Les travaux de cette thèse portent sur le développement d'une méthodologie permettant le passage au continu d'une production de cristaux d'un principe actif pharmaceutique : l'acétyl-L- leucine. Le travail est centré sur l'étape de précipitation et ses interactions avec les procédés en aval de filtration / lavage et séchage. La démarche adoptée fut donc d'abord d'acquérir un ensemble de données analytiques et thermodynamiques sur les produits, sous-produits et cristaux. L'impact de plusieurs paramètres opératoires sur la solubilité du produit fut ainsi mis en évidence. D'autre part, on a montré que l'acétyl-DL- eucine cristallise sous la forme d'un conglomérat. Des études ont ensuite été réalisées sur les procédés en discontinu, notamment sur les étapes de précipitation et de lavages, afin d'identifier les paramètres permettant de contrôler au mieux la qualité finale des cristaux. Enfin, à partir de ces résultats, des essais de précipitation en continu ont pu être réalisés à l'échelle du laboratoire, puis à l'échelle industrielle sur l'ensemble de la chaîne de production du principe actif. / This study deals with the development of a methodology allowing to transform a batch precipitation process in a continuous one. The active substance considered is acetyl-L-leucine. The work is focused on the precipitation step but also considers the interactions with the downstream processes of filtration, washing and drying. In a first part, characterization methods of pure and impure solutions of active substance were developed as well as methods for determining the crystals properties (purity, size, size distribution, shape, ...). Then the solubility of the product was investigated and the influence of parameters such as temperature, pH, impurity concentration, was studied. The crystallization mode of the racemic mixtures was also determined . Finally, batch crystallization was carried out at the laboratory scale in order to determine nucleation kinetics and to understand the main operating parameters influencing the quality of the crystals. For the applied range of concentrations, the induction time was found very small and indicates that the control of the nucleation step is difficult and depends on the mixing of the reactants. The whole continuous process was finally designed and tested, from the initial reactive to the final dry ZPI powder, in industrial site to validate the feasibility of the continuous process

La myostatine est un membre de la superfamille du TGF-β (transforming growth factor-β) impliqué dans la régulation négative de la masse musculaire. En effet, l’absence de myostatine (MSTN) chez la souris est responsable d’un phénotype hypermusclé. Depuis, il a été confirmé qu’une baisse de l’activité de la MSTN conduit à une augmentation de la masse musculaire chez d’autres espèces, y compris l’Homme. L’identification de la MSTN et des conséquences de son invalidation sur le développement musculaire ouvre de nombreuses perspectives en médecine humaine. Il existe de nombreuses situations pathologiques qui conduisent à une fonte musculaire importante : c’est le cas pour des maladies génétiques telles que les dystrophies musculaires ou pour d’autres pathologies comme le cancer et le sida. Différentes approches anti-MSTN ont été développées au cours des dernières années, par exemple un anticorps anti-MSTN ou des ligands de la MSTN. L’objectif majeur de ce projet de recherche a consisté à identifier de nouveaux inhibiteurs de la MSTN, en particulier appartenant à la famille de protéines appelées SLRP (Small Leucine Rich Proteoglycans). Il a été mis en évidence que des membres de cette famille, notamment la décorine (DCN) ainsi que des fragments issus de la DCN dont le peptide 31-71, sont capables de se lier à la MSTN en présence de zinc. La DCN peut alors empêcher l’activité de la MSTN en s’opposant à la liaison de cette dernière à son récepteur. Dans ce contexte, nous avons étudié des séquences peptidiques plus restreintes de la DCN murine pouvant interagir efficacement avec la MSTN et des peptides dérivés d’autres SLRP pour leur aptitude à lier la MSTN. Afin de faciliter le criblage in vitro de ces composés, nous avons tout d’abord créé une lignée cellulaire HEK293T exprimant stablement une cassette inductible par la MSTN fusionnée au gène de la luciférase (pCAGA-Luc). Parmi les candidats testés, le peptide mDCN48-71 a été le plus intéressant de par sa forte activité anti MSTN in vitro comparée aux autres, avec un IC50 de 7 µM. Notons également que le peptide mDCN48-71 n’a pas inhibé d’autres membres de la superfamille du TGF-β : TGF-β2, activine A et GDF-11 – ce qui suggère une spécificité d’action du peptide. En outre, des études d’anisotropie de fluorescence ont permis de prouver l’interaction directe du peptide mDCN48 71 avec la MSTN et la dépendance au zinc de cette liaison. Pour finir, nous avons montré que des injections intramusculaires répétées de ce peptide chez le modèle murin dystrophique mdx, conduisent à une augmentation significative de la masse des muscles tibiaux antérieurs injectés de l’ordre de 21 % par rapport aux muscles contrôles. / Myostatin is a member of the transforming growth factor-β (TGF-β) superfamily and a negative regulator of skeletal muscle growth. In 1997, lack of myostatin (MSTN) was related to increased muscle mass in mice. Since then, MSTN has been found in other species including humans. Inhibition of this protein offers opportunities in human medicine for many pathological conditions leading to a significant muscle loss: genetic disorders such as muscular dystrophy as well as other diseases like cancer and AIDS. Recently, several anti-MSTN approaches have been developed such as antibodies against MSTN or naturally occurring proteins that bind to and inactivate MSTN. The aim of this research was to identify novel inhibitors of MSTN, especially belonging to the SLRP (Small Leucine Rich Proteoglycans) family of proteins. Members of this family, including decorin (DCN) and fragments thereof (murine derived peptide mDCN31-71) can bind to MSTN in a zinc-dependent manner. In this context, smaller peptide sequences of mouse DCN and peptides from other SLRP have been studied for their ability to bind MSTN. First, we created a HEK293T stable cell line expressing the luciferase gene under control of a MSTN-inducible promoter (pCAGA-Luc) so as to screen these compounds in vitro. Here we report that the peptide mDCN48-71 shows the stronger activity anti MSTN in vitro among all the peptides tested (IC50 = 7 µM). Furthermore, other members of the TGF β superfamily: TGF β2, activin A and GDF-11 are not inhibited by the mDCN48-71 peptide - which suggests a specificity of its action. By performing fluorescence anisotropy studies, we proved the direct and zinc dependent interaction between peptide mDCN48-71 and MSTN. Finally, we showed that repeated intramuscular injections of this peptide in the dystrophic mdx mouse model led to a significant increase of the injected tibialis anterior muscle mass (21 %) compared to control muscles.

