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Autophagy plays an important role in immunity by delivering peptides for the antigen presentation. Among the effectors of autophagy, the ATG8 proteins have been described to play a key role in the elongation and maturation of the autophagosome, before its fusion with the lysosome. The mammalian ATG8 family comprises the LC3 subfamily (LC3A, B and C) and the GABARAP subfamily (GABARAP, GABARAPLl and GABARAPL2). ln 2016, a study reported that the fusion between the cancer testis antigen NY-ESO-1 and the N-ter of the autophagy protein LC3B could enhanced NY-ESO-1- restricted CD4+ T cell response. The aim of our study was to demonstrate that GABARAP/GABARAPLl, as well as LCB could be used to target antigens to the autophagie pathway. The OVALBUMIN (OVA) antigen was chosen to take advantage of the OT-1 and OT-11 mice which express a transgenic TCR specific for OVA peptides in their CD4 and CD8 T cells. B16-Fl0 stable cell lines overexpressing OVA, OVA-LC3B, OVA-GABARAP or OVA-GABARAPLl antigens have been established to confirm the ability of the ATG8 proteins to target OVA proteins into the autophagosomes and to present peptides on their class Il MHC molecules. However, B16-OVA-ATG8 overexpressing failed to activate specific CD4+ T cell response. Primary cell cultures of a more physiological antigen presenting cells i.e. bone marrow-derived dendritic cells (DC) were developed to address OVA to the autophagosomes. Here we report for the first time that other members of the ATG8 family proteins, GABARAP and GABARAPLl can target OVA to the autophagosomes and enhanced MHC class Il presentation to activate specific CD4+ T cells. The second aim of our study was to investigate the role of GABARAPLl protein in the presentation of OVA peptides in B16-OVA cells. We generated B16-OVA GABARAPLl-Knock-down (KD) using the CRISPR-Cas9 technology. ln GABARAPLl KD cells, the basal autophagie flux was significantly decreased. When injected into mice, these GABARAPLl KD cells developed a tumour faster in immunocompetent C57BL/6N mice than in immunocompromised (SCID) mice suggesting the involvement of GABARAPLl in the tumour cell proliferation control by the immune system; L'autophagie joue un rôle important dans l'immunité en fournissant des peptides pour la présentation antigénique. Parmi les acteurs essentiels de !'autophagie, les protéines ATG8 jouent un rôle clé dans l'élongation et la maturation de l'autophagosome, avant sa fusion avec le lysosome. La famille ATG8 chez les mammifères comprend la sous-famille LC3 et la sous-famille GABARAP. En 2016, une étude a montré que la fusion de l'antigène de tumeur NY-ESO-1 à la partie N-ter de ATG8/LC3B pouvait améliorer la réponse des lymphocytes T (LT) CD4+ restreints à NY-ESO-1. Le premier objectif de la thèse était de démontrer que GABARAP et GABARAPLl, comme LCB3B, pouvaient être utilisés pour cibler un antigène sur la voie autophagique pour favoriser la présentation peptidique par les molécules de CMH de classe Il. L'OVALBUMINE (OVA} a été choisie comme antigène afin de tirer parti des souris transgéniques pour leur récepteur à l'antigène OT-1 et OT-11 qui stimulent respectivement les LT CD8+ et LT CD4+ spécifiques de peptides issus de OVA. Des lignées cellulaires stables de cellules tumorales de mélanome murin B16-Fl0 surexprimant les antigènes OVA, OVA-LC3B, OVA­GABARAP ou OVA-GABARAPLl ont été établies et utilisées pour activer des LT CD4+ OT-11 spécifiques du peptide OVA. Toutefois, la surexpression de l'antigène OVA-ATG8 dans les cellules B16 n'a pas permis de déclencher une réponse CD4+. Des cellules dendritiques dérivées de moelle osseuse (DC) en culture primaire constituant un modèle physiologique de présentation antigénique ont été choisies par la suite pour adresser OVA aux autophagosomes. Dans ce modèle, les protéines GABARAP et GABARAPLl comme LC3B peuvent cibler l'antigène OVA aux autophagosomes et augmenter sa présentation par les molécules du CMH-11 améliorant la réponse CD4. Le deuxième objectif de cette thèse a été d'étudié le rôle de la protéine GABARAPLl exprimée par les cellules B16-OVA dans la croissance tumorale. L'expression de GABARAPLl a été invalidée dans ces cellules par la technologie CRISPR-Cas9 et le rôle spécifique de GABARAPLl dans la présentation des antigènes par les protéines du CMH-11 a été étudié. Chez les cellules B16-OVA GLl-/-, le flux autophagique basal est significativement diminué. Ces cellules ont montré une augmentation significative de leur croissance tumorale dans des souris immunocompétentes C57BL/6N mais pas dans des souris immunodéprimés (SCID) suggérant un rôle de GABARAPLl dans l'implication du système immunitaire contrôlant la prolifération cellulaire

