yes / Purpose: To evaluate the structure–function relationship between disc margin–based rim area (DM-RA) obtained with confocal scanning laser tomography (CSLT), Bruch's membrane opening–based horizontal rim width (BMO-HRW), minimum rim width (BMO-MRW), peripapillary retinal nerve fiber layer thickness (RNFLT) obtained with spectral-domain optical coherence tomography (SD-OCT), and visual field sensitivity. Methods: We examined 151 glaucoma patients with CSLT, SD-OCT, and standard automated perimetry on the same day. Optic nerve head (ONH) and RNFL with SD-OCT were acquired relative to a fixed coordinate system (acquired image frame [AIF]) and to the eye-specific fovea-BMO center (FoBMO) axis. Visual field locations were mapped to ONH and RNFL sectors with fixed Garway-Heath (VFGH) and patient-specific (VFPS) maps customized for various biometric parameters. Results: Globally and sectorally, the structure–function relationships between DM-RA and VFGH, BMO-HRWAIF and VFGH, and BMO-HRWFoBMO and VFPS were equally weak. The R2 for the relationship between DM-RA and VFGH ranged from 0.1% (inferonasal) to 11% (superotemporal) whereas that between BMO-HRWAIF and VFGH ranged from 0.1% (nasal) to 10% (superotemporal). Relatively stronger global and sectoral structure–function relationships with BMO-MRWAIF and with BMO-MRWFoBMO were obtained. The R2 between BMO-MRWAIF and VFGH ranged from 5% (nasal) to 30% (superotemporal), whereas that between BMO-MRWFoBMO and VFPS ranged from 5% (nasal) to 25% (inferotemporal). The structure–function relationship with RNFLT was not significantly different from that with BMO-MRW, regardless of image acquisition method. Conclusions: The structure–function relationship was enhanced with BMO-MRW compared with the other neuroretinal rim measurements, due mainly to its geometrically accurate properties.

During hemostatic and inflammatory responses, cell adhesion molecules play a major role in regulating the leukocytes and platelets adhesion to vascular surfaces under the hydrodynamic environment of the circulation. Selectin-ligand interactions (bonds) mediate leukocyte rolling on vascular surfaces. The molecular basis for differential ligand recognition by selectins is poorly understood. Using atomic force microscopy (AFM), the kinetics of three mutants L-selectin interacting with surrogates of PSGL-1 and PNAd, is compared with those of wild-type L-selectin. The interaction between glycoprotein Ib (GPIb) and von Willebrand Factor (VWF) mediates platelet translocation at the vascular vessel damage sites, which plays a critical role in initiating the platelets adhesion and thrombus formation. Translocation of platelets on VWF requires a shear threshold, suggesting a possible catch bond at work there. We characterized the kinetics of GPIbα interacting with VWF A1 domain, confirming the catch bond existed. Two type 2B VWD A1 mutants eliminated the catch bond and gave longer low force lifetimes. The prolonged lifetimes at low force resulted in more agglutination of platelets with A1 coated microspheres in flow. During the process of hemostasis, the size of prothrombotic ULVWF affects the affinity of VWF to platelets bearing GPIbα on the membrane. ADAMTS13 has been identified and characterized as a multi-domain metalloprotease that regulate the size of ULVWF. We studied how force regulated the binding and cleavage of ADAMTS13 on VWF. We found the cleavage effects could only be observed after the catastrophic structural change of A1A2A3. The unfolding exposed the ADAMTS13 cleavage site and favored the cleavage. Two protocols using different stretching molecules (GPIbα and CR1) and A1A2A3 immobilization methods revealed the cleavage effects diminished with increasing stretching force. This study elucidated mechanisms of the binding kinetics of L-selectin with different structure components from PSGL-1 and PNAd by structural variants. It also provided new insights into our current knowledge of the dynamic adhesion and regulation of GPIbα-VWF interaction in vivo. Using single molecule method, the chemical catalytic reaction between enzyme and substrate has been targeted. These results help us understand this important enzyme-substrate interaction involved in the hemostasis.

