<p>Extracellular GRP94 can elicit both innate and adaptive immune responses by interacting with endocytic and signaling receptors on professional antigen presenting cells (pAPCs). CD91 was the first receptor proposed to facilitate GRP94-mediated immune responses. Using a GRP94 affinity matrix, a CD91 fragment was isolated from the detergent-solubilized membranes of a pAPC cell line. It was then demonstrated that CD91 ligands could inhibit GRP94-mediated peptide cross-presentation, suggesting that CD91 played a critical role in this process. While these studies implied that CD91 could function as a GRP94 endocytic receptor, later works suggested that CD91 may not recognize GRP94 at the cell surface. These opposing observations have lead to a significant controversy surrounding the identity of CD91 as an endocytic receptor for GRP94. Because the ability of CD91 to directly mediate GRP94 surface binding and uptake has not been established, the studies included in this dissertation have focused on evaluating the ability of CD91 to facilitate three processes that are necessary for GRP94-mediated peptide cross-presentation: surface binding, internalization, and processing.</p><p>These studies utilized a recombinantly-expressed N-terminal domain of GRP94 (GRP94.NTD), which was previously shown to have nearly identical biological activity to full length GRP94. The ability of CD91 to directly bind and internalize GRP94.NTD was examined using murine embryonic fibroblast (MEF) cell lines whose expression of CD91 was either reduced via siRNA, or eliminated by genetic disruption of the CD91 locus. Binding competition experiments were also conducted. Together, these studies reveal that CD91 does not directly interact with GRP94 at the cell surface. The ability of CD91 to directly facilitate GRP94 internalization was examined using various internalization and internalization competition assays. These studies demonstrated that GRP94.NTD and the CD91 ligand RAP were internalized through spatially and kinetically distinct pathways, that CD91 was not necessary for GRP94.NTD internalization, and that RAP did not inhibit GRP94 endocytosis. Together, these studies strongly suggest that CD91 does not directly facilitate GRP94 internalization. When these studies were extended to DC2.4 mouse dendritic cells, the CD91 ligand RAP reduced GRP94.NTD internalization/process by ~15%. This suggests that CD91 may indirectly facilitate GRP94 internalization in pAPC cell lines. Lastly, cross-presentation studies were utilized to examine the ability of various CD91 ligands to influence GRP94.NTD-mediated peptide cross-presentation through a post-uptake mechanism using the DC2.4/OT-1 system. Although it was discovered that DC2.4 cells can internalize and process GRP94.NTD/peptide complexes through fluid-phase endocytosis, CD91 ligands did not significantly inhibit GRP94-mediated peptide cross-presentation by DC2.4 cells. These studies demonstrate that CD91 does not play a primary role in GRP94-mediated peptide cross-presentation.</p><p>In the course of these studies, cell surface heparan sulfate proteoglycans (HSPGs) were identified as novel cell surface binding sites for GRP94.NTD on MEF cells. This conclusion was established using three distinct experimental approaches. GRP94.NTD surface binding was significantly decreased following heparin pre-treatment, following incubation with the sulfation inhibitor sodium chlorate, and following digestion with extracellular heparinase II. Conversely, these treatments did not significantly influence GRP94.NTD binding to RAW264.7 mouse macrophage-like cells. This suggested that GRP94.NTD-HSPG cell surface interactions may require the expression of a specific type of cell surface HSPG that is not expressed by RAW264.7 cells. However, additional studies strongly suggested that GRP94.NTD-HSPG cell surface interactions were mediated by the heparan sulfate-containing side chains rather than the presence of a specific cell surface HSPG core protein.</p><p>This dissertation focuses on the critical re-examination of CD91 functions in GRP94 surface binding, uptake, and cross-presentation. Together, these results clarify conflicting data on CD91 function in GRP94 surface binding and endocytosis. This dissertation also describes the identification of cell surface HSPGs as GRP94 binding sites on MEF cells. These studies extend the diversity of surface receptors that recognize of GRP94, and suggest that cell surface HSPG-dependent interactions may contribute to the biology of GRP94-elicited immune responses.</p> / Dissertation