La perte de masse et de force musculaires liée à l’âge, ou sarcopénie, pourrait être partiellementexpliquée par un défaut de récupération de masse musculaire après des épisodes générateursd’atrophie musculaire. Ainsi, les périodes d’immobilisation qui augmentent avec l’âge (alitement,convalescence, fracture) pourraient être suivies d’une absence de récupération musculaire etcontribuer à la fonte musculaire au cours du vieillissement. Les causes de ce défaut de récupérationimpliquent notamment un déséquilibre du taux de renouvellement protéique et du taux derenouvellement cellulaire. L’objectif de cette thèse a donc été de mettre en évidence les mécanismesresponsables de l’atrophie musculaire chez le rat âgé au cours de l’immobilisation et ceux quiseraient défaillants afin de déceler les mécanismes à cibler pour favoriser la récupérationmusculaire.Des rats âgés ont été immobilisés pendant 8 jours par plâtrage unilatéral de la patte arrière, puislaissés en récupération pendant 40 jours après le déplâtrage. Nous avons montré que chez cesanimaux nourris avec un régime contenant 13% de caséine, l’immobilisation entraîne une atrophiedes muscles immobilisés mais, contrairement au rat adulte, le rat âgé ne récupère jamais la massemusculaire perdue. L’atrophie des muscles immobilisés peut être expliquée par 1/ une augmentationde l’apoptose et de la protéolyse ubiquitine-protéasome-dépendante musculaires, 2/ une diminutionde la régénération des cellules musculaires et 3/ une diminution de la protéosynthèse musculaire àl’état nourri. Tous ces phénomènes pourraient résulter de la présence d’un fort stress oxydant etd’une importante inflammation intramusculaire. Tous ces paramètres sont normalisés dès 10 jours derécupération, ce qui permet de stopper l’atrophie mais ne permet pas d’initier la phase derécupération musculaire. Nous avons donc testé l’effet de différentes supplémentationsnutritionnelles au cours de la période de récupération afin de favoriser un gain de masse musculairepost immobilisation. Des supplémentations en leucine (acide aminé bien connu pour stimuler laprotéosynthèse et inhiber la protéolyse) ont ainsi été réalisées. Chez les rats supplémentés, uneamélioration de la synthèse protéique et une normalisation plus précoce des activités protéolytiquesdu protéasome ont été observées. Cependant cette amélioration du métabolisme protéique ne s’estpas traduite par un gain de masse musculaire. Par contre, la modulation qualitative et quantitative desapports en protéines a pu permettre d’obtenir une récupération significative de masse musculaire :ainsi des régimes contenant 13% de lactosérum et des régimes hyper-protéinés ont permis de gagner50% de la masse perdue et ce, dès 20 jours de récupération.Nos résultats montrent que l’immobilisation chez le rat âgé aggrave la sarcopénie. Une fortealtération du métabolisme protéique permet d’expliquer la perte de muscle et la seule normalisationde la protéolyse et de la protéosynthèse permet d’expliquer l’absence de récupération musculaire.Nous avons montré que la modulation des apports en protéines au cours de la phase de récupérationpouvait permettre un gain de protéines. / Sarcopenia, the age-related muscle mass loss, might be partially explained by an impairedmuscle mass recovery of skeletal muscle mass after a catabolic state. Thus, immobilization periodswhich increase with aging could induce a muscle atrophy followed by a lack of muscle massrecovery. An imbalance of protein and cellular metabolisms are certainly involved in this absenceof recovery. The aim on this Ph.D thesis was to explore the mechanisms involved in muscle massatrophy during immobilization and their possible alteration during the recovery period in old rats.Old rats were immobilized for 8 days by unilateral hind limb casting and then allowed torecover for 40 days. Our results showed that animals fed a 13% casein diet wasted muscle mass inimmobilized muscles but, contrarily to adult animals, they never recovered the muscle mass loss.Muscle atrophy was due to 1/ an increase of apoptotic and ubiquitine-proteasome-dependentproteolytic pathways, 2/ a decrease of muscle regeneration processes and 3/ a decrease of muscleprotein synthesis at the fed state. These changes paralleled an increase of intracellular inflammationand oxidative stress. As these parameters were only normalized during the recovery period, theresultant nitrogen balance was then not enough positive as required for the muscle protein gain,hence contributing to the age-related incomplete muscle mass recovery. We tested free leucinesupplementation (an amino acid known for its stimulatory effect on protein metabolism) during therecovery period to improve muscle mass gain. This supplementation induced a greater muscleprotein synthesis in supplemented animals, but without any muscle mass gain. However, wedemonstrated here for the first time that muscle protein accretion after immobilization-inducedatrophy could be achieved with whey protein or high protein diets.In conclusion, we demonstrated that immobilization in old rats induced a muscle mass atrophyfollowed by an incomplete recovery, hence contributing to the development of sarcopenia. We alsodemonstrated that this lack of recovery cannot be overcome by a dietary free leucinesupplementation, despite a positive effect on protein metabolism, contrarily to high protein andwhey protein diets.