Autophagy plays an important role in immunity by delivering peptides for the antigen presentation. Among the effectors of autophagy, the ATG8 proteins have been described to play a key role in the elongation and maturation of the autophagosome, before its fusion with the lysosome. The mammalian ATG8 family comprises the LC3 subfamily (LC3A, B and C) and the GABARAP subfamily (GABARAP, GABARAPLl and GABARAPL2). ln 2016, a study reported that the fusion between the cancer testis antigen NY-ESO-1 and the N-ter of the autophagy protein LC3B could enhanced NY-ESO-1- restricted CD4+ T cell response. The aim of our study was to demonstrate that GABARAP/GABARAPLl, as well as LCB could be used to target antigens to the autophagie pathway. The OVALBUMIN (OVA) antigen was chosen to take advantage of the OT-1 and OT-11 mice which express a transgenic TCR specific for OVA peptides in their CD4 and CD8 T cells. B16-Fl0 stable cell lines overexpressing OVA, OVA-LC3B, OVA-GABARAP or OVA-GABARAPLl antigens have been established to confirm the ability of the ATG8 proteins to target OVA proteins into the autophagosomes and to present peptides on their class Il MHC molecules. However, B16-OVA-ATG8 overexpressing failed to activate specific CD4+ T cell response. Primary cell cultures of a more physiological antigen presenting cells i.e. bone marrow-derived dendritic cells (DC) were developed to address OVA to the autophagosomes. Here we report for the first time that other members of the ATG8 family proteins, GABARAP and GABARAPLl can target OVA to the autophagosomes and enhanced MHC class Il presentation to activate specific CD4+ T cells. The second aim of our study was to investigate the role of GABARAPLl protein in the presentation of OVA peptides in B16-OVA cells. We generated B16-OVA GABARAPLl-Knock-down (KD) using the CRISPR-Cas9 technology. ln GABARAPLl KD cells, the basal autophagie flux was significantly decreased. When injected into mice, these GABARAPLl KD cells developed a tumour faster in immunocompetent C57BL/6N mice than in immunocompromised (SCID) mice suggesting the involvement of GABARAPLl in the tumour cell proliferation control by the immune system, L'autophagie joue un rôle important dans l'immunité en fournissant des peptides pour la présentation antigénique. Parmi les acteurs essentiels de !'autophagie, les protéines ATG8 jouent un rôle clé dans l'élongation et la maturation de l'autophagosome, avant sa fusion avec le lysosome. La famille ATG8 chez les mammifères comprend la sous-famille LC3 et la sous-famille GABARAP. En 2016, une étude a montré que la fusion de l'antigène de tumeur NY-ESO-1 à la partie N-ter de ATG8/LC3B pouvait améliorer la réponse des lymphocytes T (LT) CD4+ restreints à NY-ESO-1. Le premier objectif de la thèse était de démontrer que GABARAP et GABARAPLl, comme LCB3B, pouvaient être utilisés pour cibler un antigène sur la voie autophagique pour favoriser la présentation peptidique par les molécules de CMH de classe Il. L'OVALBUMINE (OVA} a été choisie comme antigène afin de tirer parti des souris transgéniques pour leur récepteur à l'antigène OT-1 et OT-11 qui stimulent respectivement les LT CD8+ et LT CD4+ spécifiques de peptides issus de OVA. Des lignées cellulaires stables de cellules tumorales de mélanome murin B16-Fl0 surexprimant les antigènes OVA, OVA-LC3B, OVA­GABARAP ou OVA-GABARAPLl ont été établies et utilisées pour activer des LT CD4+ OT-11 spécifiques du peptide OVA. Toutefois, la surexpression de l'antigène OVA-ATG8 dans les cellules B16 n'a pas permis de déclencher une réponse CD4+. Des cellules dendritiques dérivées de moelle osseuse (DC) en culture primaire constituant un modèle physiologique de présentation antigénique ont été choisies par la suite pour adresser OVA aux autophagosomes. Dans ce modèle, les protéines GABARAP et GABARAPLl comme LC3B peuvent cibler l'antigène OVA aux autophagosomes et augmenter sa présentation par les molécules du CMH-11 améliorant la réponse CD4. Le deuxième objectif de cette thèse a été d'étudié le rôle de la protéine GABARAPLl exprimée par les cellules B16-OVA dans la croissance tumorale. L'expression de GABARAPLl a été invalidée dans ces cellules par la technologie CRISPR-Cas9 et le rôle spécifique de GABARAPLl dans la présentation des antigènes par les protéines du CMH-11 a été étudié. Chez les cellules B16-OVA GLl-/-, le flux autophagique basal est significativement diminué. Ces cellules ont montré une augmentation significative de leur croissance tumorale dans des souris immunocompétentes C57BL/6N mais pas dans des souris immunodéprimés (SCID) suggérant un rôle de GABARAPLl dans l'implication du système immunitaire contrôlant la prolifération cellulaire