La gp120 du VIH-1 se fixe aux héparane sulfate (HS) cellulaires, par le biais de la boucle V3 ce qui favorise l'infectivité virale. Cependant, une polyanion solubles (HS12), conjugués à CD4 (mCD4-HS12) a des propriétés antivirales et a montré in vitro une activité contre le VIH-1 à de concentrations nM. En raison de la complexité structurale des HS, le criblage d'oligosaccharides différenciellement sulfatés pour améliorer l'activité de la molécule serait trop difficile. En vue d'obtenir une molécule plus spécifique, de plus haute affinité et plus facile à produire, des peptides mimant les HS ont été synthétisés par nos collaborateurs. Notre but était de cribler ces peptides pour leur capacité à inhiber l'entrée de VIH-1. Nous avons mis en place une plateforme permettant d'immobiliser CCR5 et CXCR4 solubilisés sur des biocapteurs pour cribler des molécules qui inhibent la liaison de gp120-CD4 aux corécepteurs. Pour contrôler le processus de solubilisation, CXCL12, le ligand naturel de CXCR4, a été injecté sur CXCR4 immobilisé. Les affinités des isoformes CXCL12 (α et γ) pour CXCR4 ont été calculées dans les fourchettes de valeurs précédemment décrites avec des techniques différentes prouvant la fonctionnalité de notre système. Nous montrons pour la première fois que les HS régulent différemment les mécanismes de liaison de ces deux isoformes et nous proposons un nouveau mode d'action pour le domaine C-terminal particulièrement basique de CXCL12 γ vis-à-vis de CXCR4. Le système a ensuite été utilisé pour cribler la capacité d'inhibition des peptides mimétiques du HS. Chaque peptide, [S(XDXS)n] contient des acides aminés qui imitent les groupes hydroxyles, carboxyles et sulfates des HS. Le peptide contenant des résidus sulphotyrosines, une fois conjugué à mCD4 (mCD4-P3YSO3), montre un IC50 de l'ordre du nM, pour l'inhibition simultanée de la liaison de gp120 aux HS, à CD4, aux anticorps, aux corécepteurs ainsi que l'infection par VIH-1 in cellulo. Il constitue le premier inhibiteur bivalent de l'entrée qui cible à la fois les virus R5 et X4 et le concept d'un peptide mimétique des HS se prête à une analyse structurale et fonctionnelle de la liaison des chaînes HS aux protéines, une nouvelle technique dans ce domaine.

Alternative bone repair strategies are frequently sought after in orthopaedic surgery to address the growing need for improved morbidity and healing rates. This thesis sought to initiate and validate such an alternative, harnessing the flexible nature of a biomaterial substrate and the unique potential of glycosaminoglycan sugars. A novel, biodegradable biomaterial polymer, PHBV, has previously been identified to have the potential to mimic the characteristics of bone necessary for tissue repair and in this study, it was hypothesized that PHBV would be able to support bone formation. When tested in vitro, PHBV was found to support osteoblast cell attachment, proliferation and differentiation, despite its rougher, more hydrophobic surface characteristics compared to tissue culture plastic (TCP). However, unlike the progression of cells on TCP, PHBV caused a developmental delay at each stage of osteogenesis, suggesting a sub-optimal cell-substrate interaction. The expression profiles of genes involved in the maintenance of the extracellular matrix were monitored to investigate this phenomenon further. The results suggested that cells cultured on PHBV appeared to preference 7 against a collagen-based ECM and, instead, trigger an increase in the expression of other factors, such as osteopontin, presumably to modify the biomaterial microenvironment to optimise