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The aim of this study was to determine the physicochemical parameters, i.e. water, protein, fat, ash, sodium and carbohydrate content and energy value, as well as fatty acid composition and pH of snail meat from two species, Cornu aspersum maximum and Cornu aspersum Müller. The snail meat was found to have relatively high protein content (13.12 g/100 g vs. 16.53 g/100 g), low fat content (0.89 g/100 g vs. 1.21 g/100 g), and low energy value (343.8 kJ vs. 379.2 kJ). The pH of snail meat was very high (8.59 vs. 8.19). On average, snail meat has a favourable fatty acid composition as it contains 27.08 wt. % saturated (SFA), 19.00 wt. % monounsaturated, and 53.92 wt. % polyunsaturated fatty acids (PUFA). It has a favourable PUFA/SFA ratio (1.99), but an unfavourable n-6/n-3 ratio (20.33) and a relatively high proportion of trans fatty acids (3.93 wt. %). The fatty acid profile of snail meat is dominated by linoleic acid (20.89 wt. %), arachidonic acid (13.27 wt. %), oleic acid (12.67 wt. %), nonadecanoic acid (10.23 wt. %), and stearic acid (10.08 wt.%). The aim of our study was also to verify the culinary value of snail meat. For this purpose, two snail meat pâtés of acceptable sensory quality were prepared. The panel evaluated the sensory quality of the pâté made from the meat (foot and liver) of the snail Cornu aspersum Müller significantly worse than the pâté made from the meat of the snail Cornu aspersum maximum, mainly due to the unpleasant aroma, in which the soapy, sweet and spicy components predominated, as well as the absence of the snail meat aroma., Cilj ovog rada bio je odrediti fizikalno-kemijske parametre, tj. udio vode, bjelančevina, masti, pepela, natrija i ugljikohidrata te energetsku vrijednost, kao i sastav masnih kiselina i pH mesa puževa dviju vrsta, Cornu aspersum maximum i Cornu aspersum Müller. Utvrđeno je da meso puža ima relativno visok udio proteina (13,12 g/100 g u odnosu na 16,53 g/100 g), nizak udio masti (0,89 g/100 g u odnosu na 1,21 g/100 g) i nisku energetsku vrijednost (343,8 kJ u odnosu na 379,2 kJ). pH mesa puževa bio je vrlo visok (8,59 naspram 8,19). Meso puževa u prosjeku ima povoljan sastav masnih kiselina jer sadrži 27,08 mas. % zasićenih (SFA), 19,00 % mononezasićenih i 53,92 % polinezasićenih masnih kiselina (PUFA). Ima povoljan omjer PUFA/SFA (1,99), ali nepovoljan omjer n-6/n-3 (20,33) i relativno visok udio transmasnih kiselina (3,93 %). Profilom masnih kiselina u mesu puževa dominiraju linolna kiselina (20,89 %), arahidonska kiselina (13,27 %), oleinska kiselina (12,67 %), nonadekanska kiselina (10,23 %) i stearinska kiselina (10,08 %). Cilj našeg istraživanja također je bio provjeriti kulinarsku vrijednost mesa puževa. U tu svrhu pripremljene su dvije paštete od puževog mesa prihvatljive senzorske kvalitete. Panel je značajno lošije ocijenio senzorsku kvalitetu paštete od mesa (stopala i jetra) puža Cornu aspersum Müller od paštete od mesa puža Cornu aspersum maximum, uglavnom zbog neugodne arome, u kojoj se prevladavaju sapunaste, slatke i ljute komponente, kao i odsustvo arome puževog mesa., Ziel dieser Studie war es, die physikalisch-chemischen Parameter, d. h. den Wasser-, Protein-, Fett-, Asche-, Natrium- und Kohlenhydratgehalt und den Energiewert, sowie die Fettsäurezusammensetzung und den pH-Wert von Schneckenfleisch zweier Arten, Cornu aspersum maximum und Cornu aspersum Müller, zu bestimmen. Das Schneckenfleisch wies einen relativ hohen Proteingehalt (13,12 g/100 g gegenüber 16,53 g/100 g), einen niedrigen Fettgehalt (0,89 g/100 g gegenüber 1,21 g/100 g) und einen niedrigen Energiewert (343,8 kJ gegenüber 379,2 kJ) auf. Der pH-Wert von Schneckenfleisch war sehr hoch (8,59 vs. 8,19). Im Durchschnitt hat Schneckenfleisch eine günstige Fettsäurezusammensetzung, da es 27,08 Gew.-% gesättigte (SFA), 19,00 Gew.-% einfach ungesättigte und 53,92 Gew.-% mehrfach ungesättigte Fettsäuren (PUFA) enthält. Es hat ein günstiges PUFA/SFA-Verhältnis (1,99), aber ein ungünstiges n-6/n-3-Verhältnis (20,33) und einen relativ hohen Anteil an trans-Fettsäuren (3,93 Gew.-%). Das Fettsäureprofil von Schneckenfleisch wird dominiert von Linolsäure (20,89 Gew.-%), Arachidonsäure (13,27 Gew.-%), Ölsäure (12,67 Gew.-%), Nonadecansäure (10,23 Gew.-%) und Stearinsäure (10,08 Gew.-%). Das Ziel unserer Studie war es auch, den kulinarischen Wert von Schneckenfleisch zu überprüfen. Zu diesem Zweck wurden zwei Schneckenfleischpasteten von akzeptabler sensorischer Qualität zubereitet. Die Jury bewertete die sensorische Qualität der Pastete aus dem Fleisch (Fuß und Leber) der Schnecke Cornu aspersum Müller deutlich schlechter als die Pastete aus dem Fleisch der Schnecke Cornu aspersum maximum, was vor allem auf das unangenehme Aroma zurückzuführen war, bei dem die seifigen, süßen und würzigen Komponenten überwogen, sowie auf das Fehlen des Schneckenfleischaromas., El objetivo de este trabajo fue determinar los parámetros físico-químicos, es decir, la proporción de agua, proteínas, grasas, cenizas, sodio y carbohidratos, así como el valor energético, la composición de ácidos grasos y el pH de la carne de caracoles de dos especies, Cornu aspersum maximum y Cornu aspersum Müller. Se encontró que la carne de caracol tiene un contenido de proteína relativamente alto (13,12 g/100 g frente a 16,53g / 100g), bajo contenido de grasa (0,89g / 100g frente a 1,21g /100g) y bajo valor energético (343,8g / 100g kJ frente a 379,2 kJ). El pH de la carne de caracol era muy alto (8,59 frente a 8,19). En promedio, la carne de caracol tiene una composición de ácidos grasos favorable, ya que contiene 27,08% en peso de saturados (SFA), 19,00% monoinsaturados y 53,92% de ácidos grasos poliinsaturados (PUFA). Tiene una relación PUFA/SFA favorable (1,99), pero una relación n-6/n-3 desfavorable (20,33) y una proporción relativamente alta de ácidos grasos trans (3,93% en peso). El perfil de ácidos grasos en la carne de caracol está dominado por ácido linoleico (20,89 %), ácido araquidónico (13,27 %), ácido oleico (12,67 %), ácido nonadecanoico (10,23 %) y ácido esteárico (10,08 %). El objetivo de nuestra investigación también fue comprobar el valor culinario de la carne de caracol. Para ello fueron preparados dos patés de carne de caracol de aceptable calidad sensorial. El panel calificó la calidad sensorial del paté de carne de caracol Cornu aspersum Müller (patas e hígado) significativamente peor que la carne de paté Cornu aspersum maximum, principalmente debido al aroma desagradable, en el que predominan los componentes jabonosos, dulces y picantes, así como la ausencia de aroma a carne de caracol., Questo studio aveva lo scopo di determinare i parametri fisico-chimici, cioè il contenuto di acqua, proteine, grassi, ceneri, sodio e carboidrati, nonché il valore energetico, la composizione degli acidi grassi e il pH della carne di chiocciola (impropriamente chiamata lumaca di terra) di due specie, Cornu aspersum maximum e Cornu aspersum Müller. È stato riscontrato che la carne di chiocciola ha un contenuto proteico relativamente elevato (13,12 g/100 g contro 16,53 g/100 g), un basso contenuto di grassi (0,89 g/100 g contro 1,21 g/100 g) e un basso valore energetico (343,8 kJ rispetto a 379,2 kJ). Il pH della carne di chiocciola è risultato molto alto (8,59 contro 8,19). In media, la carne di chiocciola ha una composizione favorevole di acidi grassi, poiché contiene il 27,08 % di acidi grassi saturi (SFA), il 19,00 % di acidi grassi monoinsaturi e il 53,92 % di acidi grassi polinsaturi (PUFA). Ha un rapporto PUFA/SFA favorevole (1,99), ma un rapporto n-6/n-3 sfavorevole (20,33) e una percentuale relativamente alta di acidi grassi trans (3,93%). Il profilo degli acidi grassi nella carne di chiocciola è dominato dall’acido linoleico (20,89%), seguito dall’acido arachidonico (13,27%), dall’acido oleico (12,67%), dall’acido nonadecilico o nonadecanoico (10,23%) e dall’acido stearico (10,08%). L’obiettivo della nostra ricerca era anche quello di verificare il valore culinario della carne di chiocciola. A tale scopo sono stati preparati due pâté di carne di chiocciola di qualità sensoriale accettabile. Il panel ha valutato la qualità sensoriale del pâté di carne di chiocciola Cornu aspersum Müller (zampe e fegato) significativamente peggiore rispetto al pâté di carne Cornu aspersum maximum, principalmente a causa dell’aroma sgradevole, in cui predominano le componenti saponose, dolci e piccanti, nonché per l’assenza dell’aroma della carne di chiocciola.