We have studied the modulation of gating properties of the Ca2+-permeable, cation channel TRPV4 transiently expressed in HEK293 cells. The phorbol ester 4αPDD transiently activated a current through TRPV4 in the presence of extracellular Ca2+. Increasing the concentration of extracellular Ca2+ ([Ca2+]e) reduced the current amplitude and accelerated its decay. This decay was dramatically delayed in the absence of [Ca2+]e. It was also much slower in the presence of [Ca2+]e in a mutant channel, obtained by a point mutation in the 6th transmembrane domain, F707A. Mutant channels, containing a single mutation in the C-terminus of TRPV4 (E797 , were constitutively open. In conclusion, gating of the 4αPDD-activated TRPV4 channel depends on both extra- and intracellular Ca2+, and is modulated by mutations of single amino acid residues in the 6th transmembrane domain and the C-terminus of the TRPV4 protein.

Emergent infectious diseases are a major driver of the accelerated rates of biodiversity loss that are being documented around the world. Global losses of amphibians provide evidence of this, especially those associated with chytridiomycosis, a lethal skin disease caused by the fungus Batrachochytrium dendrobatidis (Bd). Amphibian skin can harbor diverse bacterial communities that, in some cases, can inhibit the growth of Bd. Thus, there is interest in using skin bacteria as probiotics to mitigate Bd infections in amphibians. However, experiments testing this conservation approach have yielded mixed results, suggesting a lack of understanding about the ecology of these microbial communities. My dissertation research aimed to assess basic ecological questions in microbial ecology and to contribute to the development of probiotics using amphibian skin bacteria. First, to assess whether environmental conditions influence the function of amphibian skin bacterial communities, I conducted a field survey across low and high elevation populations of an amphibian host to assess their skin bacterial communities and metabolite profiles. I found that similar bacterial communities produced different metabolites at different locations, implying a potential functional plasticity. Second, since culturing is critical for characterizing bacteria, I aimed to identify the culture media (low vs high nutrient concentration) that recovers the most representative fraction of the amphibian skin bacterial community. I found that media with low nutrient concentrations cultured a higher diversity and recovered a more representative fraction of the diversity occurring on amphibian skin. I also determined that sampling more individuals is critical to maximize culture collections. Third, I assessed the diversity of the amphibian skin fungal community in relation to Bd infection across eight amphibian species. I determined that amphibian species was the most important predictor of fungal diversity and community structure, and that Bd infection did not have a strong impact. My dissertation highlights the importance of environmental conditions in the function of amphibian skin bacteria, expands our knowledge of the understudied fungal component of the amphibian skin microbiome, and complements current efforts in amphibian conservation. / Ph. D. / In light of the global losses of amphibian diversity due to, in part, the skin disease chytridiomycosis (caused by the fungus Batrachochytrium dendrobatidis [Bd]); the discovery that some amphibian-skin bacteria can inhibit Bd growth provides hope for amphibian conservation via their use as probiotics to control Bd infections. However, experiments testing these bacteria have yielded inconsistent results, suggesting a limited understanding about the factors influencing the diversity of amphibian-skin microbes and their ability to inhibit Bd. Also, efforts to identify effective candidates for probiotic therapy are still premature. Thus, my dissertation had an ecological emphasis and focused on complementing conservation efforts focused on probiotics. First, I assessed whether environmental conditions influence bacteriallyproduced products, which can have antifungal properties. Specifically, I surveyed low and highelevation populations of an amphibian species to assess the skin-bacteria and their products. I determined that, while skin bacterial communities were similar across an environmental gradient, their products differed, suggesting potential different antifungal properties. Second, I assessed the ability of different culture media types (low vs high nutrient concentrations) to grow a high portion and most representative fraction of the amphibian-skin bacteria. I found that culture media with low nutrient concentrations allowed the growth of a higher diversity of the bacteria occurring on the amphibian-skin, including the abundant members, and also determined that including a large number of amphibians is the best way to improve culture collections. Third, I assessed the fungal diversity occurring in the skin of different amphibian species and how it might response to Bd infections, and examined whether skin-fungi interact with co-occurring bacteria. I found that the amphibian species was the most important driver of the fungal diversity, and that Bd infection did not influence the diversity of these communities. Moreover, I identified the most diverse fungal phyla occurring in the amphibian-skin and determined that these fungi might interact with co-occurring bacteria. My dissertation contributes to our understanding about the influence of the environmental conditions in the amphibian-skin bacteria, expands our limited knowledge on the amphibian-skin fungi, and complement current amphibian conservation efforts.