Carnobacterium maltaromaticum est une bactérie lactique atypique isolée de fromages à pâte molle. Les caractéristiques majeures de cette bactérie sont i) sa capacité à produire un arôme de type malté ou chocolaté, le 3-méthylbutanal à partir du catabolisme de la leucine ii) sa capacité à produire des bactériocines, peptides à activité antibactérienne vis-à-vis de flores pathogènes et/ou d'altération fréquemment rencontrées en industries alimentaire, telle que Listeria monocytogenes. Une caractérisation phénotypique et génotypique réalisée sur six souches de C. maltaromaticum isolées du même biotope a montré que celles-ci ne sont pas phylogénétiquement identiques. Même si ces souches n'ont pas la capacité à coaguler rapidement le lait, elles sont acidotolérantes. Elles n'influent pas sur la capacité ni la rapidité de coagulation des starters, Lactococcus lactis et Streptococcus thermophilus utilisés en industrie laitière. L'étude de l'impact de C. maltaromaticum sur la flore bactérienne du fromage a révélé que sa présence provoque la diminution de la concentration de Psychrobacter, germe pouvant être responsable d'une accélération du phénomène de vieillissement du fromage. Un plan d'expérience a été réalisé pour mettre en évidence ces inhibitions et les éventuelles interactions entre différents facteurs (concentration cellulaire initiale, concentration en NaCl, pH, durée d'incubation). Deux modèles ont été choisis i) Psychrobacter sp. et ii) L. monocytogenes. La concentration cellulaire de C. maltaromatiucm est le facteur le plus important pour inhiber les bactéries testées. Cette espèce opportuniste pourrait être considérée comme un auxilliaire de fabrication intéressant et pourrait être retenue comme flore bactérienne d'affinage. Toutes les souches de C. maltaromaticum testées sont capables de produire du 3-méthylbutanal pouvant conférer une flaveur maltée au fromage. Les études menées sur la biosynthèse du 3-méthylbutanal ont montré la présence et la fonctionnalité de deux voies métaboliques; la voie directe impliquant l'alpha-cétoacide décarboxylase et la voie indirecte passant par l'alpha-cétoacide déshydrogénase. L'oxygénation du milieu de culture a un impact positif sur la formation du 3-méthylbutanal et du 3-méthylbutanol avec la stimulation des voies directes et indirectes / Carnobacterium maltaromaticum is a lactic acid bacterium isolated from atypical soft cheeses. The major characterstics of this bacterium are i) its ability to produce a malty or chocolate-like flavor 3-methylbutanal from leucine catabolism ii) its ability to produce bacteriocins against pathogenic and spoilage bacteria for instance, Listeria monocytogenes. Phenotypic and genotypic characterization of six strains of C. maltaromaticum isolated from the same habitat showed that they were not phylogenetically identical. Although these strains lacked the ability to coagulate the milk quickly, they were acid tolerant. They did not affect the milk coagulation capacity of startes, Lactococcus lactis and Streptococcus thermophilus used in dairy industry. The study on the impact of C. maltaromaticum on the bacterial flora of cheese showed that its presence resulted in the decrease of the concentration of Psychrobacter, which might be responsible for accelerating the aging phenomenon of cheese. An experimental plan was realized to highlight these inhibitions and possible interactions between factors (cell concentration, NaCl, pH, incubation time) by choosing two models i) Psychrobacter sp. and ii) L. monocytogenes. The cell concentration of C. maltaromatiucm was the factor more significant for the inhibitions of the tested bacteria. Being psychrotrophic, alkalinophilic, malty or chocolate flavor producing and biopreservative agent, this species could play a role as a ripening flora of cheese. All strains of C. maltaromaticum tested were able to produce 3-methylbutanal conferring any malt flavor to cheese. The results on the physiology involved in the biosynthesis of 3-methylbutanal showed the presence and functionality of both metabolic pathways; the direct by alpha-ketoacid dacarboxylase enzyme and indirect comprising alpha-keto acid dehydrogenase enzyme. The oxygenation of culture medium had a positif impact on the formation of 3-methylbutanal and 3-methylbutanol with the stimulation of both direct and indirect metabolic routes

Chan, Ka Man Carmen. / "October 2009." / Thesis (M.Phil.)--Chinese University of Hong Kong, 2010. / Includes bibliographical references (leaves 160-165). / Abstracts in English and Chinese. / Table of Contents --- p.I / Acknowledgements --- p.IV / Abstract --- p.V / Abstract (Chinese version) --- p.VIII / List of Figures --- p.X / List of Tables --- p.XVIII / Chapter Chapter One. --- Introduction / Chapter 1.1 --- Pulmonary drug delivery --- p.1 / Chapter 1.2 --- Inhalation drug delivery systems --- p.4 / Chapter 1.3 --- Dry powder inhalation aerosols --- p.5 / Chapter 1.3.1 --- Principle of operation of DPIs --- p.5 / Chapter 1.3.2 --- Aerodynamic diameter --- p.6 / Chapter 1.3.2.1 --- Fine particle fraction --- p.8 / Chapter 1.3.3 --- Dispersibility --- p.8 / Chapter 1.3.4 --- Factors that affect dispersibility --- p.9 / Chapter 1.3.4.1 --- Particle Size --- p.9 / Chapter 1.3.4.2 --- Particle Density and Morphology --- p.10 / Chapter 1.3.4.3 --- Interparticulate interactions一Cohesion and adhesion --- p.11 / Chapter 1.3.4.3.1 --- Surface energetics --- p.11 / Chapter 1.3.4.3.2 --- Effect of hygroscopicity and electrostatic charges --- p.12 / Chapter 1.4 --- Particle formation techniques for DPI formulation --- p.14 / Chapter 1.4.1 --- Spray-drying --- p.14 / Chapter 1.4.2 --- Surface modification --- p.16 / Chapter 1.5 --- Physical characterization --- p.17 / Chapter 1.5.1 --- Laser diffraction --- p.17 / Chapter 1.5.2 --- X-ray powder diffraction --- p.18 / Chapter 1.5.3 --- Thermal analysis --- p.19 / Chapter 1.5.4 --- Particle morphology and surface area --- p.20 / Chapter 1.5.5 --- In vitro aerosol performance --- p.21 / Chapter 1.6 --- Surface characterization --- p.21 / Chapter 1.6.1 --- X-ray photoelectric spectroscopy (XPS) --- p.21 / Chapter 1.6.2 --- Inverse gas chromatography --- p.22 / Chapter 1.7 --- Atomic force microscopy in pharmaceutical science --- p.23 / Chapter 1.7.1 --- Principle of operation --- p.24 / Chapter 1.7.1.1 --- Tapping mode --- p.27 / Chapter 1.7.1.2 --- Contact mode --- p.27 / Chapter 1.8 --- Scope of thesis --- p.29 / Chapter Chapter Two. --- Materials and Methods / Chapter 2.1 --- Materials --- p.32 / Chapter 2.2 --- Methods --- p.32 / Chapter 2.2.1 --- Optimization of spray-drying