continued growth and differentiation. This finding led to the next hypothesis that functionalisation of PHBV with suitable compounds could optimise and enhance the osteogenic development at the implant site by facilitating the desired and appropriate cell-substrate interactions. Non-protein factors are often preferred for functionalisation to material scaffolds over proteins, as they are relatively robust and can survive many of the processes used in the manufacture of biomaterials. Glycosaminoglycan (GAG) sugars were appropriate candidates for this purpose, as they are not only abundantly expressed in bone, but more importantly, they are capable of binding and facilitating the activity of growth factors. Furthermore, they are resistant to several environmental influences including changes in pH, heat and desiccation. To identify a GAG that could be integrated with PHBV or any other biomaterial substrate, GAGs were extracted from phenotypically-distinct stages of MG-63 osteosarcoma cells. These GAGs were identified to display gross structural differences, as well as differences in the enzymes synthesising them, between immature and mature osteoblastic cells, with the increased production of a larger GAG species observed as the cells differentiated. Unexpectedly, however, when these GAGs were subsequently dosed back into the media of growing MG-63 cells, their bioactivity did not match the stage at which they had been harvested: all GAG species were able to influence cell survival and growth to varying degrees but were not capable of affecting cell differentiation. However, if these same GAGs were exposed to cells by first being attached to the growth substrate, they induced varying degrees of aggregation in human mesenchymal stem cells (hMSCs), with more mature GAGs producing the most profound effects. Interestingly, a similar phenomenon was not observed when MG-63 cells where cultured in a similar manner. A direct correlation between the GAGs expressed by osteoblasts and the specific cellular processes they functionally influence has yet to be identified. While the experiments presented here demonstrate an effect of GAGs in osteoblastic cell survival, a role for GAGs in the progression of bone formation was not revealed. Loss-of-function studies were therefore necessary to determine the role of GAGs in bone, but this was hampered by the limited availability of procedures that allow the alteration of GAGs and the subsequent detection of these effects. Therefore, a tool to screen the efficacy of a loss of GAG function was developed. TAT-EGFP, a purpose-designed fluorescent GAG-binding peptide, was able to confirm that treatment with sodium chlorate was an effective 8 strategy to hinder GAG expression in MG-63 cells with minimal cytotoxicity to the cells. Following more extensive studies with chlorate treatment, it was found that a recoverable disruption to both proliferation and mineralisation could be induced in MG-63 cells. This suggested a role for GAGs in osteogenesis. A series of experiments then carried out following gene expression microarray analysis indicated that GAG de-sulfation by chlorate gives rise to an S-phase block in the cell cycle and a disruption to the actin cytoskeleton, which appeared to be associated with a change in the activity of cell-surface proteoglycans, most likely syndecan 4. It was also found that cells up-regulated plasma membrane ALP activity and cholesterol synthesis, presumably in an attempt to recover from a chlorate-induced loss in GAG function. Cholesterol is known to be important in establishing connections between membrane elements and the actin cytoskeleton, and its up-regulation here may reflect dysfunctions in these units and a dysfunction in syndecan 4 activity. With further confirmation, this would suggest that syndecan 4 plays a pivotal role in maintaining osteogenesis, in at least MG-63 cells, and that sulfated GAGs function principally to facilitate this role. The effective use of GAGs in bone repair strategies will require further understanding of GAG/syndecan 4/osteogenesis relationship.