β-Glucans have long been used as an immunostimulant in aquaculture. However, the relationship of its structure to its immunomodulatory properties are poorly understood. In this study, the particle size and chemical structure of β-glucans extracted from wild-type strain of baker's yeast (Saccharomyces cerevisiae) and its null-mutant yeasts Gas1 were characterised. Using Sigma β-glucan as a reference, the immunomodulatory properties of these polysaccharides in the germ-free Artemia franciscana model system in the presence of Vibrio harveyi bacterial challenge were investigated. The survival of the A. franciscana nauplii, upon challenge with V. harveyi, was significantly higher in all three glucan-treated groups compared to the control. The glucan Gas1 with a lower degree of branching and shorter side chain length had the most prominent V. harveyi-protective effects. The particle size did not affect the nauplii survival when challenged with V. harveyi. Results also showed that the salutary effect of the tested glucans was associated with the upregulation of innate immune genes such as lipopolysaccharide and β-1,3-glucan-binding protein (lgbp), high mobility group box protein (hmgb), and prophenoloxidase (proPO). Interestingly, the up-regulation of superoxidase dismutase (sod) and glutathione-s-transferase (gst) was only observed in Gas1 treated group, indicating that Gas1 could function to induce higher reactive oxygen species and stronger immunomodulatory function in A. franciscana, and therefore higher survival rate. The expression of heat shock protein 70 (hsp70), peroxinectin (pxn), and down syndrome cell adhesion molecule (dscam) remain unaltered in response to glucan treatment. Taken together, this study provides insights into the structure-function relationship of β-glucan and the results confirmed that β-glucan can be an effective immunostimulant in aquaculture, especially the Gas1 glucan.

No description available.

Les grandes propriétés interactives des HS dépendent de leur profil de sulfatation, finement régulé au cours de la biosynthèse du polysaccharide ainsi qu'à la surface cellulaire par l'action d'endosulfatases nommées Sulfs. Ces enzymes ôtent spécifiquement les groupements 6-O-sulfates d'un certain nombre de disaccharides trisulfatés, éléments clés de nombreuses interactions protéines/HS. Ainsi, ces enzymes sont impliquées dans de nombreux processus, autant physiologiques que pathologiques. En utilisant différentes approches (biochimique et biophysique), nous avons révélé récemment que les isoformes humain HSulf-1 et -2 agissent de façon processif et orientées pour désulfater les chaines d’HS ce qui régule différemment les facteurs de croissances FGF-1 et -2 (Seffouh et al., FASEB J. 2013). Nous avons également identifié deux motifs impliqués dans l’interaction HSulf-2/HS et qui se sont avérés importants pour l’activité 6-O-endosulfatase [Manuscrit en préparation]. Par ailleurs, HSulf-2, et contrairement à HSulf-1, s’est révélé porteuse d’une O-glycosylation capable de moduler considérablement son activité à la surface cellulaire [Manuscrit en préparation]. Enfin, et en collaboration avec l’'institut Karolinska (Stockholm, Sweden), une éventuelle implication de Sulf-1 dans le mésothéliome malin a été suggéré (Heidari-Hamedani et al., Cellular Signalling J. 2015). / Heparan sulfate (HS) is a polysaccharide able to bind and modulate a wide variety of proteins. HS large interactive properties are essentially governed by precise sulfation patterns of the polysaccharide. Sulf-1 and Sulf-2 are two endosulfatases able to cleave specific 6-O sulfate groups within HS chains. Their action can modulate critical intracellular signaling processes, many of which with key relevance for cancer development. However, little is known on their structure, substrate specificities, and on the way their catalytic activity directs the subtle modifications of HS 6-O-sulfation profile.In this work, we have identified an original enzymatic mechanism by which Sulfs catalyze the processive and orientated desulfation of HS and finely regulate the polysaccharide biological properties. We also identified two basic motifs within the N-terminal domain of HSulf-2. Recombinant enzymes mutated for these sites were then produced and characterized. We found that these epitopes are necessary for its endosulfatase activity. Altogether, these results provide further insights into the enzyme/substrate recognition process and contribute to the understanding of the fundamental role played by these enzymes in the regulation of HS activity. The biochemical study of the purified HSulf-2 allowed to highlight a O-glycosylation able to significantly modulate its activity at the cell surface. Otherwise, and in collaboration with the Karolinska Institute (Stockholm, Sweden), the study of malignant mesothelioma (MM) provided new evidence that enhanced Syndecan-1 expression also finely modulates HS structure by interfering with HS-modifying enzymes HSulf-1. These results suggest important roles for Syndecan-1 and HSulf-1 in MM.