parameters --- p.32 / Chapter 2.2.2 --- Preparation of spray-dried salbutamol sulphate powders containing different concentrations of amino acid additive --- p.33 / Chapter 2.2.3 --- Physical characterization of spray-dried powders --- p.34 / Chapter 2.2.3.1 --- Particle size and size distribution --- p.34 / Chapter 2.2.3.2 --- Specific surface area --- p.35 / Chapter 2.2.3.3 --- X-ray powder diffraction --- p.35 / Chapter 2.2.3.4. --- Scanning electron microscopy --- p.36 / Chapter 2.2.3.5. --- Thermal analysis --- p.36 / Chapter 2.2.3.5.1 --- Thermogravimetric analysis (TGA) --- p.36 / Chapter 2.2.3.5.2 --- Differential scanning calorimetry (DSC) --- p.36 / Chapter 2.2.3.6 --- Water vapour sorption isotherm --- p.37 / Chapter 2.2.3.7 --- Density measurements --- p.37 / Chapter 2.2.3.8 --- In vitro particle deposition (MSLI) --- p.38 / Chapter 2.2.4 --- Surface characterization of the spray-dried powders --- p.39 / Chapter 2.2.4.1 --- X-ray photoelectric spectroscopy (XPS) --- p.39 / Chapter 2.2.4.2 --- Surface energy measurement by inverse gas chromatography (IGC) --- p.40 / Chapter 2.2.4.2.1 --- Calculation of standard free energy of adsorption --- p.41 / Chapter 2.2.4.2.2 --- Dispersive component of surface free energy and related thermodynamic parameters --- p.42 / Chapter 2.2.4.2.3 --- Specific interactions and associated acid-base properties --- p.43 / Chapter 2.2.5. --- Atomic Force Microscopy (AFM) --- p.43 / Chapter 2.2.5.1. --- Imaging --- p.43 / Chapter 2.2.5.2. --- Force measurements --- p.44 / Chapter 2.2.5.2.1 --- Adhesion force measurements --- p.44 / Chapter 2.2.5.2.2 --- Force curve data conversions --- p.44 / Chapter Chapter Three. --- "Optimal Spray-drying Conditions, Physical Characterization and Aerosol Performance of Additive-modified Spray-dried Salbutamol Sulphate particles" / Chapter 3.1 --- Optimization of spray-drying conditions --- p.46 / Chapter 3.2 --- Effect of phenylalanine on the spray-dried SS particles --- p.52 / Chapter 3.2.1. --- Phenylalanine as the additive --- p.52 / Chapter 3.2.1.1 --- In vitro aerosol performance --- p.53 / Chapter 3.2.1.2 --- Particle morphology --- p.55 / Chapter 3.2.1.3 --- Crystallinity --- p.62 / Chapter 3.2.1.4 --- Particle size distribution and specific surface area --- p.63 / Chapter 3.2.1.5 --- Density --- p.65 / Chapter 3.2.1.6 --- Thermal analysis --- p.66 / Chapter 3.2.1.7 --- Water vapour isotherm --- p.70 / Chapter 3.3 --- Effect of leucine on the spray-dried SS particles --- p.77 / Chapter 3.3.1. --- L-Leucine as the additive --- p.77 / Chapter 3.3.1.1 --- In vitro aerosol performance --- p.78 / Chapter 3.3.1.2 --- Particle morphology --- p.80 / Chapter 3.3.1.3 --- Crystallinity --- p.86 / Chapter 3.3.1.4 --- Particle size distribution and specific surface area --- p.87 / Chapter 3.3.1.5 --- Density --- p.90 / Chapter 3.3.1.6 --- Thermal analysis --- p.92 / Chapter 3.3.1.7 --- Water vapour isotherm --- p.95 / Chapter Chapter Four. --- Surface Characterization of Additive-modified Spray-dried Salbutamol Sulphate Particles / Chapter 4.1 --- X-ray photoelectric spectroscopy --- p.103 / Chapter 4.1.1 --- Phenylalanine --- p.103 / Chapter 4.1.2 --- Leucine --- p.104 / Chapter 4.2 --- Inverse gas chromatography --- p.105 / Chapter 4.2.1 --- Phenylalanine --- p.105 / Chapter 4.2.2 --- Leucine --- p.107 / Chapter 4.3 --- Atomic force microscopy --- p.109 / Chapter 4.3.1 --- Surface topography --- p.109 / Chapter 4.3.2 --- Adhesive force measurements --- p.118 / Chapter Chapter Five. --- Conclusions and Suggestions for Future Works / Chapter 5.1 --- Conclusions --- p.139 / Chapter 5.1.1 --- Physical properties --- p.139 / Chapter 5.1.2 --- Surface characteristics and aerosol performance --- p.140 / Chapter 5.2 --- Future studies --- p.142 / Appendix --- p.143 / References --- p.160

Une cellule dendritique (CD) qui exprime le facteur de transcription GILZ durant l’apprêtement de l’antigène et sa présentation aux cellules effectrices, génère des lymphocytes T régulateurs (LTregs) CD25high Foxp3+ sécréteurs d’IL-10. La production de GILZ est dépendante de l’action des glucocorticoïdes, de l’IL-10 et du TGF-.Nous avons mis en évidence chez l’homme qu’une corticothérapie orale de 48h induit l’expression de GILZ dans les cellules présentatrices de l’antigène circulantes (CPAs) de sujets allergiques. Les CPAs isolées après la corticothérapie génèrent des LTregs CD25high Foxp3+ IL-10+ spécifiques de l’allergène.Nous également constaté in vitro que les mastocytes participent à l’activation des CDs au cours des réactions allergiques en régulant l’expression de GILZ. Les médiateurs d’origine mastocytaire, dont l’histamine, diminuent l’expression de GILZ dans les CDs et altèrent ainsi leur capacité à activer des LTregs. Nous avons identifié le mécanisme par lequel l’histamine diminue l’expression de GILZ dans les CDs humaines. L’histamine inhibe l’activité transcriptionnelle de Foxo3, un facteur de transcription régulant l’expression de GILZ. Enfin, nous avons démontré que des souris transgéniques qui surexpriment GILZ constitutivement dans les CDs sont protégées contre le développement de l’asthme allergique. L’ensemble de ces résultats permet d’envisager de nouvelles stratégies d’immunomodulation dans l’allergie, centrée sur la régulation de l’expression de GILZ dans les CDs. / We previously showed in vitro that DCs with a high level of GILZ activate regulatory T cells (Tregs) whereas DCs with low level of GILZ trigger effector T lymphocytes. Glucocorticoids (GCs), IL-10 and TGF- are potent inducers of GILZ expression. The aim of this thesis is to extend the above findings to induction of tolerance to allergens. Modulation of GILZ expression by DCs should induce allergen-specific Tregs able to inhibit the activation and proliferation of allergen specific T cell clones. In order to validate this concept we demonstrated that:- allergen-specific tolerance can be achieved in allergic patients treated with oral GC through the induction of GILZ expression in their antigen-presenting cells, and the role of allergen-specific Tregs in this effect,- mast cells play a role in the activation of DCs by inhibiting their expression of GILZ and thus their ability to stimulate Tregs against harmless environmental allergens,- GILZ-expressing DCs protect against allergic asthma in a model of transgenic mice over-expressing GILZ in their DCs.The present study supports the concept of an immune regulation of allergic responses through the modulation of GILZ expression by DCs and opens new perspectives in the development of innovative immunotherapies in the treatment of allergic diseases.