National audience; Changing the structure of foods can modulate their nutritional quality by modifying the digestion process, a complex process that is still imperfectly understood. In particular, digestion dynamics have been poorly studied, although it can has significant metabolic consequences. The objective of this study is to model the proteolytic cascade by pepsin, and to highlight a potential "structural effect". Egg white is an interesting model to meet this dual objective, as it offers the possibility of obtaining gels with different structures. In a probabilistic modeling approach of the peptide cleavage dynamics, we sought to identify leverages for the cleavage of a peptide after a given digestion time. For this purpose, peptides are identified and quantified at different time points in an in vitro digestion experiment. A Generalized Additive Modeling of the probability of a cleavage based on the physicochemical profile of peptides is proposed, considering either all kinds of cleavages made by the pepsin or only those made on preferential peptide bonds. The most significant variables are related to the length of the peptide and its location on the ovalbumin sequence. These results suggest that the action of pepsin depends more on structural criteria than on the presence of specific cleavage sites.

RésuméThe objective of this thesis is the fabrication of a magnetic microdevice for the detection and manipulation of biological elements based on magnetic nanoparticles under microfluidic conditions. It aims to integrate basic functions of control and magnetic detection, to achieve specific, stable, fast and reproducible measurements. Indeed, the immunoassay technique coupled to magnetic nanoparticles, well known in the literature, requires a control of the displacement of magnetic nanoparticles to effectively functionalize them and create a biological complex to encapsulate a target molecule (biomarker). In our case, a model molecule for the field of biodefense was used: ovalbumin. To control the magnetic field which is necessary for the capture of magnetic complexes, we opted for the use of microcoils integrated in the fluidic devices and compared this original technique with other more conventional ones. To detect a biological complex, fluorescence is widely used in biology, but this technique does not allow a complete integration for an autonomous device. In this context, we propose the detection of complexes based on magnetic nanoparticles by observing the variation of the inductance of a magnetic microcircuit closing a microfluidic chamber containing these immunological complexes. The design of the control microcoils by simulation made it possible to determine the parameters allowing to obtain the most adapted magnetic field to the control of the biological complexes. In the case of microcoils used for detection, micrometric magnetic branches were inserted around the microcoils to create an even more sensitive magnetic sensing circuit. The realization of these devices involved the integration of materials and structures of highly heterogeneous nature, and their assembly has required to solve many technological locks. The challenge was to determine all the successive and necessary steps for a reliable and reproducible microfabrication process. To show the interest of magnetic nanoparticle capture devices, immunoassays were first performed in microtubes to compare with those made in a fluid circuit using external magnet and integrated microcoils. In the latter case, considerable optimization has been validated in terms of reduction of incubation time, reproducibility of measurements and detection limits equivalent to the state of the art for ovalbumin. For the magnetic detection device, first experiments of electrical characterization as well as concentration studies of magnetic nanoparticles were carried out and compared to the results obtained by simulation. For the proof of concept, a demonstrator of detection of magnetic complexes was also finalized validating the possibility of integration of the magnetic microcircuit in a fluidic device. It has also validated obtaining a remarkable range of sensitivity correlated with the presence of magnetic complexes. Its characteristics were compared to those obtained by the simulations and discussed taking into account all the critical steps of the microfabrication process.; L’objectif de cette thèse est la fabrication d’un microdispositif magnétique pour la détection et la manipulation d’éléments biologiques à base de nanoparticules magnétiques en conditions microfluidiques. Il a pour but d’intégrer des fonctions de base de contrôle et détection magnétique, pour atteindre des mesures spécifiques, stables, rapides