1 zu generieren und diese zur weiteren Antikör¬per¬produktion einzusetzen.

La technologie Lithium-air développée par EDF utilise une électrode à air qui fonctionne avec un électrolyte aqueux ce qui empêche l’utilisation de lithium métal non protégé comme électrode négative. Une membrane céramique (LATP:Li1+xAlxTi2-x(PO4)3) conductrice d’ion Li+ est utilisée pour séparer le milieu aqueux de l’électrode négative. Cependant, cette céramique n'est pas stable au contact du lithium, il est donc nécessaire d'intercaler entre le lithium et la céramique un matériau conducteur des ions Li+. Celui-ci devant être stable au contact du lithium et empêcher ou fortement limiter la croissance dendritique. Ainsi, ce projet s'est intéressé à l'étude d'électrolytes copolymères à blocs (BCE).Tout d'abord, l'étude des propriétés physico-chimiques spécifiques de ces BCEs en cellule lithium-lithium symétrique a été réalisée notamment les propriétés de transport (conductivités, nombre de transport), et la résistance à la croissance dendritique du lithium. Puis dans un second temps, l'étude des composites BCE-céramique a été mise en place. Nous nous sommes en particulier focalisés sur l'analyse du transfert ionique polymère-céramique.Plusieurs techniques de caractérisation ont été utilisées telles que la spectroscopie d'impédance électrochimique (transport et interface), le SAXS (morphologies des BCEs), la micro-tomographie par rayons X (morphologies des interfaces et des dendrites).Pour des électrolytes possédant un nombre de transport unitaire (single-ion), nous avons obtenus des résultats remarquables concernant la limitation à la croissance dendritique. La micro-tomographie des rayons X a permis de montrer que le mécanisme de croissance hétérogène dans le cas des single-ion est très différent de celui des BCEs neutres (t+ < 0.2). / The lithium-air (Li-air) technology developed by EDF uses an air electrode which works with an aqueous electrolyte, which prevents the use of unprotected lithium metal electrode as a negative electrode. A Li+ ionic conductor glass ceramic (LATP:Li1+xAlxTi2-x(PO4)3) has been used to separate the aqueous electrolyte compartment from the negative electrode. However, this glass-ceramic is not stable in contact with lithium, it is thus necessary to add between the lithium and the ceramic a buffer layer. In another hand, this protection should ideally resist to lithium dendritic growth. Thus, this project has been focused on the study of block copolymer electrolytes (BCE).In a first part, the study of the physical and chemical properties of these BCEs in lithium symmetric cells has been realized especially transport properties (ionic conductivities, transference number), and resistance to dendritic growth. Then, in a second part, the composites BCE-ceramic have been studied.Several characterization techniques have been employed and especially the electrochemical impedance spectroscopy (for the transport and the interface properties), the small angle X-ray scattering (for the BCE morphologies) and the hard X-ray micro-tomography (for the interfaces and the dendrites morphologies). For single-ion BCE, we have obtained interesting results concerning the mitigation of the dendritic growth. The hard X-ray micro-tomography has permitted to show that the mechanism involved in the heterogeneous lithium growth in the case of the single-ion is very different from the one involved for the neutral BCEs (t+ < 0.2).