<p>Für das Verständnis der Strukturbildung bei Proteinen ist es wichtig, allgemein geltende Prinzipien der Stabilität und Faltung zu verstehen. Bisher wurde viel Arbeit in die Erörterung von Gesetzmäßigkeiten zu den Faltungseigenschaften von globulären Proteinen investiert. Die große Proteinklasse der solenoiden Proteine, zu denen z. B. Leucine-Rich Repeat- (LRR-) oder Ankyrin-Proteine gehören, wurde dahingegen noch wenig untersucht. Die Proteine dieser Klasse sind durch einen stapelförmigen Aufbau von sich wiederholenden typischen Sequenzeinheiten gekennzeichnet, was in der Ausbildung einer elongierten Tertiärstruktur resultiert. In der vorliegenden Arbeit sollte versucht werden, die Stabilität und Faltung eines LRR-Proteins mittels verschiedener biophysikalischer Methoden zu charakterisieren. Als Untersuchungsobjekt diente die für die Infektion ausreichende zentrale LRR-Domäne des Invasionsproteins Internalin B (InlB₂₄₁) des Bakteriums <i>Listeria monocytogenes</i>. Des weiteren sollten die Integrität und die Stabilitäts- und Faltungseigenschaften der sogenannten Internalin-Domäne (InlB₃₂₁) untersucht werden. Hierbei handelt es sich um die bei allen Mitgliedern der Internalinfamilie vorkommende Domäne, welche aus einer direkten Fusion des C-terminalen Endes der LRR-Domäne mit einer Immunglobulin (Ig)-ähnlichen Domäne besteht.</p> <p>Von beiden Konstrukten konnte eine vollständige thermodynamische Charakterisierung, mit Hilfe von chemisch- bzw. thermisch-induzierten Faltungs- und Entfaltungsübergängen durchgeführt werden. Sowohl InlB₂₄₁ als auch InlB₃₂₁ zeigen einen reversiblen und kooperativen Verlauf der chemisch-induzierten Gleichgewichtsübergänge, was die Anwendung eines Zweizustandsmodells zur Beschreibung der Daten erlaubte. Die zusätzliche Ig-ähnliche Domäne im InlB₃₂₁ resultierte im Vergleich zum InlB₂₄₁ in einer Erhöhung der freien Enthalpie der Entfaltung (8.8 kcal/mol im Vergleich zu 4.7 kcal/mol). Diese Stabilitätszunahme äußerte sich sowohl in einer Verschiebung des Übergangsmittelpunktes zu höheren Guanidiniumchlorid-Konzentrationen als auch in einer Erhöhung der Kooperativität des Gleichgewichtsübergangs (9.7 kcal/mol/M im Vergleich zu 7.1 kcal/mol/M). Diese Beobachtungen zeigen dass die einzelnen Sequenzeinheiten der LRR-Domäne nicht unabhängig voneinander falten und dass die Ig-ähnliche Domäne, obwohl sie nicht direkt mit dem Wirtszellrezeptor während der Invasion interagiert, eine kritische Rolle für die <i style='mso-bidi-font-style:normal'>in&nbsp;vivo</i> Stabilität des Internalin B spielt. Des weiteren spiegelt die Kooperativität des Übergangs die Integrität der Internalin-Domäne wieder und deutet darauf hin, dass bei beiden Proteinen keine Intermediate vorliegen.</p> <p>Kinetische Messungen über Tryptophanfluoreszenz und Fern-UV<span style='color:red'> </span>Circulardichroismus deuteten auf die Existenz eines relativ stabilen Intermediates auf dem Faltungsweg der LRR-Domäne hin. Faltungskinetiken aus einem in pH&nbsp;2 denaturierten Zustand zeigten ein reversibles Verhalten und verliefen über ein Intermediat. Eine Erhöhung der Salzkonzentration des sauer-denaturierten Proteins führte zu einer Kompaktierung der entfalteten Struktur und resultierte im Übergang zu einem alternativ gefalteten Zustand. Bei der Internalin-Domäne deuteten kinetische Messungen des Fluoreszenz- und Fern-UV Circulardichroismus-Signals während der Entfaltung möglicherweise auf die Präsenz von zwei Prozessen hin. Der erste langsame Entfaltungsprozess kurz nach dem Übergangsmittelpunkt zeigte eine starke Abhängigkeit von der Temperatur, während der zweite schnellere Prozess der Entfaltung stärker von der Guanidiniumchlorid-Konzentration abhing. Renaturierungskinetiken zeigten das Auftreten von mindestens einem Faltungsintermediat. Kinetische Daten aus Doppelsprungexperimenten lieferten für die Erklärung der langsamen Faltungsphase zunächst keinen Hinweis auf dass Vorliegen einer Prolinisomerisierungsreaktion. Die vollständige Amplitude während der Renaturierung konnte nicht detektiert werden, weswegen von einer zweiten schnellen Phase im Submillisekundenbereich ausgegangen werden kann.</p> <p>Die Ergebnisse der Faltungskinetiken zeigen, dass die InlB-Konstrukte als Modelle für die Untersuchung der Faltung von Solenoidproteinen verwendet werden können.<span lang=EN-GB style='mso-ansi-language: EN-GB'><o:p></o:p></span></p> / <p class=MsoBodyText><span lang=EN-GB style='mso-ansi-language:EN-GB'>To understand the processes of protein structure formation, it is necessary to investigate protein stability and protein folding kinetics. The focus of many folding studies has been directed at small, globular proteins. The larger class of solenoid proteins, including leucine-rich repeat (LRR) and ankyrin proteins, has not been extensively investigated. These proteins contain tandem repeat motifs, and their tertiary structure consists of a regular linear array of modules that stack to form non-globular elongated or supercoiled structures. In the present work, the folding and stability of the central LRR domain of the invasion protein internalin B (InlB₂₄₁) from the bacterium <i>Listeria monocytogenes</i> was characterized using different biophysical techniques. In addition, the integrity, stability and folding behavior of the so-called internalin-domain (InlB₃₂₁) was investigated. In this single domain, which is found in all members of the internalin-family, an immunoglobulin (Ig)-like domain is directly fused to the C-terminal end of the LRR domain.