Tissue repair is a complex process that is difficult to emulate. The addition of the glycosaminoglycan heparan sulfate (HS), a multi-potential regulator of numerous growth factors and cytokines endogenously expressed during the repair process, may represent a valuable tool for tissue engineering. The addition of exogenous HS into wound site has previously been shown to promote tissue repair in a number of models, however, the incorporation of HS into controlled release systems or biomaterials for tissue engineering had not been explored prior to the work presented here. Thus, this thesis explores the incorporation of HS and its analogue heparin into synthetic biodegradable polymer biomaterials with different potential applications, either as a slow releasing drug reservoir, or as a drug releasing cell scaffold. Polycaprolactone was used to make microcapsules and electrospun fibers for HS or heparin entrapment. These materials were characterized for their drug release profiles, biocompatibility and bioactivity. Microcapsules encapsulating heparin or HS were made by the oil - in - water solvent evaporation method which allowed fabrication of slow releasing drug reservoirs. Either pure water or a poly(vinyl alcohol) solution was used in the drug phase which resulted in capsules with similar size and drug loading. However the internal morphology and drug release profiles showed differences depending on the drug phase, in either case release was sustained for over 30 days. These capsules elicited no pro-inflammatory response from macrophages in vitro, and the released HS retained its bioactivity to induce the proliferation of human mesenchymal stem cells, an important cell type for bone tissue engineering. Heparin and HS were incorporated into electrospun fibers as a drug releasing scaffold for two different tissue engineering applications. Heparin fibers were studied as a drug releasing membrane that could be used in vascular repair to prevent the unwanted proliferation of vascular smooth muscle cells. Heparin release was sustained from the fibers for at least 2 weeks. The fibers did not induce a pro-inflammatory response from macrophages in vitro and the released heparin retained the ability to inhibit the proliferation in vascular smooth muscle cells. HS fibers were studied as a tissue engineering scaffold for bone repair using human mesenchymal stem cells. HS release was maintained for over 30 days which is thought to be an appropriate time for bone repair applications. The release profiles depended on the HS concentration in the spinning solution which affected the morphology of the fibers. The fibers did not elicit a pro-inflammatory response in cultured macrophages and supported the proliferation and mineralization of human mesechymal stem cells. The HS fibers were then taken through to an in vivo model to study ectopic bone formation of pre-osteoblast cells on HS releasing scaffolds. The fibers produced a chronic inflammatory response in vivo, which lead to the clearance of implanted cells and no mineralization of the scaffold. The HS and heparin materials made in this work showed sustained release over appropriate time frames for different tissue repair applications. The released HS and heparin maintained bioactivity and showed good biocompatibility in vitro, however, further in vivo studies are required to fully test their efficacy for tissue engineering.

Il est bien établi que la gp120 du VIH-1 se fixe aux héparane sulfate (HS) cellulaires, par le biais de la boucle V3 ce qui favorise l'infectivité virale. Cependant, une variété de polyanions solubles, conjugués à CD4 (mCD4-HS12) a des propriétés antivirales et a montré in vitro une activité contre le VIH-1 à de très faibles concentrations (nM). En raison de la complexité structurale des HS, le criblage d'oligosaccharides différenciellement sulfatés pour améliorer l'activité de la molécule serait trop difficile. En vue d'obtenir une molécule plus spécifique, de plus haute affinité et plus facile à produire, des peptides mimant les HS ont été synthétisés par nos collaborateurs. Notre but était de cribler ces peptides pour leur capacité à inhiber l'entrée de VIH-1. À cette fin, nous avons mis en place une plateforme permettant d'immobiliser CCR5 et CXCR4 solubilisés sur des biocapteurs (Biacore) pour cribler des molécules qui inhibent la liaison de gp120-CD4 aux corécepteurs. Pour contrôler le processus de solubilisation, CXCL12, le ligand naturel de CXCR4, a été injecté sur CXCR4 immobilisé. Les affinités des isoformes