La bio-modélisation numérique est un domaine hautement multidisciplinaire à la frontière entre la biologie, la physique, les mathématiques et l'informatique. Il s'agit d'un domaine en pleine effervescence grâce à une habile exploitation des avancées informatiques et algorithmiques. Parmi ses sujets d'étude, on retrouve les protéines, des molécules de grand intérêt. En effet, elles sont des nanomachines jouant des fonctions primordiales pour la survie de tout organisme. En plus de leurs fonctions naturelles, certaines protéines sont associées au développement de diverses maladies ou pourraient servir de molécules thérapeutiques. La vision traditionnelle de la biologie moléculaire stipule que les fonctions des protéines sont étroitement liées à leur structure tri-dimensionnelle elle-même déterminée par les propriétés physico-chimiques de la séquence en acides aminés. Ainsi, l'étude de la structure est indispensable. Les méthodes de la bio-modélisation numérique, en partenariat avec l'expérience, sont particulièrement appropriées pour l'étude des protéines. Cette thèse s'articulera donc autour de trois classes de méthodes qui permettent d'étudier divers aspects des protéines. Le premier chapitre présentera les améliorations apportées à PEP-FOLD, une méthode simplifiée pour la prédiction structurelle \emph{de novo} des petits peptides. Deux des trois éléments-clés de PEP-FOLD ont été peaufinés, l'alphabet structurel et le potentiel gros-grain sOPEP, avec comme résultat une amélioration de la qualité des prédictions. Cette nouvelle version est comparée aux méthodes de prédictions utilisant les plus récents développements de l'apprentissage machine. Le second chapitre présentera les résultats de simulations numériques sut deux petites molécules thérapeutiques contre la COVID-19, grâce à des méthodes basées sur la physique. En collaboration avec les résultats expérimentaux, nos simulations montrent que nos deux molécules pourraient prévenir des interactions cruciales pour l'émergence de la maladie. Finalement, le dernier chapitre présentera quelques résultats préliminaires au développement du potentiel simplifié aaOPEP, qui permettra d'étudier les processus d'agrégation et de fibrillation de la protéine $\beta$-amyloïde, associés à l'apparition de la maladie d'Alzheimer. Ce processus étant fondamentalement multi-échelle, au niveau spatial et temporel, le développement de méthodes simplifiées est essentiel pour obtenir le portrait global du phénomène., Molecular modeling is a multidisciplinary enterprise combining the fields of biology, physics, mathematics and informatics. By utilizing improvements in both computer hardware and algorithms, the field is experiencing a spectacular growth in the past two decades. Proteins are nanomachines and play multiple essential functions in the life of every organisms. Additionally, proteins are also associated with the emergence of many diseases and could also be used as therapeutic molecules. In the classical view of molecular biology, protein's functions are closely related to its tri-dimensional structure, which is encoded by the chemical properties of the amino acid sequence. Therefore, the study of protein's structure is of fundamental importance. Tools from molecular modeling are, in partnership with experimental techniques, very well suited to study proteins. The following thesis will be divided into three main classes of techniques, each able to study a wide range of protein's characteristics. The first chapter present improvements made to PEP-FOLD, a simplified, freely-available online and successful technique for \textit{de novo} peptide-structure prediction. Two of the three key components of PEP-FOLD were revisited in this work; the structural alphabet and the coarse-grained potential sOPEP. These modifications lead to an important increase in the quality of PEP-FOLD's predictions. A thorough comparison with state-of-the-art machine learning techniques is made and we highlight key successes and possible future improvements. The second chapter present the study of potential therapeutic molecules against COVID-19 using physics-based techniques. These results, combined with experimental data from immunobinding assay and SPR microscopy, showed that our two small molecules could prevent key interactions between the wild-type/mutant SARS-CoV-2 and the cells of the host and therefore could potentially be potent therapeutic molecules against COVID-19. Finally, the last chapter present preliminary results about the development of the new aaOPEP forcefield designed to study the multi-scale process of amyloid-$\beta$ aggregation and fibrillation, associated with the Alzheimer disease. This new potential will take the core ideas of the coarse-grained potential OPEP into the all-atom regime and will allow to study bigger systems over longer time-scale.

Legumes are essential for providing protein-rich products and introducing nitrogen into cropping systems. Perennial species, mainly grown for fodder production but also as service plants, contribute to the protein autonomy of livestock. Perennial legumes also contribute to many other ecosystem services: soil fertility through the supply of nitrogen coupled with organic matter, weed management, reduction of pollution emitted by ruminant livestock, and biodiversity preservation. Able to adapt to climate change, perennial legumes remain valuable under different food transition scenarios. The article suggests avenues of research in agronomy, plant breeding and animal husbandry to further increase these services, Les légumineuses sont indispensables pour fournir des produits riches en protéines et introduire de l’azote dans les systèmes de culture. Les espèces pérennes, essentiellement cultivées pour une production fourragère, mais aussi comme plantes de services, contribuent à l’autonomie protéique des élevages. Les légumineuses pérennes contribuent aussi à de nombreux autres services écosystémiques : fertilité des sols grâce à la fourniture d’azote couplé à la matière organique, gestion des adventices, réduction des pollutions émises par les élevages de ruminants, préservation de la biodiversité. Capables de s’adapter au changement climatique, les légumineuses pérennes restent précieuses sous différents scénarios de transition alimentaire. L’article suggère des pistes de recherche en agronomie, amélioration des plantes et zootechnie pour accroître encore ces services.

Oppositely charged proteins can form soluble assemblies that under specific physical chemical conditions lead to liquid-liquid phase separation, also called heteroprotein coacervation. Increasing evidence suggests that surface charge anisotropy plays a key role in heteroprotein complexation, and coacervation. Here, we investigated complexation of an acidic protein, β-lactoglobulin (BLG), with two basic proteins, rapeseed napin (NAP) and lysozyme (LYS), of similar net charge and size but differing in surface charge distribution. Using turbidity measurements and isothermal titration calorimetry, we confirmed that LYS binds BLG as expected from previous studies. This interaction leads to two types of phase separation phenomena, depending on pH: liquid-solid phase separation in the case of strong electrostatic attraction and liquid-liquid phase separation for weaker attraction. More interestingly, we showed using dynamic light scattering that NAP interacts with BLG, resulting in formation of assemblies in the nanometer size range. The formation of assemblies was also evident when modeling the interactions using Brownian dynamics for both BLG + NAP and BLG + LYS. Similarly, to DLS, BLG and NAP formed smaller assemblies than BLG with LYS. The molecular details rather than the net charge of BLG and NAP may therefore play a role in their assembly. Furthermore, simulated BLG + NAP assemblies were larger than those experimentally detected by DLS. We discuss the discrepancy between experiments and simulations in relation to the limitations of modelling precisely the molecular characteristics of proteins.

Acacia senegal gum (Asen) is a natural exudate of Acacia trees species largely used in food as well as other industries. This natural product appears as a continuum of molecular species which shows diverse sugar and protein composition, molar masses and charge density. The presence of larger macromolecules, or aggregates, has been demonstrated to have a great influence on the Acacia gum characteristics. The present work is designed to recover and characterize one protein-rich fraction presenting a high aggregate content. With this fraction we will open the door to future works with the aim to acquire a deeper knowledge about the origin and the role of the aggregates from Asen gum. Our methodology is based on the well-known ion exchange chromatography, using DEAE Sephacel gel as stationary phase. We have separated Asen into two different fractions (fraction IEC-F1 and fraction IEC-F2), being both of them confirmed as arabinogalactan-proteins (AGP) by Yariv detection. Fraction IEC-F1 has been thoroughly characterized (sugar and amino acid composition, molar mass distribution, weight-average molar mass, number-average molar mass, polydispersity index, intrinsic viscosity, radius of gyration, Mark-Houwink-Sakurada analysis, hydrodynamic radius, partial specific volume and partial specific adiabatic compressibility). From amino acid data, we have estimated that fraction IEC-F1 theoretically corresponds to about 70% of HIC F3 and 30% of HIC F2, respectively the second and the third fractions separated by hydrophobic interaction chromatography (HIC) and largely described in literature. The obtained information indicates that fraction IEC-F1 appears as a fraction highly rich in aggregates.