The extracellular matrix (ECM) is a complex network of molecules secreted by cells, which is essential for providing structural support and facilitating cell processes including adhesion, migration and survival. Tenascin-C is an immunomodulatory ECM protein that exhibits limited expression in healthy tissues, but is transiently elevated at sites of tissue injury, and is persistently expressed in chronic inflammatory diseases and tumours. Alternative splicing of 9 of tenascin-C's fibronectin type III-like domains (FnIII- A1, A2, A3, A4, B, AD2, AD1, C and D) generates enormous diversity in form; yielding 511 possible isoforms. Post-transcriptional modification of tenascin-C has been studied in cancer and during development where disease and tissue specific isoforms exhibit distinct adhesive, migratory and proliferative effects. However, little is known of how tenascin-C is expressed or alternatively spliced during inflammation. This study characterises inflammation and disease specific tenascin-C isoforms made by immune cells and fibroblasts, and investigates their functional relevance. Biosynthesis and alternative splicing of tenascin-C was examined using standard curve qPCR, ELISA, Western blot and confocal immunocytochemistry in resting and activated primary human immune cells, dermal fibroblasts, and in synovial fibroblasts isolated from healthy controls and from osteoarthritis (OA) and rheumatoid arthritis (RA) patients. Based on these data, three recombinant proteins comprising FnIII domains AD2-AD1, B-C-D and B-AD2-AD1-C-D were cloned, expressed and purified, and their impact on cell behaviour including adhesion, morphology and migration was assessed. Basal tenascin-C expression was lower in myeloid and lymphoid cells than fibroblasts, and was induced in all following inflammatory stimulation. Tenascin-C expression was elevated in disease with RA and OA synovial fibroblasts containing higher levels than healthy controls. Alternative splicing following cell activation was cell-type specific: all FnIII except AD2 and AD1 were upregulated in dendritic cells and macrophages, in T-cells all FnIII remained unchanged with FnIII A1 absent; and no change in splicing was observed in activated dermal fibroblasts. Normal and OA synovial fibroblasts exhibited similar tenascin-C splicing patterns, but FnIII B and D were specifically elevated in RA. Functional analysis revealed differences in the adhesion, morphology and migration of myeloid cells and dermal fibroblasts cultured on FnIII AD2-AD1, B-C-D, B-AD2-AD1-C-D and full length tenascin-C substrates; FnIII B-C-D promoted MDDC migration while B-AD2-AD1-C-D promoted fibroblast adhesion, compared to full length tenascin-C. For the first time, this study reveals differences in tenascin-C biosynthesis and alternative splicing by immune cells and fibroblasts following activation with inflammatory stimuli; and starts to reveal how alternative splicing of tenascin-C may influence the behaviours of both stromal and immune cells types during inflammation and in inflammatory diseases.

Heterozygous and recently identified homozygous mutations in the SPTBN2 gene, encoding b-III spectrin, are implicated in spinocerebellar ataxia type 5 (SCA5) and spectrin-associated autosomal recessive cerebellar ataxia type 1 (SPARCA1), respectively. Our mouse model, lacking b-III spectrin (KO), mimics the progressive human phenotype displaying motor deficiencies as well as reduced Purkinje cell firing frequency followed by dendritic tree degeneration and cell death. The aims of this study were to evaluate progression of Purkinje cell degeneration following loss of b-III spectrin function and determine whether the reintroduction of C-terminus (C-trm) of b-III spectrin to the cerebellum is enough to halt, alleviate or reverse the disease phenotype. Additionally, this study investigated whether the abnormal electrophysiological and morphological phenotypes of Purkinje cells from KO mice are re-capitulated in a primary cerebellar culture and if so, whether they could be rescued by modulating calcium signaling. Morphological and histological analyses revealed that Purkinje cell degeneration is not uniform throughout the cerebellum of KO mice with Purkinje cells from posterior cerebellar regions possessing significantly smaller dendritic trees when compared to anterior cerebellum (p=0.0003, N=4-6, n=11-29). Similarly, significant reduction in Purkinje cell density was observed in posterior, not anterior regions of KO mice when compared to WT animals (p=0.014, N=3) and reduced tonic firing is most significant in Purkinje cells from the posterior cerebellum compared to WT mice (p=0.0328, N=3-6, n=11-29), with posterior KO PCs appearing to have elevated input resistance. Two-week expression of C-trm b-III spectrin in 3-month old KO animals significantly reduced Purkinje cell input resistance when compared to non-transduced