La paroi végétale est une structure complexe composée principalement de polysaccharides (cellulose, hémicellulose et pectine), de lignine et de protéines. Elle est impliquée dans de nombreuses fonctions essentielles à la vie de la cellule végétale. De plus, les constituants de cette paroi, que sont les polysaccharides et la lignine, représentent la plus grande source de carbone renouvelable de la planète. Ceci en fait des cibles de choix notamment pour la production d'énergies « vertes ». Toutefois, l'utilisation des polysaccharides tels que les hémicelluloses constituant la paroi végétale reste, à l'heure actuelle, limitée du fait de la difficulté à les dégrader. Ces dernières années, un effort important a été mis en œuvre pour identifier et caractériser de nouvelles enzymes, telles que les glycosides hydrolases, permettant de dégrader efficacement la biomasse végétale. Dans le but de découvrir de nouvelles enzymes impliquées dans la dégradation de la biomasse végétale, des chercheurs de l'équipe « Catalyse et Ingénierie Moléculaire Enzymatiques » du LISBP ont décidé d'explorer le métagénome d'organismes connus pour dégrader la biomasse végétale. Deux espèces animales ont fait l'objet d'analyses : tout d'abord les termites qui sont considérés comme les champions de la dégradation de la biomasse végétales et souvent comparés à des bioréacteurs, et le ver de terre. Des banques métagénomiques de trois espèces différentes de termites ainsi qu'une banque métagénomique de ver de terre ont ainsi été créées. Dans ces travaux de thèse deux des banques métagénomiques de termites, celle de Nasutitermes corniger et celle de Termes hispaniolae, ont fait l'objet d'une étude afin de comparer le potentiel hémicellulolytique de ces deux espèces. Après sélection de nombreux clones positifs sur substrats chromogéniques de chacune des deux banques, séquençage puis annotation taxonomique et fonctionnelle, un grand nombre d'enzymes et principalement des glycosides hydrolases, a pu être identifié. Les résultats montrent que le métagénome de Nasutitermes corniger présente majoritairement des enzymes à activité endoglycosidase alors que le métagenome de Termes hispaniolae possède plutôt des enzymes à activité exoglycosidase. Toutes les activités trouvées dans chacune des espèces de termite sont en bonne corrélation avec l'alimentation du termite. De plus, nous avons observé que le microbiote intestinal des deux termites ne possèdent pas les mêmes embranchements bactériens majoritaires et nous avons pu voir que le microbiote de Termes hispaniolae est plus diversifié ce qui corrèle aussi avec l'alimentation des deux termites. D'autre part, dans la banque métagénomique du ver de terre, l'annotation fonctionnelle a révélé une enzyme intéressante. Il s'agit d'une enzyme annotée par B. Henrissat (responsable de la base de données CAZy) comme étant une glycoside hydrolase putative mais n'appartenant à aucune des 135 familles de glycosides hydrolases existantes. Cette enzyme putative, appelée GH* présente des similitudes avec les GH de la famille 5 sans pour autant appartenir à cette famille du fait notamment de l'absence du résidu catalytique nucléophile conservé. Une étude structurale et fonctionnelle de GH* a donc été menée. Les expériences ont permis de prouver que GH* est une endo-xylanase ayant une préférence pour les arabinoxylanes et les xylooligosaccharides de degré de polymérisation d'au moins 5 ou 6. La structure tridimensionnelle de GH* à 1,6Å de résolution a été obtenue par cristallographie des rayons X par remplacement moléculaire à l'aide d'une