<span style='color:red'><o:p></o:p></span></span></p> <p class=MsoBodyText><span lang=EN-GB style='mso-ansi-language:EN-GB'>A complete thermodynamic characterization of the stability of both constructs was performed, using chemical- and temperature-induced folding and unfolding transitions. The reversible and cooperative equilibrium transition of InlB₂₄₁ and InlB₃₂₁ allowed the use of a two-state model for the description of the data points. The additional Ig-like domain present in InlB₃₂₁ resulted in an increase of the unfolding free energy (8.8 kcal/mol compared to 4.7 kcal/mol). This resulted both, from a shift of the transition midpoint to higher denaturant concentration, and from an increase in the <i>m</i>-value, the denaturant dependence of the unfolding free energy (9.7 kcal/mol/M compared to 7.1 kcal/mol/M). These observations suggest that the unravelling of the individual structural repeats in the LRR region is a cooperative process and that the tight fusion with the Ig-like domain leads to a dramatically increased stability <i>in vivo</i> without interfering with the functionality of the protein. In addition, the cooperativity of the equilibrium transition reflects the integrity of the internalin-domain, and suggests that both InlB fragments unfold without significantly populated equilibrium intermediates.<o:p></o:p></span></p> <p class=MsoBodyText><span lang=EN-GB style='mso-ansi-language:EN-GB'>Kinetic measurements with tryptophan fluorescence and far-UV circular dichroism are indicative for the existence of a relative stable intermediate on the folding pathway of the LRR domain. Refolding kinetics from an acid-denatured state showed a reversible behavior and passes off an intermediate. An increase in the salt concentration of the acid-denatured protein results in a transition of the unfolded structure to a compact and alternatively folded state. Unfolding kinetics of the internalin-domain measured by fluorescence and far-UV circular dichroism are indicative for the possible presence of two processes. The first slow unfolding process after the transition midpoint showed a strong dependence on temperature, whereas the second and faster unfolding process showed a stronger dependence on the denaturant concentration. Renaturation kinetics indicated the existence of at least one folding intermediate. Preliminary double-mixing experiments revealed no evidence for a rate-limiting proline isomerization reaction. It was not possible to detect the complete amplitude of the renaturation reaction, suggesting existence of a second faster phase occuring in the submillisecond range.<o:p></o:p></span></p> <p class=MsoBodyText><span lang=EN-GB style='mso-ansi-language:EN-GB'>The results on folding kinetics prove the InlB constructs to be suitable models for the investigation of solenoid protein folding by techniques of high structural resolution.<o:p></o:p></span></p>

Native states of globular proteins typically show stabilization in the range of 5 to 15 kcal/mol with respect to their unfolded states. There has been a considerable progress in the area of protein stability and folding in recent years, but increasing protein stability through rationally designed mutations has remained a challenging task. Current ability to predict protein structure from the amino acid sequence is also limited due to the lack of quantitative understanding of various factors that defines the single lowest energy fold or native state. The most important factors, which are considered primarily responsible for the structure and stability of the biological active form of proteins, are hydrophobic interactions, hydrogen bonding and electrostatic interactions such as salt bridges as well as packing interactions. Several studies have been carried out to decipher the importance of each these factors in protein stability and structure via rationally designed mutant proteins. The limited success of previous studies emphasizes the need for comprehensive studies on various aspect of protein stability. An integrated approach involving thermodynamic and structural analysis of a protein is very useful in understanding this particular phenomenon. This approach is very useful in relating the thermodynamic stability with the structure of a protein. A survey of the current literature on thermodynamic stability of protein indicates that the majority of the model proteins which have been used for understanding the determinants of protein stability are small, monomeric, single domain globular proteins like RNase A, Lysozyme and Myoglobin. On the other hand large proteins often show complex unfolding transition profiles that are rarely reversible. The major part of this thesis is focused on studying potential stabilizing/destabilizing interactions in small and large globular proteins. These interactions have been identified and characterized by exploring the effects of various rationally designed mutations on protein stability. Spectroscopic, molecular biological and calorimetric techniques were employed to understand the relationships between protein sequence, structure and stability. The experimental systems used are Leucine binding proteins, Leucine isoleucine valine binding protein (LIVBP), Maltose binding protein (MBP), Ribose binding protein (RBP) and Thioredoxin (Trx). The last section of the thesis discusses thermodynamic properties of molten globule states of the periplasmic protein LBP, LIVBP, MBP and RBP. The amino acid Pro is unique among all the twenty naturally occurring amino acids. In the case of proline, the Cδ of the side chain is covalently linked with the main chain nitrogen atom in a five membered ring. Therefore, Pro lacks amide hydrogen and it is not able to form a main chain hydrogen bond with a carbonyl oxygen. Hence Pro is typically not found in the hydrogen bonded, interior region of α-helix. There have been several studies which showed that introduction of the Pro residue into the interior of an α-helix is destabilizing. Although, it is not common to find Pro residue in the interiors of an α-helix, it has been reported that it occurs with appreciable frequency (14%). The thermodynamic effects of replacements of Pro residue in helix interiors of MBP were investigated in Chapter 2 of this thesis. Unlike many other small proteins, MBP contains 21 Pro residues