cells (p=0.0139, N=4-5, n=15), but no effect was seen 9 months after viral injection. In contrast, a difference in cell surface area was no longer detected between WT and KO animals at 12 months of age following 9-months of viral expression. Nevertheless, using the elevated beam test motor deterioration was still observed 5 months after surgery (p=0.0023, N=4). In contrast, earlier stereotaxic injections at 6-weeks of age had a positive effect on mice motor performance with no deterioration in performance detected 5 months after the surgery. Latency to stay on the rotarod at 3 rpm was also significantly extended 6 months after stereotaxic injections at 6-weeks of age with slower motor deterioration (p=0.0348, N=6). In primary cerebellar cultures, Purkinje cells from KO animals exhibit an abnormal morphology with significantly more dendritic branches (p < 0.0001, N=4-7, n=35-69) and a larger total dendritic length (p=0.0079). Chronic application of 2 μM mibefradil, a T-type calcium channel blocker, was observed to reduce total dendritic length and branching in KO animal cultures bringing these morphological measurements closer to WT Purkinje cell levels. Finally although after 14 days in vitro 40% of Purkinje cells were found to be spontaneously firing, no significant difference in firing frequency (p=0.9434) or input resistance (p=0.8434, N=4, n=6-10) was detectable between WT and KO cultures. In summary, Purkinje cells in posterior cerebellar regions of KO mice were found to be more susceptible to dendritic degeneration and cellular death than cells in the anterior cerebellum. Expression of C-trm b-III spectrin at 3 months of age had an immediate effect on cell input resistance and a modest effect on Purkinje cell morphology but no effect on motor decline. Viral injections at 6-weeks of age, however, significantly slowed motor decline. Although an abnormal KO cell morphology could be successfully recapitulated in primary cell culture, it was not possible to discern any differences in electrophysiological properties. Nevertheless, the abnormal cell morphology was successfully modified in vitro by manipulating calcium signaling via T-type calcium channels.

Ventricular dysfunction and dilated cardiomyopathy (DCM) develop among untreated HIV-infected people at much higher rates than among HIV-negative individuals, resulting in significant contributions to morbidity and mortality. Mechanisms underlying development of HIV-associated cardiomyopathy (HIVCM) are as yet poorly understood. The well-characterized simian immunodeficiency virus (SIV) model of HIV infection provides a unique context for HIVCM pathogenesis studies in that SIV-infected rhesus monkeys develop myocardial lesions and contractile dysfunction similar to those described in HIV-infected people, suggesting a shared disease mechanism. Lymphocytic myocarditis is a commonly reported finding in AIDS patients at autopsy and constitutes one of several conditions known to predispose to development of DCM, irrespective of HIV-infection status. As lymphocytic myocarditis also occurs with high frequency among SIV-infected rhesus monkeys, a retrospective analysis of rhesus monkey cardiac tissue collected at necropsy was performed to examine viral and cellular correlates of lymphocytic inflammation within myocardial tissue. One subpopulation of macrophages, which has been reported by other groups to be associated with an anti-inflammatory phenotype, was found to correlate inversely with lymphocytic infiltration and positively with numbers of virus infected cells, suggesting effects of an anti-inflammatory cytokine production profile. In contrast, the detrimental effects of inflammatory cytokines on myocardial structure and function are well-recognized and HIV infection in general is characterized by chronic immune activation and inflammatory cytokine dysregulation. To further investigate a role for myocardial cytokine production in development of HIVCM, a prospective study was conducted in which SIV-infected rhesus monkeys and uninfected controls were treated with recurrent administration of inactivated Mycobacterium aviumcomplex bacteria (MAC). SIV-infected, MAC-treated animals rapidly developed significant ventricular systolic dysfunction and chamber dilatation not seen in control groups, suggesting an exaggerated myocardial sensitivity to exogenous antigenic stimulation. Concurrent treatment with the TNFα antagonist etanercept completely abrogated development of these changes, strongly implicating a causative role for TNFα in evolution of the contractile dysfunction and chamber remodeling. Findings reported from the current studies suggest that characteristics of local myocardial macrophage populations and the myocardial tissue cytokine milieu may play more important roles than lymphocytic infiltration, cardiomyocyte damage, or viral proteins in the pathogenesis of HIVCM.