Background 3D printing or additive manufacturing (AM) now provides enormous freedom to design, manufacture and innovate in various domains, even in foodstuffs development. Given the immense potential applications related to AM, many authors are even talking about a new industrial revolution. Scope and approach In this article, we review the state of the science in applied AM methods for developing biobased products in the medical and food sectors, with these two sectors having similar points. We were therefore interested in the technological locks encountered in the various studies carried out on the subject. Consideration has also been given to the possibility of using alternative sources of protein, such as animal by-products, to address resource management and sustainable development issues. One of the strengths of 3D printing is personalization, so we chose to evaluate the impact of this technology on target populations and evaluate the possible evolutions. Key findings and conclusions In order to design food in optimal conditions, the development of new 3D printers is fundamental 1) to ensure the sanitary quality (both microbiological and chemical) of these products, and 2) to control the structure and texture of these 3D-printed foods. From there, it will be possible to propose personalized foods, adapted to different categories of population (e.g. seniors or young people …). The major challenge in the next years will be to develop, using 3D printing, meat products or products blending alternative protein sources that remain perfectly structured without having to use additives. The final step will be to garner consumer acceptance for these 3D-printed foods.

Fungi are surrounded by a thick layer of carbohydrates and proteins, which is essential for the cell’s viability – the fungal cell wall. Proteins are incorporated into this organelle in different ways: some are covalently linked to the carbohydrate moiety of the cell wall via Glycosylphosphatidylinositol (GPI)-anchors or alkali-sensitive linkages, others are indirectly attached to the cell wall via disulfide bonds. Cell wall proteins are involved in various cellular functions, such as cell wall biosynthesis, adhesion to external surfaces, or sensing. The GPI-anchored cell wall proteome of the thermophilic model organism Chaetomium thermophilum was identified in the first part of this thesis. First, a prediction of GPI-proteins, anchored to the cell wall and the plasma membrane was done. The prediction was then complemented by mass-spectrometric identification of GPI-anchored cell wall proteins in isolated cell walls. The detected proteins were then analyzed concerning their functions and putative roles and interesting targets for pharmaceutical applications and fundamental research were established, including Gel1/2, Kre9/Knh1, and Ecm33. In addition, the ultrastructure of the C. thermophilum cell wall was analyzed via transmission electron microscopy, revealing short microfibrils in its outer layer and its similarity to the cell wall of S. cerevisiae. The thesis then advances to the analysis of the A-domains of the Candida glabrata adhesins Awp1 and Awp3, which are members of adhesin cluster VI. Although the fungal pathogen lacks certain virulence factors – such as hyphae formation – C. glabrata infections are commonly observed; its large repertoire of adhesins is believed to be the reason therefore. Awp1A and Awp3A both consist of a β helix domain and an α crystallin domain. They are structurally similar to carbohydrate binding proteins, e. g. polysaccharide lyases, but carbohydrate binding could not be observed. A sequence similarity network (SSN) elucidates their high similarity to cluster V adhesins Awp2 and Awp4 and thereby reinforces previous classifications. The structures of Awp1 and Awp3 provide first insights into new types of adhesins in C. glabrata that include the adhesin clusters V and VI. Furthermore, the G-protein coupled receptor Pth11 from C. thermophilum was analyzed. It contains an N-terminal CFEM domain – a domain exclusively found in fungal cell wall and plasma membrane proteins – that is predicted to be the ligand binding site. The CtPth11 CFEM domain consists of five α helices and reveals two potential binding sites, divided by F48. Distinct conformers of F48 allow formation of a tunnel through the domain. A model of the CtPth11 CFEM domain and transmembrane region – based on prediction of neighboring residues via sequence covariation analysis – shows that both potential binding sites are accessible. In a fragment screen, four fragments were bound in the same cavity; three of them could be fitted into their respective electron densities. These hydrophobic fragments are placed in the hydrophobic cavity, with only few additional interactions, which is in accordance with the proposal that Pth11 senses hydrophobic cues on the plant surface., Pilze sind von einer dicken Schicht aus Kohlenhydraten und Proteinen umgeben, die für die Lebensfähigkeit der Zelle essentiell ist – der pilzlichen Zellwand. Proteine sind auf unterschiedliche Arten in dieses Organell integriert: einige sind kovalent an den Kohlenhydratanteil der Zellwand gebunden, entweder über Glycosylphosphatidylinositol (GPI)-Anker oder alkaliempfindliche Bindungen, andere indirekt über Disulfidbindungen. Zellwandproteine sind an unterschiedlichen zellulären Funktionen beteiligt, wie der Zellwandbiosynthese, der Adhäsion an Oberflächen oder der Sensorik. Im ersten Teil dieser Arbeit wurden die GPI-verankerten Proteine des thermophilen Modellorganismus Chaetomium thermophilum identifiziert. Zunächst wurde eine Vorhersage der an Zellwand und Plasmamembran befindlichen GPI-Proteine durchgeführt. Die Vorhersage wurde durch den massenspektrometrischen Nachweis der GPI-verankerten Zellwandproteine in isolierten Zellwänden ergänzt. Die detektierten Proteine wurden hinsichtlich ihrer Funktionen und mutmaßlichen Rollen analysiert. Interessante Targets für pharmazeutische Anwendungen und Grundlagenforschung konnten ermittelt werden, u. a. Gel1/2, Kre9/Knh1 und Ecm33. Zusätzlich wurde die Ultrastruktur der Zellwand von C. thermophilum mittels Transmissionselektronenmikroskopie analysiert, wobei kurze Mikrofibrillen in der äußeren Zellwandschicht und Ähnlichkeit zu der Zellwand von S. cerevisiae festgestellt werden konnten. Die Arbeit behandelt im zweiten Teil die Analyse der A-Domänen der Candida glabrata Adhäsine Awp1 und Awp3, die Mitglieder des Adhäsinclusters VI sind. Obwohl diesem humanpathogenen Pilz bestimmte Virulenzfaktoren - z. B. zur Hyphenbildung - fehlen, werden C. glabrata Infektionen häufig beobachtet, wobei sein großes Repertoire an Adhäsinen einer der wesentlichen Gründe sein sollte. Awp1A und Awp3A bestehen beide aus einer β-Helix-Domäne und einer α-Kristallin-Domäne. Sie ähneln strukturell kohlenhydratbindenden Proteinen, z. B. Polysaccharid-Lyasen. Allerdings konnte keine Bindung von Kohlenhydraten an Awp1-Typ Adhäsinen nachgewiesen werden. Ein Sequenzähnlichkeitsnetzwerk leitet eine hohe Ähnlichkeit zu den Adhäsinen Awp2 und Awp4 des Adhäsinclusters V ab und untermauert damit frühere Klassifizierungen. Die Strukturen von Awp1 und Awp3 geben erste Einblicke in neue Typen von Adhäsinen in C. glabrata, zu denen Adhäsine der Cluster V und VI gehören. Weiterhin wurde der G-Protein-gekoppelte Rezeptor Pth11 aus C. thermophilum analysiert. Er enthält eine N-terminale CFEM-Domäne - diese Domäne kommt ausschließlich in Pilzzellwand- und Plasmamembranproteinen vor -, die als Ligandenbindungsstelle vorhergesagt wurde. Die CFEM-Domäne von CtPth11 besteht aus fünf α-Helices und weist zwei potenzielle Bindungsstellen auf, die durch F48 geteilt werden. Bestimmte Orientierungen des Aminosäurerestes F48 ermöglichen die Bildung eines Tunnels durch die Domäne. Ein Modell der CtPth11-CFEM-Domäne und der Transmembranregion - basierend auf der Vorhersage benachbarter Reste mittels Sequenzkovarianzanalyse - zeigt, dass beide potenziellen Bindungsstellen zugänglich sind. In einem Fragment-Screen wurden vier Fragmente an der gleichen Bindestelle gebunden; drei davon konnten in die jeweiligen Elektronendichten modelliert werden. Diese hydrophoben Fragmente sind in der hydrophoben Bindestelle platziert und weisen nur wenige zusätzliche Interaktionen auf, was zu der Hypothese passt, dass Pth11 hydrophobe Charakteristika auf der Pflanzenoberfläche wahrnimmt.