de CXCL12 (α et γ) pour CXCR4 ont été calculées dans les fourchettes de valeurs précédemment obtenues avec des techniques différentes, prouvant ainsi la fonctionnalité de notre système et nous permettant d'étudier les mécanismes de fixation de ces deux isoformes sur CXCR4 ainsi que leur régulation par HS. Le système a ensuite été utilisé pour cribler la capacité d'inhibition des peptides mimétiques du HS. Chaque peptide, [S(XDXS)n] contient des acides aminés qui imitent les groupes hydroxyles, carboxyles et sulfates des HS. Le peptide contenant des résidus sulphotyrosines, une fois conjugué à mCD4 (mCD4-P3YSO3), montre un IC50 de l'ordre du nM, pour l'inhibition simultanée de la liaison de gp120 aux HS, à CD4, aux anticorps, aux corécepteurs ainsi que l'infection par VIH-1 in cellulo. Il constitue le premier inhibiteur bivalent de l'entrée qui cible à la fois les virus R5 et X4 et le concept d'un peptide mimétique des HS se prête à une analyse structurale et fonctionnelle de la liaison des chaînes HS aux protéines, une nouvelle technique dans ce domain / It is well-established that cell-associated Heparan Sulphate (HS) binds the V3 loop of gp120 of HIV-1 thus aiding in viral infectivity. However, a variety of soluble polyanions have antiviral properties once conjugated to CD4 and a CD4-conjugated HS (mCD4-HS12), showed nM activity against HIV-1 in vitro. Due to the structural complexity of HS, screening differently sulphated oligosaccharides to improve the molecule's activity would be too cumbersome, thus in order to obtain a more specific, higher affinity and easier to produce moiety, collaborators synthesized HS mimetic peptides. We aimed to screen these peptides and other anionic molecules for their capacity to inhibit HIV-1 entry. To this end we set-up a platform whereby solubilised CCR5 and CXCR4 were immobilized on biosensors (biacore) and used to screen for molecules that inhibited gp120-CD4 binding to the coreceptors. To control the solubilization process, CXCL12, the natural ligand of CXCR4, was injected over the immobilized CXCR4. The affinities of CXCL12 isoforms (α and γ) for CXCR4 were calculated within the ranges of values that have been previously described with different techniques, thus proving the functionality of our system and enabling us to investigate the binding mechanisms of these two isoforms with CXCR4 and their regulation by HS. The system was subsequently used to screen the inhibitory capacity of the HS mimetic peptides. Each peptide, [S(XDXS)n], contained amino acids that mimic the hydroxyl, carboxyl and sulphate groups on HS chains. The peptide containing sulphotyrosine residues, when conjugated to mCD4 (mCD4-P3YSO3), displayed nM IC50 for simultaneously inhibiting gp120 binding to HS, CD4, antibody, coreceptors and HIV-1 infection in vitro. This is the first bivalent entry inhibitor that targets both R5 and X4 viruses and the concept of a HSmimetic peptide lends itself to structural-functional analysis of HS chains binding to proteins, a novel technique in this field.

L’analyse du mécanisme d’interaction de l’héparane sulfate protéoglycane (HSPG) avec lesprotéines amyloïdes et l’effet de paramètres physiologiques (pH,…) sur ce mécanisme nouspermettent une meilleure compréhension des mécanismes menant à l’amyloïdogenèse.La transthyrétine (TTR), molécule circulant à la fois dans le plasma et dans le liquidecéphalo-rachidien, est une des protéines impliquée dans les amyloses. Elle est responsable desamyloses à la TTR et elle joue un rôle dans la maladie d’Alzheimer en séquestrant la protéinebêta-amyloïde (Aβ). La biochromatographie est un outil très efficace pour analyser lemécanisme entre un ligand et son récepteur dans des conditions modulables et se rapprochantdes conditions biologiques. De plus, les nanotubes de carbones (NTCs) peuvent être utiliséspour détecter ou transporter des molécules qui se lient sur leur surface externe et peuventinteragir avec d’autres composés. Au cours de ce travail, un support particulaire a été utilisé où l’HSPG est immobilisé sur desparticules de silice pré-activées par des résidus amines. Ce support remplissant une colonnechromatographique a permis dans un premier temps d’étudier et de comparer les mécanismesd’association entre l’HSPG et une forme sauvage de la TTR et une forme sénile de la TTRextraite d’un patient décédé des suites d’une amylose sénile à la TTR. Cette étude a montréque pour la TTR sauvage, l’association avec l’HSPG est indépendante du pH et implique desinteractions faibles. Pour la TTR