GH5. Cette structure a permis de confirmer l'identité du résidu acide/base identifié par alignement de séquences et d'émettre une hypothèse sur l'identité du résidu nucléophile. Enfin des mutants de GH* pour ces deux résidus ont été obtenus et ont confirmé leur implication dans l'activité de l'enzyme. / Plant cell wall is a complex structure surrounding plant cells mainly composed by polysaccharides (cellulose, hemicellulose and pectin), lignin and proteins. The plant wall maintains and imposes the size and shape of cells. It is also important for exchanges between cells and extra cellular medium. The polysaccharides of this cell wall are the largest renewable carbon source on the earth, which makes them good targets to produce green energies. Because plant cell wall is difficult to degrade, its use for biofuels for is still limited. However, some organisms are able to efficiently degrade this biomass. Exploring the diversity of the living word to discover new effective biocatalysts has grown considerably last years, because of the emergence of metagenomics. In this context and to discover new enzymes involved in the degradation of plant biomass, the team « Catalyse et Ingénierie Moléculaire Enzymatiques » of LISBP decided to explore metagenome of organisms known to degrade plant biomass. Two animal families were chosen for metagenomics analysis, the termite and earthworm. Metagenomics banks of three different species of termite and one metagenomics bank of an earthworm were created. In this thesis project, two of the three metagenomics banks of termites, the one from Nasutitermes corniger and the other one from Termes hispaniolae, were studied to compare the hemicellulolytic potential of these two species. After selection of many positive clones on chromogenic substrates of both banks, sequencing, taxonomic and functional annotations, a large number of enzymes and mainly glycoside hydrolases, could be identified. The results obtained shown that the trends observed during functional screens were maintained. Indeed, it appears that Nasutitermes corniger has a majority of endoglycosidases while Termes hispaniolae has mainly exoglycosidases. Thereby, families of enzymes highlighted allowed correlating their hydrolytic activities with the diet of these species. Furthermore, we observed that the intestinal microbiota of each termite is different. Indeed, both termites do not have the same majority bacterial phyla and the microbiota of Termes hispaniolae is more diverse than the one of Nasutitermes corniger. On the other hand, functional annotation of the metagenomics bank of the earthworm revealed an enzyme annotated as a glycoside hydrolase no belonging to any of the 135 glycoside hydrolase existing families. This enzyme, named GH*, seems to be close to GH5 but does not shown the nucleophilic catalyst residue perfectly conserved in this glycoside hydrolase family. A functional and structural study of GH* was then done. We have shown that GH* is an endo-xylanase which prefers arabinoxylans and xylooligosaccharides having a polymerization degree greater than 5. In addition, we determined the crystal structure of GH* at 1.6Å resolution. This 3D structure has confirmed the presence of the acid/base residue identified by sequence alignment and allowed us to hypothesize about the identity of the nucleophilic residue. Finally, mutants of GH* for these two residues were obtained and confirmed their involvement in the activity of the enzyme. We were able to progress in the understanding of structure/function relationships of this protein.