Due to climate change, drought periods will occur more frequently in the future. They will have a strong negative impact on crop yields. Drought stress leads to an osmotic imbalance and causes the closure of stomata to reduce water loss of transpiration. However, this reduces photosynthesis and ultimately leads to the formation of oxygen radicals, which may damage cell structure and function. If drought stress continues, a dramatic lack of energy is caused, which threatens plant life. To prevent irreversible damage, plants adapt their entire metabolism to resist drought stress at an early stage. This dissertation is dedicated to the adaptation of plants upon drought stress and the specific contribution of amino acid metabolism during this process. An in vitro experiment was performed to investigate the implications of a short but severe water deficit (Chapter 2.1). The Arabidopsis seedlings showed a strong decrease in protein content within 24h and at the same time a strong accumulation of the amino acids L-proline and GABA. Proteome analyses revealed that the aromatic amino acids were primarily used for the synthesis of stress mitigating secondary metabolites, such as flavonoids and anthocyanins, which are known to scavenge reactive oxygen species. Furthermore, a general induction of amino acid catabolism was observed, which provides sufficient amounts of L-glutamate for the synthesis of L-proline and GABA. Simultaneously, the catabolic pathways could represent an alternative source of reduction equivalents, which may fuel mitochondrial ATP production under carbon starvation conditions. In soil experiments were performed to investigate the plant drought stress response in a more physiological context (Chapter 2.2). In both, the in vitro and the in soil system, the plant stress response can be divided into distinct phases. The osmotically active amino acids, L-proline and GABA, are already produced in early phases of the water deficit and allow keeping the cellular water content constant for several days. Shortly before plants become irreversibly impaired by drought, a massive protein degradation takes place. This marks the beginning of the severe stress phase. Based on the proteome data and theoretical considerations, an experimental strategy was developed, which allows calculating absolute contents, concentrations and even copy numbers of individual proteins per leaf cell. As a result, the dynamic interconnection of protein homeostasis and amino acid homeostasis could be monitored and quantified on absolute scales. Our approach reveals the energy content of the released amino acids and indicates that their complete oxidation would cover the energy demand of the plant for several hours. In a review article, the regulatory properties of amino acids during the plant stress response were summarized and discussed (Chapter 2.3): Amino acids can be used as signal molecules, e.g. for inducing stomatal closure, as sensors of the nutrient content of cells or regulators for inducing their own catabolism. Our findings contribute to a general understanding of the effects of drought stress on the plant metabolism and shed light on the versatile and important roles of amino acids beyond their role in representing building blocks for protein biosynthesis.

Neue Sequenzierungstechnologien haben eine überwältigende Menge an Daten für Protein-Datenbanken generiert, die nicht länger von Menschenhand verarbeitet werden können und rechnergestützte Methoden sind noch nicht akkurat und zuverlässig genug um ihnen die Aufgabe vollständig zu überlassen. Diese Arbeit versucht einen Überblick darüber zu geben, was mit den Daten gemacht werden muss, welcher Fehler und Problem dabei auftreten, die Lösungen die verfügbar und vorgeschlagen sind und die Möglichkeiten, die die Daten mit sich bringen. Next-generation sequencing technologies have created overwhelming amounts of data for protein databases, that can no longer be handled by manual curation and computational methods are not yet accurate and reliable enough to leave the task completely to them. This thesis tries to give an overview about what needs to be done with the data, the errors and problems that wait along the way, the solutions that are available and proposed and the opportunities the data brings.

The present dissertation covers the usage of technical enzyme preparations (TEPs) for vegetable and insect protein hydrolysis, due to the mounting interest in alternative protein sources to cover the increasing demand for food from a growing world population. The TEPs, as defined in this study, are enzyme preparations that include side activities and are used in food processing. Today, TEPs are used by food manufacturers based on the supplier’s information that usually states the main enzyme activity and includes information on side activities only in some cases. However, knowledge about the activity profile is crucial as side activities can contribute to the properties of the protein hydrolysates produced (e.g. degree of hydrolysis [DH], liberation of amino acids) and the final food product quality. In the first study, an automated photometric analyzer (GalleryTM Plus, Thermo Fisher Scientific) was introduced for the comprehensive activity determination of TEPs. The new setup of the analyzer covered 32 synthetic and natural substrates in order to determine aminopeptidase, carboxypeptidase, dipeptidylpeptidase and endopeptidase activities distinguishably. Accordingly, the overall proteolytic activity of TEPs was quantified and detailed information about the substrate spectra and peptidase side activities was generated. Furthermore, several batches of the industrial TEP Flavourzyme1000L were measured. By determining 32 peptidase activities, batch variations were shown. As Flavourzyme1000L is standardized by its supplier Novozymes on only one activity (leucine aminopeptidase), the additional 31 new peptidase activities determined showed differences of the side activities of the batches. In addition, the study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting lupine protein hydrolysates (DH and liberation of amino acids). Due to the determination of 32 peptidase activities (so-called “activity fingerprint”), TEPs were selected specifically to increase, for example, the DH. The two TEPs P278 and DZM were selected due to their complementary peptidase activities, as an example of this study. The combination of these two TEPs resulted in an increase of the DH of 47%. Now, TEPs can be selected targeted more on the basis of their peptidase activities to, for example, increase the hydrolysis efficiency of lupine protein by combining complementary peptidase activities. In the second study, six food-grade TEPs (Flavourzyme1000L, ProteaseP “Amano”6SD, DeltazymAPS-M-FG, Promod278, ProteAX-K and PeptidaseR) were investigated regarding their influence on the hydrolysis of soy, pea and canola protein. The hydrolysates were investigated analytically concerning their DH and free amino acid profiles and sensorically concerning the taste attributes umami and bitter. By using a random forest model, the taste attributes bitter and umami were connected to specific peptidase activities (exo- and endopeptidase activities). Furthermore, out of the six selected TEPs, the usage of ProteAX-K showed high umami and low bitter taste of the vegetable protein hydrolysates (soy, pea and canola). In line with the first study, the second study showed that the detailed information of the peptidase activities of the TEPs could explain the properties of the resulting vegetable protein hydrolysates. Based on these new insights, TEPs can be selected more specifically based on their peptidase activity profiles to direct the formation of desired taste attributes of the protein hydrolysates. In the third study, two TEPs with various peptidase activities (Flavourzyme1000L, ProteaseA “Amano”2SD) were applied for the hydrolysis of insect proteins. This study investigated the potential of cricket and mealworm protein and their hydrolysates regarding their sensory potential. The sensory profiles of the insect proteins were altered by, firstly, applying proteolytic hydrolysis and then a Maillard reaction (30 min, T = 98°C, 1% (w/v) xylose) to the hydrolysates. The initially earthy-like flavor of the insect proteins resulted in modified taste profiles described by e.g., savory-like attributes, due to both processing steps. Furthermore, 38 odor-active molecules (1 alcohol, 5 acids, 11 aldehydes, 5 ketones and 16 heterocyclic compounds) were identified by gas chromatography-olfactometry (GC-O). The identified molecules are also found in meat and edible seafood products. The third study showed that the flavoring profile of insect proteins was modified and can be developed further by the respective processing. Die vorliegende Dissertation befasst sich mit der Verwendung von technischen Enzympräparaten (TEP) zur Hydrolyse von Pflanzen- und Insektenproteinen. Alternative Proteinquellen sind von zunehmendem Interesse, um die steigende Lebensmittelnachfrage der wachsenden Weltbevölkerung zu decken. In dieser Studie werden TEP als Enzympräparate definiert, die zur Herstellung von Lebensmitteln verwendet werden und meist Nebenaktivitäten aufweisen. In der Regel werden TEP von Lebensmittelherstellern basierend auf den Informationen der Produzenten verwendet. Diese nennen vorwiegend die Hauptenzymaktivität und selten weitere Nebenaktivitäten der Präparate. Detaillierte Informationen über Nebenaktivitäten sind allerdings wesentlich, da diese die Eigenschaften der erzeugten Proteinhydrolyse (z. B. Hydrolysegrad, Freisetzung von Aminosäuren) und die finale Lebensmittelqualität beeinflussen können. In der ersten Studie wurde ein automatisiertes photometrisches Analysengerät (GalleryTM Plus, Thermo Fisher Scientific) vorgestellt, um TEP umfangreich hinsichtlich ihrer Peptidaseaktivitäten zu bestimmen. Die neue Installierung des Analysengerätes beinhaltete 32 synthetische und natürliche Substrate, um Aminopeptidase-, Carboxypeptidase-, Dipeptidylpeptidase- und Endopeptidaseaktivitäten von TEP differenzierbar zu messen. Dabei wurden die allgemeine proteolytische Aktivität der TEP quantifiziert, sowie detaillierte Informationen über das Substratspektrum und über proteolytische Nebenaktivitäten generiert. Zudem wurden mehrere Chargen des industriellen TEP Flavourzyme1000L gemessen. Durch die Bestimmung der 32 Peptidaseaktivitäten wurden Batchvariationen aufgezeigt. Da Flavourzyme1000L vom Hersteller Novozymes auf nur eine Aktivität (Leucin Aminopeptidase) standardisiert wird, haben die weiteren 31 neu bestimmten Peptidaseaktivitäten die Unterschiede der Nebenaktivitäten aufgezeigt. Ferner hat diese Studie gezeigt, dass die detaillierten Informationen über die Peptidaseaktivitäten der TEP Auswirkungen auf den resultierenden Hydrolysegrad und die Aminosäurefreisetzung von Lupinprotein-hydrolysaten haben. Durch die Bestimmung der 32 Peptidaseaktivitäten (egl. „activity fingerprint“) wurden TEP gezielt ausgewählt, um beispielsweise den Hydrolysegrad zu erhöhen. Unter anderem wurden die zwei TEP Promod278 und DeltazymAPS-M-FG auf Grund ihrer komplementären Peptidaseaktivitäten ausgewählt und kombiniert, was zu einem Anstieg des Hydrolysegrades um 47% führte. Von nun an können TEP gezielter aufgrund ihrer Peptidaseaktivitäten ausgewählt werden, um durch die Kombination von komplementären Aktivitäten z. B. eine effizientere Hydrolyse zu erzielen. In der zweiten Studie wurde der Einfluss von sechs lebensmitteltauchglichen TEP (Flavourzyme1000L, ProteaseP “Amano”6SD, DeltazymAPS-M-FG, Promod278, ProteAX-K, PeptidaseR) auf die Hydrolyse von Soja-, Erbsen- und Canolaprotein untersucht. Die Hydrolysate wurden hinsichtlich des Hydrolysegrads, der Aminosäurefreisetzung und der Geschmacksattribute umami und bitter sensorisch untersucht. Mit Hilfe eines Random Forest Algorithmus wurden diese beiden Geschmacksattribute bestimmten Peptidaseaktivitäten (Exo- und Endopeptidase-aktivitäten) zugeordnet. Ferner zeigte, von den sechs untersuchten TEP, der Einsatz von ProteAX-K einen stark umami und schwach bitteren Geschmack der Pflanzenproteinhydrolysate (Soja, Erbse und Canola). Die Ergebnisse der ersten Studie konnten somit untermauert werden, auch hier konnten die detaillierten Informationen über die Peptidaseaktivitäten der TEPs die Eigenschaften der resultierenden Pflanzenproteinhydrolysate erklären. Durch diese neuen Erkenntnisse können TEP auf Grund ihrer Peptidaseaktivitäten künftig gezielter ausgewählt werden, um die Entwicklung von Geschmacksattributen der Hydrolysate zu beeinflussen. In der dritten Studie wurden zwei TEP mit verschiedenen Peptidaseaktivitäten (Flavourzyme1000L, ProteaseA “Amano”2SD) zur Hydrolyse von Insektenprotein eingesetzt. Diese Studie hat das sensorische Potential von Grillenprotein (Acheta domesticus) und Mehlwurmprotein (Tenebrio molitor) und deren Hydrolysate untersucht. Mittels proteolytischer Hydrolyse und anschließender Maillard Reaktion (30 min, T = 98°C, 1% (w/v) Xylose) wurden die Geschmacksprofile der Insektenproteine verändert. Die zunächst erdig schmeckenden Insektenproteine zeigten nach der technologischen Verarbeitung veränderte Geschmacksprofile durch z. B. herzhafte Attribute auf. Ferner wurden 38 geruchsaktive Moleküle (1 Alkohol, 5 Säuren, 11 Aldehyde, 5 Ketone and 16 hetero-zyklische Verbindungen) mittels olfaktorischer Gaschromatographie (GC-O) bestimmt. Die identifizierten Moleküle kommen auch in Fleisch- und essbaren Meerestieren vor. Die dritte Studie hat gezeigt, dass das Geschmacksprofil der Insektenproteine durch technologische Verarbeitung verändert und weiterentwickelt werden kann.