sénile, cette association est dépendante du pH. A pH<6,5, laprotonation d’un résidu histidine est observée. De plus, l’étude des paramètresthermodynamiques et de la compensation enthalpie/ entropie montrent un changement dans lemécanisme de fixation avec l’apparition d’interactions ioniques à pH<6,5. Un pH acide estnécessaire pour dissocier et dénaturer partiellement la TTR. L’affinité de la TTR avec l’HSPGdépend de la structure tétramérique quaternaire de la TTR qui présente alors des résiduscapables de créer des interactions. Dans un deuxième temps, cette colonne a permis d’évaluerl’effet de la TTR et du pH sur la liaison Aβ/HSPG. Comme précédemment, la protonationd’un résidu histidine présent sur la Aβ est observée à pH<6,5. Ce résultat confirme des étudesmenées auparavant sur le précurseur de la protéine Aβ. Les résultats thermodynamiques ontmis en évidence que l’affinité de Aβ avec l’HSPG diminuait avec la concentration croissanteen TTR et l’étude des chromatogrammes associés a montré que la TTR ne séquestrait passeulement Aβ mais la clivait en fragments plus courts qui diminuent son affinité avecl’HSPG. Dans la maladie d’Alzheimer, la TTR exerce une activité protéolytique vis-à-vis deAβ.2. Dans un troisième temps, l’effet de la fonctionnalisation de la TTR par des nanotubes decarbone sur la liaison TTR/HSPG a été étudié. Les résultats obtenus montrent que lesinteractions entre l’HSPG et la TTR-NTC sont de type van der Waals et hydrogène. Lesparamètres thermodynamiques des liaisons TTR/HSPG et TTR-NTC/HSPG sont similairespour des pH>6. A pH<6, il n’existe quasiment pas de différences entre les valeurs obtenues àpH >6 et celles obtenues à pH<6. Les NTCs empêcheraient la formation de liaisons ioniques / The analysis of the heparan sulfate proteoglycan (HSPG) association mechanism withamyloid proteins and the effect of physiological parameters (pH,…) on this mechanism allowa better understanding of the mechanisms leading to amyloidogenesis.Transthyretin (TTR), which circulates in both plasma and cerebrospinal fluid, is one of theproteins involved in amyloidosis. It leads to TTR amyloidosis and plays a role in Alzheimer’sdisease in sequestrating the beta-amyloid (Aβ) protein. Biochromatography is an effectivetool for the analysis of the mechanism involved between a ligand and its receptor inadjustable conditions which could be close to biological conditions. Moreover, carbonnanotubes can be used to detect or to carry molecules which bind on its external surface andcould interact with other molecules. In this work, a particulate support was used where the HSPG was immobilized on the silica particles preactivated by amine residues. This support filling a column was used to study andcompare association mechanisms between HSPG and a wild type TTR form and a senile formof the TTR which was was extracted from a patient who died of cardiac failure with a senilesystemic amyloid. This study showed that the association between wild type TTR and HSPGwas independent of the pH and involved weak interactions.For the senile TTR, this association was dependent on pH. At pH<6,5, a histidine residue wasprotonated. Moreover, the study of both thermodynamical parameters and enthalpy-entropycompensation showed a change in the association mechanism with involvement of more ionicinteractions at pH<6,5. An acidic pH was necessary to dissociate and partially denaturate theTTR. The affinity of the TTR with HSPG depends on the tetrameric quaternary structure ofthe TTR which presents some residues which are able to create interactions.In a second time, this column was used to evaluate the effect of both the TTR and the pH onAβ/HSPG binding. As previously, the protonation of a histidine residue present on Aβ wasobserved at pH<6,5. This result confirmed studies on the Aβ precursor. Thermodynamicalvalues highlighted that the affinity of Aβ for l’HSPG decreased with the increase of TTRconcentration, and the study of the chromatograms associated showed that TTR sequestratedAβ and cut Aβ in smaller fragments which decreased its affinity for HSPG. In Alzheimer’sdisease, TTR has a proteolytic activity on Aβ.In a third time, the effect of the carbon nanotubes (CNTs), immobilized on TTR, on theTTR/HSPG binding was studied. Results showed that interactions between TTR-NTC andHSPG are van der Waals and hydrogen. Thermodynamical data of TTR/HSPG et TTRNTC/HSPG bindings are similar at pH>6. At pH<6, no differences between values obtainedat all pH values for TTR-NTC. NTCs would avoid ionic interactions formations.