Der Komplex aus Kohlenmonoxid-Dehydrogenase und Acetyl-CoA-Synthase (CODH/ACS Komplex) des thermophilen Bakteriums Carboxydothermus hydrogenoformans katalysiert die Fixierung von CO2 in Acetyl-CoA und ist damit ein potenzieller Katalysator für die Erzeugung erneuerbarer Kraftstoffe aus CO2. Die Katalyse erfolgt an zwei verschiedenen Stellen: CO2 wird am Cluster C in der CODH-Untereinheit zu CO reduziert, das dann durch einen Tunnel innerhalb des Proteins zum Cluster A in der ACS-Untereinheit wandert, wo es mit einer Methylgruppe und CoA zu Acetyl-CoA reagiert. Die Art und Weise, wie die beiden katalytischen Aktivitäten zusammenwirken, sind noch unklar. Um hier mehr Licht ins Dunkel zu bringen, verfolgte diese Arbeit drei Ziele: die Bestimmung der Struktur des CODH/ACS-Komplexes von C. hydrogenoformans, die Untersuchung der CO2-Reduktionsaktivität von CODHasen und die Analyse der Rolle des internen Tunnels im CODH/ACS-Komplex. Die Struktur des CODH/ACS-Komplexes von C. hydrogenoformans wurde durch Röntgenkristallographie mit einer Auflösung von 2,04 Å bestimmt. Die CO2-Reduktion am Cluster C wurde kinetisch untersucht. Es zeigte sich, dass die CO2-Reduktion durch einen Ping-Pong-Mechanismus mit zwei Reaktionsstellen erfolgen könnte, der in früheren Studien vorgeschlagen wurde, aber auch durch andere Mechanismen. Um eine Struktur-Funktionsbeziehung für CODHs zu ermitteln, wurde die CO2-Reduktionsaktivität für drei CODHasen von C. hydrogenoformans untersucht, deren Strukturen bekannt sind: CODH-II, CODH-IV, und der CODH/ACS-Komplex. Das Tunnelsystem im CODH/ACS-Komplex ist viel enger als in den anderen CODHs und könnte somit der Grund für die vergleichsweise geringe Aktivität des CODH/ACS-Komplexes sein. Dies wurde auch durch die Manipulation und Analyse des internen Tunnels des CODH/ACS-Komplexes unterstützt. Die Ergebnisse deuten darauf hin, dass der Hauptzweck des Tunnels im CODH/ACS-Komplex die Kompartimentierung von CO und nicht der schnelle Substrattransport ist. / The complex of carbon monoxide dehydrogenase and acetyl-CoA synthase (CODH/ACS complex) of the thermophilic bacterium Carboxydothermus hydrogenoformans catalyses the fixation of CO2 into acetyl-CoA and is thus a potential catalyst for the production of renewable fuels from CO2. Catalysis occurs at two different sites: CO2 is reduced to CO at cluster C in the CODH subunit, which then travels through a tunnel within the protein to cluster A in the ACS subunit, where it reacts with a methyl group and CoA to form acetyl-CoA. The way in which the two catalytic activities interact is still unclear. To shed more light on this, this work pursued three goals: to determine the structure of the CODH/ACS complex of C. hydrogenoformans, to investigate the CO2 reduction activity of CODHases and to analyse the role of the internal tunnel in the CODH/ACS complex. The structure of the CODH/ACS complex of C. hydrogenoformans was determined by X-ray crystallography at 2.04 Å resolution. The CO2 reduction at cluster C was investigated kinetically. It was found that CO2 reduction could occur by a two-site ping-pong mechanism proposed in previous studies, but also by other mechanisms. To establish a structure-function relationship for CODHs, CO2 reduction activity was investigated for three CODHases of C. hydrogenoformans whose structures are known: CODH-II, CODH-IV, and the CODH/ACS complex. The tunnel system in the CODH/ACS complex is much narrower than in the other CODHs and could thus be the reason for the comparatively low activity of the CODH/ACS complex. This was also supported by the manipulation and analysis of the internal tunnel of the CODH/ACS complex. The results suggest that the main purpose of the tunnel in the CODH/ACS complex is to compartmentalise CO and not to rapidly transport substrate.

Les trois projets que j’ai développés au cours de ma thèse s’inscrivent dans un même contexte, celui de l’étude de l’acétylcholinestérase (AChE) et des molécules apparentées à l’AChE au plan structural ou fonctionnel. Existence, identification et caractérisation du fonctionnement d’une « porte arrière » dans l’AChE. Caractérisation de la « protéine X », un récepteur non AChE de la fasciculine. Recherche d’un partenaire protéique endogène de l’AChE dans le cerveau de rat. / The three projects I developed during my thesis are related to the study of acetylcholinesterase (AChE) and of molecules related to AChE in their function or structure. Existence, identification and characterization of the functioning of a back door in AChE. Characterization of “protein X”, a non-AChE receptor for fasciculin. Search for an endogenous proteic partner of AChE in rat brain.