The renewed interest in plant proteins has stimulated research aimed at developing markers to aid in the selection of legume varieties better adapted to nutritional needs. Among the traits to be improved and stabilized is the amino acid balance of seeds, the essential amino acids methionine and tryptophan being particularly under-represented in legume seeds. This thesis focuses on seed protein composition, which is a major determinant of the seed amino acid balance. The objective was to explore the genetic and environmental variability in this trait and to identify genes potentially involved in its plasticity when subjected to environmental stresses. In the first part of the thesis, the protein composition of seeds from 200 Medicago truncatula genotypes cultivated in four contrasted environments (sufficient water supply, water stress, and two time-shifted sowing) was analyzed by one-dimensional electrophoresis. The plasticity indices calculated for each protein band made it possible to identify, by genome-wide association studies (GWAS), sequence polymorphisms associated with variations in the plasticity of the major storage proteins, the 7S and 11 globulins. The list of candidate genes is enriched in genes related to transcription, DNA repair and signal transduction. Other over-represented genes are involved in sulfur and aspartate metabolism leading to the synthesis of methionine and lysine. By exploiting publicly available expression datasets in Medicago truncatula, and by localizing these genes in the corresponding metabolic pathways, we discovered that methionine recycling pathways are likely to contribute to the plasticity of seed protein composition. This recycling involves the homocysteine S-methyltransferase 3 enzyme that regenerates methionine from S-methylmethionine, a long-distance transport form of methionine in the phloem. This part of the thesis has led to the identification of new genes to be targeted to improve and stabilize the amount of sulfur amino acids in seeds, opening prospects for translational research aimed at improving the quality of seeds in protein crops such as pea.The second part of the thesis aimed at exploring the variability of response of a collection of pea ecotypes to sulfur deficiency, at the levels of yield components and of seed protein composition, and to identify by GWAS genes potentially involved in this variability of response. Two experiments were carried out in two successive years with respectively 304 and 198 genotypes developed under two conditions: sufficient or deficient in sulfur. Since both year effect and sulfur deficiency effect were significant on the variables, plasticity indices reflecting the amplitude of the sulfur deficiency response were calculated for each variable. The results obtained by GWAS have revealed candidate genes for controlling the protein composition of seeds in low sulfur environments. Several are associated with transport mechanisms (e.g. amino acids) and post-translational regulation, while others play a role in sulfur metabolism or in the cross-talk between nitrogen and sulfur metabolisms. This work has shed light on pea genes that might control the nitrogen and sulfur status of seeds, and thus modulate the balance between the 7S and 11S globulins. The same GWAS approach conducted on yield component traits has led to the proposal of relevant candidate genes in particular for seed weight control. Altogether, these results offer prospects for varietal improvement to stabilize yield and the protein composition of legume seeds in fluctuating environments, in particular for the availability of sulfur., Le regain d’intérêt pour les protéines végétales stimule les recherches visant à développer des marqueurs d’aide à la sélection de variétés de légumineuses mieux adaptées aux besoins nutritionnels. Parmi les caractères à améliorer et à stabiliser est l’équilibre en acides aminés des graines, les acides aminés essentiels méthionine et tryptophane étant particulièrement sous-représentés dans les graines de légumineuses. Cette thèse est centrée sur la composition protéique des graines, un déterminant majeur de l’équilibre en acides aminés. L’objectif était d’explorer la variabilité génétique et environnementale de ce caractère et d’identifier des gènes potentiellement impliqués dans sa plasticité vis-à-vis de l’environnement.Dans le premier volet de la thèse, la composition protéique des graines de 200 génotypes de Medicago truncatula cultivés dans quatre environnements contrastés (apport suffisant en eau, stress hydrique, et deux semis décalés) a été analysée par électrophorèse mono-dimensionnelle. Les indices de plasticité calculés pour chaque bande protéique ont permis d’identifier, par génétique d’association à l’échelle du génome (GWAS), des polymorphismes de séquence associés à des variations de plasticité des protéines de réserve majeures, les globulines 7S et 11S. La liste des gènes candidats est enrichie en gènes liés à la transcription, à la réparation de l'ADN et à la transduction du signal. D’autres gènes sur-représentés sont impliqués dans les métabolismes du soufre et de l'aspartate conduisant à la synthèse de méthionine et de lysine. En exploitant des données d’expression publiquement disponibles chez Medicago truncatula, et en replaçant ces gènes dans les voies métaboliques correspondantes, nous avons découvert l'importance des voies de recyclage de la méthionine dans le contrôle de la plasticité de la composition protéique des graines. Ce recyclage fait intervenir l’enzyme homocystéine S-méthyltransférase 3 impliquée dans la régénération de méthionine à partir de S-méthylméthionine, une forme de transport de méthionine dans le phloème. Cette section de la thèse a fait émerger de nouveaux gènes à cibler pour améliorer et stabiliser le contenu en acides aminés soufrés, ouvrant des perspectives de recherches translationnelles visant à améliorer la qualité des graines de protéagineux comme le pois. Le second volet de la thèse avait pour objectifs d’explorer la variabilité de réponse d’une collection d’écotypes de pois à une carence en soufre, aux niveaux des composantes de rendement et de la composition protéique des graines, et d’identifier par GWAS des gènes potentiellement impliqués dans cette variabilité de réponse. Deux expériences ont été conduites deux années successives avec respectivement 304 et 198 génotypes développés dans deux conditions : apport suffisant ou carencé en soufre. L’effet année et l’effet de la carence en soufre étant significatifs sur les variables étudiées, des indices de plasticité reflétant l’amplitude de réponse vis-à-vis d’une carence en soufre ont été calculés pour chaque variable. Les résultats obtenus par GWAS ont mis en évidence des gènes candidats pour contrôler la composition protéique des graines dans les environnements pauvres en soufre. Plusieurs sont associés aux mécanismes de transport (ex. d’acides aminés) et de régulation post-traductionnelle, tandis que d’autres jouent un rôle dans le métabolisme du soufre ou sont au carrefour entre les métabolismes azotés et soufrés. Ce travail a permis de mettre en lumière des gènes de pois susceptibles de contrôler le statut azoté et soufré des graines, et ainsi de moduler l’équilibre entre les globulines 7S et 11S. La même approche GWAS menée au niveau des composantes de rendement a conduit à proposer des gènes candidats pertinents en particulier pour le contrôle du poids d’une graine.