Les endosulfatases (H-Sulf 1 et 2) catalysent l’hydrolyse des sulfates en position 6 des résidus glucosamine des chaines d’héparane sulfate. Elles modulent ainsi l’activité des héparanes sulfate vis à vis de nombreuses protéines. Plusieurs études ont montré que l’endosulfatase H-Sulf 2 est produite en quantité plus importante dans de nombreux cancers (poumons, sein, pancréas etc …). La réduction de l’activité de cette enzyme chez des souris malades permet la diminution de la tumeur ainsi que le prolongement de la durée de vie. Ce travail de thèse vise à synthétiser une série d’inhibiteurs spécifiques des endosulfatases humaines. Une étude préliminaire a été menée sur un monosaccharide portant une fonction sulfamate, cette fonction étant connue pour inhiber les sulfatases. Ce projet de thèse repose sur la synthèse d’oligosaccharide de type héparane sulfate de tailles différentes portant tous cette fonction inhibitrice. Pour ce faire il a fallu mettre au point une stratégie itérative incluant des réactions de glycosylations stéréosélectives. Puis l’ouverture régiosélective d’acétal de benzylidène ainsi que la sulfamoylation ont été examinées l’optimisation des fonctionnalisations et des déprotections sont encore en cours afin de compléter cette librairie de molécules inhibitrice des endosulfatases humaines. / Human endosulfatase (H-Sulf 1 and 2) catalyse heparan 6-O-sulfate hydrolysis. Thanks to this hydrolysis they change the heparan sulfate proteoglycan activity. Many studies have shown that H-Sulf 2 are surexpressed in many cancer (lung, breast, pancreas etc...) The decrease of the activity of this enzyme in the case of the mouse allows a decrease of the size of the tumor and an extension of the mouse life. The aim of this thesis is to synthetise a library of endosulfatase specific inhibitors. A preliminary study was made on a monosaccharide carrying a sulfamate. This fonctional group have shown that it is able to inhibate the sulfatases. This project thesis is to synthetise heparan sulfate oligsaccharides carrying this sulfamate moeity. We apply an iterative process to make this oligosaccharide, thanks to a high stereoselective glycosylation. Finaly a reductive opening of benzylidène acetals, a sulfamoylation have been examinated, functionalization and deprotection are still in progress for get the full library.

L’AVC représente la première cause d’handicap acquis chez l’adulte. L’AVC ischémique, représentant 87% des AVCs, est une pathologie complexe dont le premier facteur de risque aggravant est l’hypertension artérielle. À l’heure actuelle les seuls traitements disponibles sont la thrombolyse et la thrombectomie. Cependant, ces traitements présentent de nombreuses contre-indications et effets secondaires limitant leurs applications chez les patients. L’objectif des travaux menés dans cette thèse est l’évaluation d’un traitement pharmacologique, le RGTA (ReGeneraTing Agent), combiné ou non à un traitement cellulaire utilisant les cellules souches mésenchymateuses (CSMs), chez des rats normo- et hyper-tendus. Les résultats obtenus dans cette thèse montrent qu’à la suite d’une ischémie cérébrale, les traitements évalués offrent une neuroprotection et une récupération fonctionnelle persistantes, chez les animaux noromo- et hyper-tendus. Cette récupération est expliquée par la réduction du volume lésionnel, par une meilleure plasticité cérébrale (angiogenèse, neurogenèse), ainsi par la potentialisation de l’effet des CSMs par le RGTA. En conclusion, nos études démontrent l’efficacité d’une thérapie robuste de neurorprotection chez le rongeur à la suite d’une ischémie cérébrale. / Stroke is the leading cause worldwide of adult severe disability. The limited available treatments for ischemic stroke, which accounts for 87% of strokes, makes it necessary to develop new therapeutical approaches. Stroke is a complex pathology and chronic hypertension (CAH) represents the first aggravating risk factor for ischemic stroke. At the present time, the only two available treatments for ischemic stroke, thrombolysis and thrombectomy, present several side effects limiting their clinical use. Here we evaluate the effect of a molecular RGTA (ReGeneraTing Agent) based therapy combined or not to a cellular therapy based on the use of mesenchymal stem cells (MSCs) for the treatment of ischemic stroke in normo- and hyper-tensive rats. The results demonstrate that the evaluated therapies confer a long lasting neuroprotection accompanied by animals’ functional recovery. Further analysis suggest that RGTA enhances brain plasticity (angiogenesis, and neurogenesis), protects the extracellular matrix structure, and potentiates MSCs’ beneficial effects. In conclusion, our studies demonstrate the efficacy of a molecular and cellular combined therapy conferring a persistent neuroprotection and functional recovery for the treatment of ischemic stroke.