et reproductibles. En effet, la technique d’immunodosage couplée à des nanoparticules magnétiques, bien connue dans la littérature, nécessite un contrôle du déplacement de ces dernières pour les fonctionnaliser efficacement et créer un complexe biologique encapsulant une molécule cible (biomarqueur). Dans notre cas une molécule modèle pour le domaine de la biodéfense a été utilisée : l’ovalbumine. Pour contrôler le champ magnétique nécessaire pour la capture des complexes magnétiques, nous avons opté pour l’utilisation de microbobines intégrées aux dispositifs fluidiques et comparé cette technique originale avec d’autres plus conventionnelles. Pour détecter un complexe biologique, la fluorescence est largement utilisée en biologie, mais cette technique ne permet pas une intégration complète pour un dispositif autonome. Dans cette optique, nous proposons la détection des complexes à base de nanoparticules magnétiques en relevant la variation de l’inductance d’un microcircuit magnétique refermant une chambre microfluidique contenant ces complexes immunologiques. Le dimensionnement des microbobines de contrôle par simulation a permis de déterminer les paramètres permettant d’obtenir le champ magnétique le plus adapté au contrôle des complexes biologiques. Dans le cas des microbobines utilisées pour la détection, des branches magnétiques micrométriques ont été insérées autour des microbobines pour créer un circuit de détection magnétique encore plus sensible. La réalisation de ces dispositifs a impliqué l’intégration de matériaux et de structures de nature fortement hétérogène, et leur assemblage a nécessité de résoudre de nombreux verrous technologiques. L’enjeu a été de déterminer l’ensemble des étapes successives et nécessaires pour un procédé de microfabrication fiable et reproductible. Pour montrer l’intérêt des dispositifs de capture des nanoparticules magnétiques, des tests immunologiques ont été réalisés tout d’abord en microtubes pour les comparer à ceux réalisés dans un circuit fluidique à l’aide d’aimant externe puis de microbobines intégrées. Dans ce dernier cas, une optimisation considérable a été validée en termes de réduction de temps d’incubation, de reproductibilité des mesures et de limites de détection équivalentes à l’état de l’art pour l’ovalbumine. Pour le dispositif de détection magnétique, des premières expériences de caractérisation électrique ainsi que des études en concentration de nanoparticules magnétiques ont été réalisées et comparées aux résultats obtenus par simulation. Pour la preuve de concept, un démonstrateur de détection de complexes magnétiques a été également finalisé validant la possibilité d’intégration du microcircuit magnétique dans un dispositif fluidique. Il a validé également l’obtention d’une gamme de sensibilité remarquable corrélée à la présence des complexes magnétiques. Ses caractéristiques ont été confrontées à celles obtenues par les simulations et discutées en tenant compte de toutes les étapes critiques du procédé de microfabrication.

International audience; From their amphiphilic nature, proteins in solution adsorb spontaneously at hydrophobic interfaces. Proteins are thus able to change their conformation, which modifies their surface properties. In the confined space near the interface, these modifications favor the formation of many interactions of weak energy (hydrophobic interactions, hydrogen bonds, Van der Waals interactions), and thus the creation, in the interface plane, of a network of intermolecular interactions. Adsorbed proteins can thus, in favorable conditions, constitute at the interface a continuous film with visco-elastic properties. These mechanical properties contribute to the stabilization of the interface created in multiphasic systems such as foams. Our work focuses on the behavior at the air-water interface and, if needed the foam properties, of some model globular proteins: ovalbumin, lysozyme, ovotransferrin, β-lactoglobulin… The aim of our work is to understand, using these models, how protein properties (structure, biochemistry, physico-chemistry), potentially adjusted by the physico-chemical environment, chemical changes or thermo-mechanical treatments, take part in the interfacial behavior, film characteristics and macroscopic proprieties of foams (overrun, stability, texture). Besides, in systems where several proteins with different properties (size, hydrophobicity, charge, conformation flexibility,…) coexist, we look to understand how the interactions between proteins enhance behaviors that cannot be considered as the sum of the single behaviors. The issue over time is to control technological parameters to improve texture properties in relation to foaming capacity.