Proteins are highly studied molecules because of their different roles in living organisms and the impact of their dysfunction on the state of health of living organisms. In recent years, studies have shown that a protein doesn’t have a single conformation (3D structure) to respond to its function, but several. As of today, different techniques exist to detect and determine the protein conformation, no technique can reconstitute all the conformations studied by a protein by individually screening the conformations that constitute it. The work of this memory is to determine if the combination of nanoconfinement and broadband dielectric spectroscopy can be used to screen different protein conformations and thus reconstitute the conformational set. Nanoconfinement is used to approximate the dimensions of proteins and reduce the conformational heterogeneity that exists within a population. Broadband dielectric spectroscopy characterizes a confined protein conformation via its molecular dynamics. My thesis work demonstrated that the combination of nanoconfinement and broadband dielectric spectroscopy could be used to screen different protein conformations by modifying the dimensions of the confinement. Different conformations of pentameric B subunits of cholera toxin (CtxB5), heat labile enterotoxin (LTB5), and lysozyme have been observed. The differences between the dielectric signals of the two toxins reveal local structures with distinct dynamics, while the differences with lysozyme reveal global structures with distinct dynamics. These results open perspectives for diagnosing structural and / or dynamic impacts linked to mutations, the implication of which in the development of diseases or the design of drugs can be explored within the framework of personalized therapies, Les protéines sont des molécules fortement étudiées à causes de leurs différents rôles dans les organismes vivants et de l’impact de leur disfonctionnement sur l’état de santé des organismes vivants. Ces dernières années, des études ont montré qu’une protéine ne possède pas une seule conformation (structure 3D) pour répondre à sa fonction, mais plusieurs. Au jour d’aujourd’hui, différentes techniques existent pour détecter et déterminer la conformation protéique, toutefois aucune technique ne permet de reconstituer l’ensemble des conformations adoptées par une protéine en criblant individuellement chacune des conformations qui le constitue. Le travail de ce mémoire est de déterminer si la combinaison du nanoconfinement et de la spectroscopie diélectrique à large bande est utilisable pour cribler différentes conformations protéiques et ainsi reconstituer l’ensemble conformationel. Le nanoconfinement est utilisé pour se rapprocher des dimensions des protéines et réduire l’hétérogénéité conformationelle qui existe au sein d’une population. La spectroscopie diélectrique à large bande caractérise une conformation protéique confinée via sa dynamique moléculaire. Mon travail de thèse a démontré que la combinaison du nanoconfinement et de la spectroscopie diélectrique à large bande pouvait être utilisée pour cribler différentes conformations protéiques en modifiant les dimensions du confinement. Différentes conformations des sous-unités B pentamériques de la toxine du choléra (CtxB5), de l’enterotoxine thermolabile (LTB5), et du lysozyme ont été observées. Les différences entre les signaux diélectriques des deux toxines révèlent des structures locales de dynamiques distinctes, alors que les différences avec le lysozyme révèlent des structures globales de dynamiques distinctes. Ces résultats ouvrent des perspectives pour diagnostiquer des impacts structuraux et/ou dynamiques liés à des mutations, dont l’implication dans le développement de maladies ou la conception de médicaments pourra être explorées dans le cadre de thérapies personnalisées

Nuclear Magnetic Resonance (NMR) provides valuable structural and dynamic information at the atomic scale, however, the low sensitivity and resolution of signals rapidly preclude investigations of larger molecular objects. We present three isotopic labeling strategies for different protein-solution NMR experiments and demonstrate their potential for the structural study of biomolecules in solution. Among the strategies considered, two are based on the use of in vitro protein expression to obtain selectively labeled proteins of a certain chemical group and/or amino acid in a perdeuterated environment. Perdeuteration is essential for the optimal use of Transverse Relaxation Optimized Spectroscopy pulse sequences. They allowed significant spectral gains when samples were specifically labeled on amide groups or on the methylene of glycines while maintaining a very high rate of deuteration on the other chemical functions of the proteins. The third protein labeling strategy employed is based on in vivo protocols but used in innovative NMR applications: a technique of hyperpolarization of nuclei in solution which increases their sensitivity by several orders of magnitude. The lifetime of this hyperpolarization is governed by the longitudinal relaxation time of nuclei, which are reduced for proteins at room temperature. By isolating the nuclei of interest in a perdeuterated environment, dipolar interactions created by neighboring protons were eliminated and hyperpolarized nuclei relaxed much more slowly. Hyperpolarization of a small protein domain was successfully undertaken at 1K but the dissolution conditions need to be improved in order to preserve a homogeneous aqueous phase.; La résonance magnétique nucléaire (RMN) fournit des informations structurelles et dynamiques précieuses à l'échelle atomique, cependant, la faible sensibilité et résolution des signaux empêchent l’étude d'objets moléculaires plus importants. Nous présentons 3 stratégies de marquage isotopique pour différentes expériences RMN des protéines en solution et démontrons leur potentiel pour l'étude structurale des biomolécules. Parmi les stratégies envisagées, 2 utilisent l'expression in vitro pour obtenir des protéines marquées sélectivement sur un groupe chimique et/ou acide aminé dans un environnement perdeutéré. Avec l’utilisation de séquences d'impulsions TROSY, ces échantillons ont permis des gains spectraux importants lorsque ils étaient spécifiquement marqués sur des groupes amide ou sur le méthylène des glycines tout en maintenant un taux de deutération élevé sur les autres fonctions chimiques des protéines. La troisième stratégie de marquage protéique utilise des protocoles in vivo pour des applications RMN innovantes: l'hyperpolarisation de noyaux en solution qui augmente leur sensibilité de plusieurs ordres de grandeur. La durée de vie de cette hyperpolarisation est régie par le temps de relaxation longitudinale des noyaux, qui est réduit pour les protéines à température ambiante. En isolant les noyaux d'intérêt dans un environnement perdeutéré, les interactions dipolaires créées par les protons voisins sont éliminées et les noyaux hyperpolarisés relaxent beaucoup plus lentement. L'hyperpolarisation d'un petit domaine protéique a été entreprise avec succès mais les conditions de dissolution doivent encore être améliorées pour conserver une phase aqueuse homogène.