Ce travail de thèse a eu pour thème principal le protéoglycane syndecan-1 au cours du myélome multiple, une hémopathie maligne caractérisée par la présence d'un clone de plasmocytes tumoraux au sein de la moelle osseuse. Syndecan-1 est un élément majeur de la physiopathologie du myélome multiple, ce protéoglycane étant au coeur d'un réseau complexe d'interactions moléculaires conditionnant le devenir des cellules plasmocytaires tumorales.Les chaînes de glycosaminoglycanes présentes sur le core protéique de syndecan-1sont responsables d'une grande partie de son activité. Nous avons ainsi caractérisé, par une approche transcriptomique, 100 gènes codant les protéines impliquées dans la synthèse et la modification de ces chaînes. Nous avons de cette manière identifié des cibles moléculairesen vue de moduler, voire d'inhiber leur activité.Dans le but d'identifier les métalloprotéinases des familles ADAM et ADAMTS susceptibles d'interagir avec syndecan-1, nous avons réalisé l'étude du profil d'expression des gènes codant ces reprolysines et leurs inhibiteurs dans les cellules de la différentiation lymphocytaire B, les cellules plasmocytaires normales et tumorales ainsi que dans l'environnement médullaire.Dans une dernière partie, nous avons évalué l'efficacité d'une approche d'inhibitiondes chaînes héparanes sulfates via l'utilisation de l'héparine. Nous observons que certaines lignées myélomateuses sont inhibées par l'héparine et ses dérivés et que ces mêmes lignées sont stimulées par l'antidote de l'héparine, le sulfate de protamine. Les mécanismes mis enjeu sont en relation avec la modulation de la biodisponibilité des facteurs permettant la croissance des cellules. / Multiple myeloma is a hematological malignancy characterized by the expansion of aclone of malignant plasma cells in the bone marrow compartment. Syndecan-1 is a majorproteoglycan involved in a complex network of molecular interactions in multiple myelomaphysiopathology. As heparan sulfate and chondroitin sulfate chains are the bioactive components ofsyndecan-1, we first analysed the signature of genes encoding 100 proteins involved in thesynthesis of these chains, from precursor uptake to post-translational modifications, usingAffymetrix microarrays.In order to identify the metalloproteinases belonging to ADAM and ADAMTS familiespotentially implicated in the interactions with syndecan-1, we performed a gene expressionprofile focused on the genes encoding these reprolysines and their inhibitors.In a last part, we evaluated the efficacy of an inhibitory approach based on theutilization of heparin in human myeloma cell lines in vitro, inhibitory effects being in relationwith a modulation of the biodisponibility of heparin-binding factors.This work led us to identify targets of interest in relation with syndecan-1 biology inmultiple myeloma. They could be used to design new therapeutic strategies.

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