distributed throughout the structure. It contains three residues in the interiors of α-helices, at positions 48, 133 and 159. These Pro residues were replaced with an alanine and serine amino acids using site directed mutagenesis. Stabilities of all the mutant and wild type proteins have been studied via isothermal chemical denaturation at pH 7.4 and thermal denaturation as a function of pH ranging from pH 6.5 to 10.4. It has been observed that replacement of a proline residue in the middle of an α-helix does not always stabilize a protein. It can be stabilizing if the carbonyl oxygen of residue (i-3) or (i-4) is well positioned to form a hydrogen bond with the ith (mutated) residue and the position of mutation is not buried or conserved in the protein. Partially exposed position have the ability to form main chain hydrogen bonds and Ala seems to be a better choice to substitute Pro than Ser. Unlike other amino acids, the pyrolidine ring of Pro residue imposes rigid constraints on the rotation about the N---Cα bond in the peptide backbone. This causes conformational restriction of the φ dihedral angle of Pro to -63±15º in polypeptides. Therefore, introduction of a rigid Pro residue into an appropriate position in a protein sequence is expected to decrease the conformational entropy of the denatured state and consequently lead to protein stabilization. In Chapter 3 of this thesis, the thermodynamic effects of Pro introduction on protein stability has been investigated in LIVBP, MBP, RBP and Trx. Thirteen single and two double mutants have been generated in the above four proteins. Three of the MBP mutants were characterized by X-ray crystallography to confirm that no structural changes had occurred upon mutation. In the remaining cases, CD spectroscopy was used to show the absence of structural changes. Stability of all the mutant and wild type proteins was studied via isothermal chemical denaturation at neutral pH and thermal denaturation as a function of pH. The mutants did not show enhanced stability with respect to chemical denaturation at room temperature. However, six of the thirteen single mutants showed a small but significant increase in the free energy of thermal unfolding in the range of 0.3-2.4 kcal/mol, two mutants showed no change and five were destabilized. In five of the six cases, the stabilization was because of a reduced entropy of unfolding. Two double mutants were constructed. In both cases, the effects of the single mutations on the free energy of thermal unfolding were non-additive. In addition to the hydrogen bond, hydrophobic and electrostatic interactions, other interactions like cation-π and aromatic-aromatic interactions etc. are also considered to make important contributions to protein stability. The relevance of cation-π interaction in biological systems has been recognized in recent years. It has been reported that positively charged amino acids (Lys, Arg and His) are often located within 6 Å of the ring centroids of aromatic amino acids (Phe, Tyr and Trp). The importance of cation-π interaction in protein stability has been suggested by previous theoretical and experimental studies. We have attempted to determine the magnitude of cation-π interactions of Lys with aromatic amino acids in four different proteins (LIVBP, MBP, RBP and Trx) in Chapter 4 of the thesis. Cation-π pairs have been identified by using the program CaPTURE. We have found thirteen cation-π pairs in five different proteins (PDB ID’s 2liv, 1omp, 1anf, 1urp and 2trx). Five cation-π pairs were selected for the study. In each pair, Lys was replaced with Gln and Met. In a separate series of experiments, the aromatic amino acid in each cation-π pair was replaced by Leu. Stabilities of wild type (WT) and mutant proteins were characterized by similar methods, to those discussed in previous chapters. Gln and Aromatic → Leu mutants were consistently less stable than the corresponding Met mutants reflecting the non-isosteric nature of these substitutions. The strength of the cation-π interaction was assessed by the value of the change in the free energy of unfolding (ΔΔG0=ΔG0 (Met) - ΔG0(WT)). This ranged from +1.1 to –1.9 kcal/mol (average value – 0.4 kcal/mol) at 298 K and +0.7 to –2.6 kcal/mol (average value –1.1 kcal/mol) at the Tm of each WT. It therefore appears that the strength of cation-π interactions increases with temperature. In addition, the experimentally measured values are appreciably smaller in magnitude than the calculated values with an average difference |ΔG0expt -ΔG0calc|avg of 2.9 kcal/mol. At room temperature, the data indicate that cation-π interactions are at best weakly stabilizing and in some cases are clearly destabilizing. However at elevated temperatures, close to typical Tm’s, cation-π interactions are generally stabilizing. In Chapter 5, we have attempted to characterize molten globule states for the periplasmic proteins LBP, LIVBP, MBP and RBP. It was observed that all these proteins form molten globule states at acidic pH (3 - 3.4). All these molten globule states showed cooperative thermal transitions and bound with their ligand comparable to (LBP and LIVBP) or with lower (MBP and RBP) affinity than the corresponding native states. Trp, ANS fluorescence and near-UV CD spectra for ligand bound and free forms of molten globule states were found to be very similar. This shows that molten globule states of these proteins have the ability to bind to their corresponding ligand without conversion to the native state. All four molten globule states showed destabilization relative to the native state. ΔCp values indicate that these molten globule states contain approximately 29-67% of tertiary structure relative to the native state. All four proteins lack prosthetic groups and disulfide bonds. Therefore, it is likely that molten globule states of these proteins are stabilized via hydrophobic and hydrogen bonding interactions.