Metalla-bis-dicarbollides, such as the COSAN anion [Co(C2B9H11)2]-, have attracted much attention in biology but a deep understanding of their interactions with cell-components is still missing. For this PhD work, we first studied the interactions of COSAN with glucose moiety, a neutral chemical function that is ubiquitous at biological interfaces. Octyl-glucopyranoside surfactant (C8G1) was chosen as a model as it self-assembles in water into micelles and creates a hydrated glucose-covered interface. Using complementary scattering and spectroscopic techniques, we have deduced from experiments that at low COSAN content and below the critical micellar concentration (CMC) of C8G1, COSAN binds to C8G1 monomers through the hydrophobic effect. Above the CMC of C8G1, COSAN adsorbs onto C8G1 micelles through the superchaotropic effect. At high COSAN concentrations, COSAN disrupts C8G1 micelles and the assemblies become similar to COSAN micelles but with small amount of solubilized C8G1. Therefore, COSAN binds in a versatile way to C8G1 upon either the hydrophobic or the superchoatropic effect depending on their relative concentrations. Then we extended this study to another type of interface, a compressed Langmuir monolayer containing glycolipids over a subphase containing various concentration of COSAN and also other types of nano-ions such as polyoxometalates. Varying the headgroup chemistry (one or two galactoside functions) and the alkyl chain saturation we observed different behaviour of nano-ion in interaction with the subsurface of the glycolipids and this as a function of the surface pressure. Using synchrotron techniques, we found superchaotrope nano-ions interact preferentially with species in gas or in the liquid expended phases than with the solid phase whose crystalline structure is not affected. Lower the charge density of the nano-ions and weaker the interaction between the sugar head groups, stronger is the interaction between the nano-ions and the glycolipids. Finally, in a last part, more complex interfaces were considered and investigated such as protein’s. Using electrophoretic methods, we observed some preferential interaction of COSAN with proteins characterized by various secondary structures, a research opening that will be explored for another PhD subject., Les métalla-bis-dicarbollides, tels que l'anion COSAN [Co(C2B9H11)2]-, intéressent beaucoup les biologistes, mais il manque encore une compréhension approfondie de leurs interactions avec les composants cellulaires. Pour ce travail de thèse, nous avons d'abord étudié les interactions de COSAN avec une fonction sucre neutre type glucose qui est omniprésente aux interfaces biologiques. Le tensioactif octyl-glucopyranoside (C8G1) a été choisi comme molécule modèle car il s'auto-assemble sous la forme de micelle directe dans l'eau et crée une interface recouverte de glucose hydraté. En utilisant des techniques complémentaires de diffusion et de spectroscopie, nous avons déduit des expériences qu'à une faible teneur en COSAN et en dessous de la concentration micellaire critique (CMC) du C8G1, l’anion COSAN se lie aux monomères C8G1 par l'effet hydrophobe. Au-dessus de la CMC de C8G1, le COSAN s'adsorbe sur les micelles de C8G1 par effet superchaotropique. À des concentrations élevées de COSAN, l’anion COSAN impacte les micelles C8G1 pour former des assemblages plus petits, similaires aux micelles de COSAN et solubilisant une partie du C8G1. Par conséquent, COSAN se lie de manière polyvalente à C8G1 par un effet hydrophobe ou superchotropique selon leurs concentrations relatives. Nous avons ensuite étendu cette étude à un autre type d'interface, une monocouche de Langmuir contenant des glycolipides et plus ou moins comprimée avec en sous-phase aqueuse différentes concentrations de COSAN mais également d'autres types de nano-ions tels que les polyoxométalates. En variant la chimie du groupe de tête (une ou deux fonctions galactosides) et la saturation de la chaîne alkyle, nous avons observé un comportement différent du nano-ion en interaction avec les têtes polaires glycolipidiques et ce en fonction de la pression de surface. En utilisant les techniques X de synchrotron, nous avons constaté que les nano-ions superchaotropes interagissent préférentiellement avec les espèces dans les phases gazeuses ou dans les phases liquides expansées plutôt qu'avec les phases solides dont la structure cristalline n'est pas affectée par la présence des nao-ions. Plus la densité de charge des nano-ions est faible et plus l'interaction entre les groupes de tête de sucre en surface est faible, plus l'interaction entre les nano-ions et les glycolipides est forte. Enfin, dans une dernière partie, des interfaces plus complexes ont été étudiées, comme celle des protéines. En utilisant des méthodes électrophorétiques, nous avons observé une interaction préférentielle du COSAN avec des protéines caractérisées par diverses structures secondaires, une ouverture de recherche qui sera explorée dans un autre sujet de doctorat.

Protein functions in living organism are governed by their 3D structure. This thesis focuses on the characterization of protein conformation and conformational dynamics by means of mass spectrometry, and participates to the development of tools for structural biology. Mass spectrometry coupled to time resolved optical spectroscopy can set light on mechanisms associated with conformational dynamics: photon absorption, electronic relaxation, photo-induced charge transfer, etc. Experimental approaches combining mass spectrometry, optics and molecular biology (protein expression, gel electrophoresis, circular dichroism, etc.) will be presented in the first section of this manuscript. This thesis work was initiated by the study of the photo-induced activation of the chromophores which were used, at ILM, for action-FRET and more generally for the exploration of the proteins structure. The mechanisms of photon absorption and non-radiative relaxation were studied for chromophores grafted on multiple peptides. We then probed the photo-induced intramolecular charge transfer and associated conformational dynamics in series of small peptides in order to understand the influence of the system size and composition on its structural dynamics. The latter experiment required the implementation of a two-color pump-probe setup. Finally, we used ion mobility spectrometry to probe and investigate the conformational space and dynamics of the PUMA peptide, ubiquitous in mammalian organisms and associated to the regulation of apoptosis; Les protéines sont des molécules dont la structure est liée à leur fonction dans les organismes vivant. Cette thèse porte sur la caractérisation de ces structures et de leur dynamique et s'inscrit dans le développement de l'analyse par spectrométrie de masse en biologie structurale. La spectrométrie de masse couplée à des techniques de spectroscopie optique résolue en temps permet de mettre en lumière les mécanismes d'absorption, de relaxation et de transfert de charges photo-induits impliqués dans la dynamique conformationnelle et électronique de ces molécules biologiques. Ces approches expérimentales avec la combinaison de techniques de biologie moléculaire (expression, gel électrophorèse, dichroïsme circulaire, etc.) sont présentées en début de manuscrit. C'est par l'étude de l'activation photo-induite de chromophores utilisés pour l'analyse de la structure de protéines qu'a commencé ce travail de thèse. Les mécanismes d'absorption de photons et de relaxation non radiative de chromophores greffés sur des peptides ont été sondés. Nous avons ensuite sondé les mécanismes de transferts de charges photo-induits au sein de petits peptides afin de comprendre l'influence de la taille de ces systèmes et de la composition en acides aminés sur la dynamique conformationnelle de ces peptides. Ceci a nécessité le montage d'un dispositif pompe-sonde pour l'étude de la dynamique de transfert de charge. Enfin nous nous sommes intéressés à un processus de changement de conformation du peptide PUMA, présent dans les organismes de mammifères et impliqué dans la régulation de l'apoptose, en utilisant la spectrométrie de mobilité ionique comme sonde de conformation et de dynamique structurale

Ficus species have adapted to diverse environments and pests by developing physical or chemical protection strategies. Physical defences are based on the accumulation of minerals such as calcium oxalate crystals, amorphous calcium carbonates and silica that lead to tougher plants. Additional cellular structures such as non-glandular trichomes or laticifer cells make the leaves rougher or sticky upon injury. Ficus have also established structures that are able to produce specialized metabolites (alkaloids, terpenoids, and phenolics) or proteins (proteases, protease inhibitors, oxidases, and chitinases) that are toxic to predators. All these defence mechanisms are distributed throughout the plant and can differ depending on the genotype, the stage of development or the environment. In this review, we present an overview of these strategies and discuss how these complementary mechanisms enable effective and flexible adaptation to numerous hostile environments.