Résumé : Les phases concentrées de protéines sont au centre de nombreuses études visant à identifier et caractériser les interactions et transitions de phases mises en jeu, en utilisant le large corpus de connaissances acquis sur les phases concentrées de colloïdes. Ces phases concentrées de protéines possèdent en outre une grande importance dans des domaines aussi variés que l’industrie agroalimentaire, l’industrie pharmaceutique et la médecine. L’établissement d’équations d’état présentant la pression osmotique (Π) en fonction de la fraction volumique (Φ) est une méthode efficace de caractérisation des interactions entre les composants d’un système. Nous l’avons appliquée à des solutions de deux protéines globulaires, le lysozyme et l’ovalbumine, en balayant une gamme de fractions volumiques allant d’une phase diluée (Φ < 0,01) à une phase concentrée, solide (Φ > 0,62). Les équations d’état obtenues, couplées à d’autres techniques (SAXS, simulations numériques), ont permis de mettre en évidence un comportement très différent des deux protéines lors de la concentration et ont montré leur complexité en comparaison avec des colloïdes modèles. La mise en relation des équations d’état et du comportement interfacial de ces deux protéines a montré des points de convergence et permis de formuler une nouvelle hypothèse expliquant certaines observations portant sur l’adsorption des protéines à l’interface air-eau., Concentrated phases of proteins are the subject of numerous studies aiming at identifying and characterizing the interactions and phase transitions at play, using the large corpus of knowledge in the field of concentrated colloids. Those concentrated phases of proteins have, in addition, a great importance in various fields, such as food industry, pharmaceutical industry and medicine. The establishment of equations of state relating osmotic pressure (Π) and volume fraction (Φ) is an efficient way of characterization of the interactions between the components of a system. We applied this method to solutions of two globular proteins, lysozyme and ovalbumin, spanning volume fractions ranging from a dilute phase (Φ < 0,01) to a concentrated, solid phase (Φ > 0,62). The equations of state, coupled to other methods (SAXS, numerical simulations), enabled us to show that the two proteins carry a very different behavior when submitted to concentration and that their complexity is beyond that of colloids. Relating equations of state and interfacial behavior of these two proteins also showed points of convergence and enabled us to formulate a new hypothesis which explains some of the results obtained in the study of adsorption of proteins at the air-water interface.

National audience; Les protéines amphiphiles se concentrent aux interfaces avec des fractions volumiques localement élevées. Ce mécanisme est le principal stabilisateur des interfaces des milieux divisés (mousses, émulsions). La nature attractive ou répulsive des interactions entre protéines est modulée par la charge de ces dernières et nous avons émis l’hypothèse que la couche interfaciale est affectée par la nature de l’interaction. Les protéines utilisées pour notre étude sont des protéines basiques (lysozyme, avidine) et acides (ovalbumine, serum-albumine bovine, alpha-lactalbumine) et nous avons modifié leur charge par le pH de la solution tampon.Nous avons mis en évidence, par suivi ellipsométrique de la quantité de protéine adsorbée à l’interface eau-air, que lorsque les interactions protéine-protéine sont répulsives, une monocouche se forme à l’interface alors que des interactions de nature attractives conduisent à la formation de multicouches. Il semble que dans ce dernier cas l’advection soit un ingrédient nécessaire.

This study aimed to investigate the impact of protein aggregate structure on the nutritional valueof proteins, and especially on the extent of digestion, the nature and amount of peptides released.Ovalbumin was chosen as a model protein source. A range of four typical aggregate structures,from linear to spherical-agglomerated, were obtained after heating under different combinationsof pH and ionic strength. The non-aggregated and aggregated ovalbumins were digested usingan in vitro digestion model that simulated gastrointestinal digestion. The extent of digestion wasdetermined based on the disappearance of intact ovalbumin and the appearance of solublepeptides. The peptide profile of the digests was analyzed using LC-MS/MS.The extent of hydrolysis differed according to the aggregate structure with linear aggregatesbeing more extensively hydrolyzed than the spherical aggregates. The results suggest that thesurface area to volume ratio, and probably the degree of unfolding of its constituent proteinbefore ingestion are the major influencing criteria. Ovalbumin aggregation appeared to render anumber of peptide bonds accessible to digestive proteases, whereas these bonds were notaccessible in the native ovalbumin; moreover, cleavage sites appeared to be specific dependingon the structure of aggregates (fig.1). Nutritional peptidomics analysis by multivariate statisticalapproaches made it possible to connect the aggregate structure and the profile of generatedpeptides. For instance, it was possible to demonstrate that bioactive peptides were significantlypromoted after digestion of spherical and spherical-agglomerated aggregates.This work highlights the existing links between food structures resulting from technologicalprocesses and their breakdown during the digestive process. Such results imply that a fine tuningof unfolding and aggregation conditions can be used for targeted structuring that can lead to animprovement of the nutritional quality of food proteins.