La leucine zipper de la rétine neurale (Nrl) joue un rôle central dans le développement et l'homéostasie des bâtonnets en activant I'expression de gènes tels que le photopigment Rhodopsine. Nrl est aussi associé à la Rétinite Pigmentaire, faisant ainsi de ce gène un modèle intéressant pour la compréhension des programmes contrôlant le développement et I'homéostasie des photorécepteurs.Ce travail de thèse vise à caractériser les mécanismes régulateurs de I'expression de Nr/ au cours du développement rétinien. L'électroporation in vivo de vecteurs rapporteurs dans des rétines de souris en développement, a révélé des séquences minimales de promoteur Nr/ nécessaires à une expression spécifique dans les photorécepteurs. Nous avons identifié RORI3 comme facteur requis pour cette expression, et montré que les facteurs OTX2, CRX et CREB s'accrochent aussi directement à des régions régulatrices particulières du promoteur. Nous avons construit un virus adéno-associé (AAV) contenant un promoteur minimal Nrl de 0.3 kb, et montré qu'il est adapté à la délivrance de gène spécifiquement dans les photorécepteurs.Nous avons montré que NRL, CRX et NR2E3, les régulateurs principaux de la Rhodopsine, ont une expression rythmique au cours de 24 h, et que l'expression cyclique de Nr/ peut être due à l'activation par RORp, un composant l'horloge circadienne. Enfin, nous avons identifié un nouveau facteur de transcription, NonO, au niveau de la région du promoteur proximal de la Rhodopsine, qui en combinaison avec NRL et CRX, active le promoteur de la Rhodopsine. L'invalidation de NonO au cours du développement rétinien a prouvé son implication pour le développement et I'homéostasie des bâtonnets. / The Neural Retina Leucine zipper transcription factor (Nrl) plays a central role in rod photoreceptor development and homeostasis, by activating the expression of rod-specific genes such as the visual photopigment, Rhodopsin. Nrlhave been also associated with Retinitis Pigmentosa, making this gene an interesting model for understanding genetic programs controlling photoreceptors development and homeostasis.This thesis work aimed at characterizing regulatory mechanisms of Nr/ expression during retinal development. Using in vivo electroporation of reporter vectors carrying distinct portions of Nrlpromoter into neonatal mouse retina, we identified minimal sequences required for expression photoreceptors-specific expression. We identified RORI3 as being required for this expression and showed that OTX2, CRX and CREB transcription factors also directly bind to the defined regulatory regions.We designed a novel adeno-associated virus (AAV) vector containing a minimal Nrl promoter fragment of 0.3 kb, and showed that it is well-suited for gene delivery specifically into photoreceptors.We also showed that NRL, CRX, and NR2E3, the main transcriptional regulators of Rhodopsin, display rhythmic expression over 24 h. and that Nrl might undergo cyclic activation by RORB which is part of the photoreceptor circadian clock. Finally, we investigated the role of a novel Rhodopsin transcriptional regulator, NonO, identified in theRhodopsin proximal promoter region. We demonstrated that NonO co-activates Rhodopsin promoter along with NRL and CRX. By knocking down this gene during retinal development we provided evidence for its role in rod development and homeostasis.

Skeletal muscle adapts differently to specific modes of exercise, where resistance training results in muscle growth and endurance training induces mitochondrial biogenesis. These are results of molecular events that occur after each exercise session, increasing the expression of specific genes and the rate of both synthesis and breakdown of protein. The rate of protein synthesis is controlled by the mTORC1 signaling pathway, which is potently stimulated by resistance exercise and amino acid, and their combined effect is needed for muscle growth. The essential amino acids (EAA) are responsible for the stimulation of protein synthesis and here leucine has been attributed specific attention, but its particular role among the EAA, and the involvement of the other branched-chain amino acids (BCAA) is unclear. Endurance exercise activates the protein AMPK which, in animal models, has been shown to inhibit mTORC1 signaling and protein synthesis.  Suggesting that concurrent endurance and resistance exercise could restrain muscle growth, but it is unknown if this mechanism is relevant in exercising human muscle. Little is known about the regulation of protein breakdown and although much attention has been given the proteins MuRF-1 and MAFbx which target proteins for degradation, their role requires further investigation. The aim of thesis was to address the mentioned uncertainties by examining how different modes of exercise and amino acids affect mTORC1 signaling, protein synthesis and markers of protein breakdown in human muscle. In study I, the influence of high intensity endurance exercise on subsequent resistance exercised induced mTORC1 signaling was examined. Despite robust activation of AMPK by the endurance exercise there was no inhibition of mTORC1 signaling or protein synthesis during recovery from resistance exercise. Study II utilized a similar set up, but with the difference that resistance exercise was performed with the triceps. The cycling exercise reduced the resistance exercise stimulated mTORC1 signaling immediately after the exercise, but during the recovery period mTORC1 signaling and protein synthesis was similar between trials. Concurrent exercise induced the mRNA expression of MuRF-1 and that of PGC-1α, the master regulator of mitochondrial biogenesis, in both studies, despite that the exercise modes in study II were separated between legs and arms. In study III, the effect of an EAA supplement with or without leucine, in the stimulation of mTORC1 signaling in connection with resistance exercise was examined. Intake of EAA robustly stimulated mTORC1 signaling after exercise, but this was only minor when leucine was excluded from the supplement. In study IV, subjects were supplied with leucine, BCAA, EAA or placebo in a randomized fashion during four sessions of resistance exercise. Leucine alone stimulated mTORC1 signaling after the exercise, but both the amplitude and extent of stimulation was substantially greater with EAA, an effect that was largely mediated by the BCAA as a group. In conclusion, endurance exercise prior to resistance exercise using the leg or arm muscles does not affect mTORC1 signaling or protein synthesis during the three hour recovery period from exercise, supporting compatibility between resistance- and endurance exercise induced signaling. Concurrent exercise increases the expression of the proteolytic marker MuRF-1 compared to resistance exercise only, which could indicate both and increased demand of cellular adaptive remodeling or a more direct detrimental proteolytic effect. Leucine is crucial among the EAA in the stimulation of mTORC1 signaling after exercise, its effect is however potentiated by intake of the remaining EAA. As a supplement a mixture of EAA must be regarded preferable, although the effect is largely mediated by the BCAA as a group.