Negli ultimi decenni, lo sviluppo di nuove tecniche di singola molecola ha creato le basi per nuovi paradigmi nel campo della biofisica. In particolare, la nanomanipolazione di singole biomacromolecole ha permesso la caratterizzazione meccanica di proteine e DNA, cercando di evidenziare la relazione fondamentale tra struttura e funzione biologica. Nel corso degli anni sono stati sviluppati diversi metodi di nanomanipolazione: tra questi, il Microscopio a Forza Atomica (AFM), il Magnetic Tweezers (MT) e il Flow-Stretching (F-S) accoppiato a fluorescenza. Queste tre tecniche sono state utilizzate per la caratterizzazione di singole biomacromolecole con tecniche di spettroscopia di forza a livello di singola molecola (SMFS) in quattro diversi progetti. In questa Tesi mi sono occupata principalmente di caratterizzare strutturalmente diverse proteine e correlarne la conformazione alla funzione biologica. In questa ottica, sono state studiate le diverse conformazioni strutturali di una proteina intrinsecamente disordinata (IDP) coinvolta nella malattia del Parkison’s (PD), chiamata alfa-synucleina (AS) e il riarrangiamento strutturale di una proteina coinvolta nel mantenimento strutturale cromosomico, la condensina, durante la condensazione di singoli filamenti di DNA. Inoltre, è stata studiata la struttura di un DNA-analogo sia caratterizzandone la stabilità termica sia attraverso studi di nanomeccanica in SMFS. Infine, è stata descritta una implementazione tecnica di un apparato di F-S accoppiato con illuminazione TIRF, per ottenere una rapida sostituzione di soluzioni durante esperimenti di microfluidica, ottenendo un setup particolarmente indicato per studi di interazione tra DNA e proteine. Durante lo studio della proteina AS in SMFS, il problema della mancanza di una struttura secondaria ben definita è stato risolto con l’impego di una singola poliproteina, contenente un modulo di AS umana. Sono state caratterizzati tre diversi stati conformazionali per AS, da uno stato totalmente destrutturato a una conformazione altamente strutturata. I riarrangiamenti conformazionali di AS sono stati studiati anche in presenza di gallato epigallocatechina (EGCG) e dopamina (DA). Una particolare attenzione è stata quindi riservata al confronto tra i risultati ottenuti da tecniche di SMFS e quelli precedentemente ricavati da analisi di spettroscopia di massa. La proteina AS è stata caratterizzata anche in presenza di tre diverse mutazioni puntuali legate al PD (A30P, A53T e E83A). Nel secondo progetto, un analogo del DNA (DAP) è stato caratterizzato sia con metodi di stabilità termica che di nanomeccanica, studiando il comportamento del DAP-DNA in presenza di forze esterne. L’estensione e la rigidità delle due molecole di DNA (DAP e WT) sono state caratterizzate a basse forze (AFM e MT) e alte forze (MT), dove è stata descritta la transizione di overstretching. Nel terzo progetto è stato studiato l’effetto della condensina sulla condensazione di singoli filamenti di DNA. In particolare, è stato effettuato uno studio di singola molecola per seguire, in tempo reale, la riduzione di estensione di DNA in presenza di condensina e ATP. Considerato che la maggior parte delle curve sono caratterizzate da improvvisi e ben evidenti salti durante la condensazione, sono stati sviluppati e validati due diversi algoritmi per l’identificazione automatica dei gradini. Infine, diverse celle fluidiche sono state testate nell’apparato di F-S, nell’ottica di studi di interazione tra proteine e DNA. Queste celle sono state caratterizzate sia in termini di rapidità di scambio di flussi laminari che di forza capaci di generare sul campione. Sono stati infine visualizzate singole molecole di DNA fluorescenti in presenza di flussi di diversa portata. In the last few decades, the constant development of novel single molecule techniques has created the basis for a new paradigm in the field of biophysics. Among all, the nanomanipulation of individual biomolecules revealed new insights into the mechanics of biological molecules, in particular proteins and DNA, improving the understanding of the fundamental relation between structural properties and biological functions. Therefore, several single-molecule nanomanipulation methods have been developed, including Atomic Force Microscopy (AFM), Magnetic Tweezers (MT) and Flow Stretching (F-S) coupled with fluorescence. All these technique were employed in this Thesis for the characterisation of biological macromolecules by single molecule force spectroscopy (SMFS). In this Thesis I focus mainly on several aspects of a few different proteins trying to depict a frame in which the strong link between proteins function and their structure can be clarified. With this aim, I study the conformational states of an intrinsically disordered protein (IDP) involved in Parkinson's Disease, the a-synuclein (AS) and the structural change driving the DNA compaction mediated by structural maintenance protein, the condensin. Finally, I present a structural study of a DNA-analogue by thermal shifting essays and single molecule experiments. I included also a technical implementation of a (F-S) combined with TIRF set up to promote the high-speed exchanging buffer for study protein DNA interactions. In the AS single molecule force spectroscopy (SMFS) study, I afford the problem of AS lacking of well defined structure by stretching and unfolding a single polyprotein containing the human AS by employing a SMFS approach. The analysis of the different unfolding pathways gives information about the structural conformation of the protein before the mechanical denaturation. The AS was found to assume three distinct conformational states ranging from a random coil to a highly structured conformation. Since ligands, such as Epigallocatechin-3-Gallate (EGCG) and Dopamine (DA), are known to affect the fibrillation process of AS, I used this single molecule technique to investigate the effect of EGCG and DA on the conformational ensemble of the WT AS. Moreover, knowing from several studies that the presence of point mutations, linked to familial PD, correlate with the gaining of structure and therefore with AS aggregation, I SMFS studies also on AS with three different single point mutations (A30P, A53T and E83A). A particular emphasis was given to the comparison between SMFS results and native mass spectrometry data for the conformational changes of AS in the presence of both DA and EGCG. In the following part, related to the DAP: diaminopurine-substituted DNA, a systematic comparison between a wild-type DNA and DAP DNA is performed, in terms of thermal stability and nanomechanical properties, measured at low and high forces. At low forces the DNA extension and bending rigidity were investigated, by using both MT and AFM, while at high forces the overstretching transition behaviour was explored. In the section related to condensin mediated DNA collapsing, I present a single-molecule MT study to measure, in real-time, the compaction of individual DNA molecules by the condensin complex in the presence of ATP. Since many compaction traces showed sudden distinct decreases in the DNA end-to-end length, I present and validate two different very conservative user-bias-independent step-finding algorithm to extract the size of these compaction steps. Finally, a DNA flow stretching implementation is presented. Briefly, several flow cells were tested to achieve a fast buffer exchange in both MT and F-S coupled with TIRF, in the frame of visualisation of DNA:proteins interactions. We validated our flow cells in term of boundary exchange and applied force. We also visualized fluorescent DNA molecules stretched in the presence of several flow rates.

International audience; Replacement of Brazilian soybean meal (BS) associated to deforestation with European protein sources (EPS) was studied to reduce the impact of feed production on climate change. This study examined four EPS: the fine fraction of rapeseed meal (EPS1), soybean meals obtained from an innovative extrusion process with dehulled soybeans (EPS2) or non-dehulled soybeans (EPS3), and Danish protein pastes extracted from green biomass (EPS4). The goal was to estimate environmental impacts of pig production resulting from incorporating EPS in feeds for fattening pigs. Environmental impacts per kg of pig liveweight at the farm gate were estimated using life cycle assessment. Potential benefits were assessed by comparing each scenario with EPS to reference scenarios with “classic” protein sources (including BS). The study considered the recent economic context of four countries (France, Germany, Spain and the Netherlands) for four contrasting years to assess the current potential for using EPS. A second, virtual, context was defined with scenarios favorable to either BS or EPS, and the potential benefit of EPS was then appraised. In the recent economic context, results showed a moderate benefit with EPS because SB had low incorporation rates in the reference formulas (

Latexes in immature fruit, young petioles and lignified trunks of fig trees protect the plant using toxic proteins and metabolites in various organ-dependent ways. Latexes from plants contain high amounts of toxic proteins and metabolites, which attack microbes and herbivores after exudation at pest-induced wound sites. The protein and metabolite constituents of latexes are highly variable, depending on the plant species and organ. To determine the diversity of latex-based defense strategies in fig tree (Ficus carica) organs, we conducted comparative proteomic, transcriptomic and metabolomic analyses on latexes isolated from immature fruit, young petioles and lignified trunks of F. carica after constructing a unigene sequence library using RNA-seq data. Trypsin inhibitors were the most abundant proteins in petiole latex, while cysteine proteases ("ficins") were the most abundant in immature fruit and trunk latexes. Galloylglycerol, a possible defense-related metabolite, appeared to be highly accumulated in all three latexes. The expression levels of pathogenesis-related proteins were highest in the latex of trunk, suggesting that this latex had adapted a defensive role against microbe attacks. Although young petioles and immature fruit are both unlignified soft organs, and potential food for herbivorous insects, unigenes for the sesquiterpenoid pathway, which likely produces defense-associated volatiles, and the phenylpropanoid pathway, which produces toxic furanocoumarins, were expressed less in immature fruit latex. This difference may indicate that while petioles and fruit protect the plant from attack by herbivores, the fruit must also attract insect pollinators at younger stages and animals after ripening. We also suggest possible candidate transcription factors and signal transduction proteins that are involved in the differential expression of the unigenes.