This study aimed to investigate how food structure, in the form of different ovalbumin aggregate morphologies,impacted the proteolysis of ovalbumin using an in vitro model that simulated digestion in the adult gastrointes-tinal tract. Four different aggregate morphologies were prepared by heating ovalbumin solution using differentcombinations of pH and ionic strength. Quantitative peptidomics (label-free) and multivariate data analysis ofthe resulting in vitro digests were performed. The 593 identified peptides were distributed in 6 homogeneousclusters based on the relative amount of peptide release from the different aggregate morphologies. Each clustergatheredpeptideswithcommonphysicochemicalcharacteristics.Theresultssuggestthatpepticandchymotryp-tic cleavages were favored by aggregation regardless of the aggregate morphology, while tryptic cleavages werefavored when ovalbumin aggregates were spherical-agglomerated. It is notable that even after extensive diges-tion, the initial aggregate morphology in fluenced the amount of each peptide released

Food structure can have a major impact on digestion in the gastrointestinal tract. Moreover, the structure of food proteins, which are typically consumed within complex food matrices, can be modified during food processing. Protein aggregation is one such modification and in the present study, the relationship between thermally-induced aggregated structures and in vitro digestion was explored using ovalbumin (the major egg white protein) as a food protein model. In order to explore the relationship between the structure of ovalbumin aggregates with their behavior during in vitro digestion, four different thermally-induced ovalbumin aggregates were prepared. The digestion of the latter aggregates and the resulting peptides profiles of the digests were investigated using an in vitro two-step model (stomach and small intestine mimicking the adult gastrointestinal tract). Different combinations of pH and ionic strength (IS; pH 5/IS 0.8M, pH 7/IS 0.3M, pH 7/IS 0.03M, pH 9/IS 0.03M) were used to generate different aggregate structures. These structures were characterized using light scattering methods, transmission electronic microscopy (TEM) and SDS-PAGE electrophoresis. Aggregate structures varied from random to linear, with particle size distributions (D[4;3]) ranging from 16nm to 90μm for linear and random aggregates, respectively. After in vitro simulated gastrointestinal tract digestion, the degree of hydrolysis of the linear aggregates was almost twice that of the random aggregates or the native ovalbumin. The overall peptide profiles of the digests of the untreated OVA and the random and linear OVA aggregate preparations were compared using spectral principal component analysis of reverse-phase HPLC chromatograms for each sample. Three distinct clusters were identified which corresponded to untreated OVA, linear aggregates and random aggregates treatments respectively. Consequently, based on an in vitro digestion model, it would appear that the type of protein aggregates present in a processed food may impact the extent and the nature of protein digestion in the gastrointestinal tract.

Food structure can have a major impact on digestion in the gastrointestinal tract. Moreover, the structure of food proteins, which are typically consumed within complex food matrices, can be modified during food processing. Protein aggregation is one such modification and in the present study, the relationship between thermally-induced aggregated structures and in vitro digestion was explored using ovalbumin (the majoregg white protein) as a food protein model. In order to explore the relationship between the structure of ovalbumin aggregates with their behavior during in vitro digestion, four different thermally-induced ovalbuminaggregates were prepared. The digestion of the latter aggregates and the resulting peptides profiles of the digests were investigated using an in vitro two-step model (stomach and small intestine mimicking the adult gastrointestinal tract). Different combinations of pH and ionic strength (IS; pH 5/IS 0.8M, pH 7/IS 0.3M, pH7/IS 0.03M, pH 9/IS 0.03M) were used to generate different aggregate structures. These structures were characterized using light scattering methods, transmission electronic microscopy (TEM) and SDS-PAGE electrophoresis. Aggregate structures varied from random to linear, with particle size distributions (D[4;3]) ranging from 16nm to 90μm for linear and random aggregates, respectively. After in vitro simulated gastrointestinal tract digestion, the degree of hydrolysis of the linear aggregates was almost twice that of the random aggregates or the native ovalbumin. The overall peptide profiles of the digests of the untreated OVA and the random and linear OVA aggregate preparations were compared using spectral principal componentanalysis of reverse-phase HPLC chromatograms for each sample. Three distinct clusters were identified which corresponded to untreated OVA, linear aggregates and random aggregates treatments respectively.Consequently, based on an in vitro digestion model, it would appear that the type of protein aggregates present in a processed food may impact the extent and the nature of protein digestion in the gastrointestinal tract.

Ovalbumin aggregation due to heating in solution increases its in vitro digestibility, compared to the native and dry-heated protein. 1 st Food Structures, Digestion & Health Conference

The avian egg, which contains the egg yolk, the egg white, and the eggshell, represents the mostly advanced amniotic egg in oviparous vertebrates. In mammals, this reproductive strategy of laying egg has gradually evolved toward placentation. In order to better understand the unique status of the avian egg in the evolution of the vertebrate reproduction, we investigated the evolution of some Gallus gallus egg-specific protein-coding genes. Based on our finding and other recent research, we have summarized here that gene formation (such as ovalbumin genes, ovocalyxin-36 and apovitellenin-1 encoding genes in the G. gallus), gene divergence between G. gallus and mammals (such as the ovocalyxin-32 gene with its ortholog, the mammalian RARRES1, and the ovocleidin-116 with its ortholog, the mammalian MEPE), and gene loss (egg-expressed genes lost during the evolution of the mammals, such as vitellogenin and RBP encoding genes) play significant roles in the evolution of egg-specific genes.

Dry heating is performed in egg product industries to pasteurise egg white powder. This treatment (55 to 80°Cduring a few days) is also used to improve egg white powder functional properties among other foamingproperties. Several studies have shown this foaming properties‟ improvement with dry heating length (Kato etal, 1989; Baron et al, 2003; Van der Plancken et al, 2007; Talansier et al, 2009) that Kato et al (1989) attributedto protein surface hydrophobicity increase. However, during these treatments, soluble and insoluble covalentaggregates were also generated (Kato, 1989; Van der Plancken et al, 2007; Talansier et al, 2009) that may beinvolved in foaming properties improveme nt. Conclusions from such a complex protein solution as egg whiteare difficult to draw; this is the reason why we choose ovalbumin, the major egg white protein (54% of totalprotein amount) and lysozyme (one of the most famous model protein) to identify the molecular speciesgenerated by dry heating that are responsible for foaming properties‟ improvement. Ovalbumin and lysozymefoaming properties are improved after dry heating (Kato, 1990a) and the protein undergoes some mildconformational changes close to the molten globule state as well as aggregation driven by hydrophobicinteractions and disulfide bonding (Kato, 1990b; Matsudomi, 2001). The present study has been performed toidentify the molecular species generated by dry heating responsible for foam ing properties improvement. Mostof the data of the literature were confirmed as we found that ovalbumin and lysozyme aggregated and thatdeamidation occurred. We also identified less negatively charged ovalbumin that we attributed todephosphorylation. We performed surface pressure and ellipsometric angle measurement on dry heatedovalbumin but also on dephosphorylated, desamidated and aggregated forms. Dry heated ovalbumin andlysozyme show faster adsorption kinetics to air water interface than non-heated one. However the equilibriumsurface pressure and surface concentration are quite close for ovalbumin whereas no equilibrium were reachedfor lysozyme. Shear elastic constant measurement showed higher values for dry heated ovalbumin during thefirst hour but no significant difference after 8 hours. Measurements of dilatational and shear modulus were alsoperformed to complete the data. Foaming properties of the different molecular species were measured whenpossible. This multidimensional approach helped in the understanding of the characteristic of the interfacial filmthat explain foaming properties. It seems that more than the values of surface pressure, ellipsometric angle orcomplex modulus at the equilibrium, it is the evolution of these values in the first few stage that is important topredict foaming properties

International audience; The variable domain of heavy-chain camelid antibodies (VHH), exclusively present in the homodimer IgGs (HC IgG), provides valuable ligands for diagnosis, imaging and therapy. These VHHs are efficiently produced from lymphocytes of immunized animals through phage display and recombination biotechnology. For VHH development it is desirable to identify animals with high affinity HC IgG response by monitoring antigen-binding in the course of immunization. The aim of this study was to propose a direct approach on whole plasma samples to distinguish between homodimer IgG and heterotetramer (IgG(1)) responses, and quantify them, using western ligand blotting (WLB). WLB consists here in electrophoretic separation of the target IgG subclasses on the basis of molecular size and binding of (125)I labeled antigen. First, we established the WLB parameters for titration of antigen-binding homodimers in relation to antigen-binding total IgGs in ovalbumin-immunized dromedary plasma samples and demonstrated that the WLB is an alternative to ELISA for IgG subclass titration. As purification of IgG subclasses or availability of IgG subclass-specific antibodies is not necessary, WLB is more direct and practical for screening a large number of samples. Second, WLB was applied to study the pattern of homodimer and heterotetramer responses during the time-course of immunizations against three different types of immunogens. As these patterns can differ between animals and immunogens, the method may be useful for identifying animals displaying the desired antigen-specific homodimer IgG response. Lastly, WLB was also described in its variant form of dot ligand blotting for identifying antigen-binding phages at the final step of a phage display experiment. (C) 2008 Elsevier B.V. All rights reserved.

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ConferenceConference: 11th European Symposium on the Quality of Eggs and Egg ProductsLocation: Doorwerth, NETHERLANDSDate: MAY 23-26, 2005; The hen's egg plays a crucial role in the embryonic development of the bird. It serves first as a source of energy and nutrients of high digestibility and, second, protects the embryonic bird against external aggressions. The unique structure of an egg with yolk containing the embryo surrounded by albumen and shell as physical barriers, is the first element of this protection. The second one consists of the specific composition of yolk, albumen and shell with many molecules possessing elevated biological properties. Thus, these molecules represent a major source of active principles usable by medical, pharmaceutical, cosmetic, nutraceutical and biotechnological industries. In this review, we will focus particularly on nutritional, health, and biotechnological activities of egg molecules and on the approaches, which are proposed by European research groups, to exploit this bioactive potential.

The conformational changes of egg-white proteins, in a ternary-protein system, at the air–water interface have been studied. Three of the major egg-white proteins, ovalbumin, ovotransferrin and lysozyme, were studied with concentration ratios reflecting those in egg-white. Results were compared to those obtained in a previous work on protein denaturation at the air–water interface in single-protein systems (Lechevalier, V., Croguennec, T., Pezennec, S., Guerin-Dubiard, C., Pasco, M., & Nau, F. (2003). Ovalbumin, ovotransferrin, lysozyme: Three model proteins for structural modifications at the air–water interface. Journal of Agricultural and Food Chemistry 51, 6354–6361). Foaming altered the protein structure more profoundly in the mixture than in single-protein systems. Strong electrostatic interactions were observed between the three proteins. Their existence at the air–water interface could ease intermolecular sulfhydryl–disulfide exchange reactions between ovalbumin and both ovotransferrin and lysozyme. This study highlighted the fact that results obtained on single-protein systems were not easily extrapolable to complex systems, such as egg-white.

Gelation of heat-treated milk protein solutions by acid fermentation was performed using a model system of micellar casein alone (systR), micellar casein and β -lactoglobulin (systB) or micellar casein and egg ovalbumin (systO) dissolved in a milk ultrafiltrate and heat-treated at 90°C for 24 min. Solubility of globular proteins at given pH values and their interactions with casein were determined. Particle size and ζ -potential of the protein systems were measured before and after heat treatment. Acid gelation of the heated systems was monitored using dynamic low-amplitude strain oscillation. Ovalbumin alone or with casein produced larger particles than casein alone or with β -lactoglobulin upon heat treatment. The systems gelled at different pH values, i.e. 4.88, 5.47 and 5.88 in systR, systB and systO, respectively. The latter two pH values were clearly related to the pH of the loss of solubility of the heated globular proteins. While the gelation pattern for systR resembled unheated milk, the pattern for systB and systO showed the usual maximum for tan δ of heated milk.

This paper is an experimental contribution to the optimization of ovalbumin elution in RP-HPLC with a C4 support. The effects of TFA concentration, gradient duration, flow rate, and the nature of the solvent were studied using an experimental design. The results highlighted the major role of TFA concentration in the recovery yield of ovalbumin, with an optimum concentration range which is very narrow and much lower (0.025%) than the levels usually applied (0.1%). In this respect, ovalbumin seems to behave in a specific way. Among the four solvents tested, i.e. acetonitrile, isopropanol, ethanol, and a mixture of these three, the first one is definitely the most satisfactory in the area of recovery yield and efficiency. By modifying the elution conditions alone, the recovery yield of ovalbumin may range from 50 to 85%. Finally, this work has enabled us to define the chromatographic conditions for quantitative egg white analysis of its major proteins in only six minutes and with minimal subsequent ghosting effects.