No description available.

No description available.

Many diseases, including Alzheimer's and Creutzfeldt-Jakob disease, are believed to be the result of protein misfolding and aggregation into insoluble, highly ordered Β-sheet structures known as amyloid fibrils. The mechanism of formation of these fibrils is unknown. The work presented in this thesis uses human cystatin B as a model system for the elucidation of the mechanism of fibril formation. Cystatin B, a cysteine protease inhibitor, readily forms amyloid fibrils in vitro. Amyloid fibrils formed from the structurally homologous cystatin C, are associated with a form of cerebral haemorrhage as well as incorporation in amyloid derived from Alzheimer's disease sufferers. The work in this thesis sheds light on the mechanism by which proteins spontaneously change conformation and self associate to form amyloid fibrils. Investigation of the state induced by conditions of fibrillisation and the effects of single point mutations highlight particular regions within the natively folded protein that are involved in the self association process. Studies on Cu2+ and Zn2+ binding to cystatin B highlight the same small regions of importance and show that the effect induced by divalent metals is very metal specific. It is also shown that fibrillisation of cystatin B is preceded by aggregation into a large insoluble structure. This may act to increase local concentration by protein adsorption and involve unknown surface interactions that induce or catalyse the initiation of fibrillisation. The interactions and conformational changes required for fibrillisation are likely to be local rather than global events which require the weakening of the co-operative properties normally associated with protein folding. Alteration of these interactions will promote alternative pathways of assembly resulting in different "strains" of amyloid. Ultimately the high resolution structure of cystatin B fibrils will answer many questions and the initial solid state NMR experiments to achieve this are presented in this thesis.

HIRA is one of the chaperones of histone variant H3.3. Deficiency of HIRA or its homologues in S. cerevisiae, S. pombe, Drosophila and chicken has been associated with both activating and repressing effects on euchromatic gene transcription, with gene silencing defects in pericentromeric heterochromatin and with mitotic defects including an extension of cell cycle length. At the organism level, homozygous Hira knockout is embryonically lethal in mice. Several previously established mouse strains have a transgene inserted into a variety of genomic locations including regions of putative facultative heterochromatin and constitutive (pericentromeric) heterochromatin where it displays position effect variegation (PEV) in that the transgene is stochastically silenced in a proportion of T cells. The effects of HIRA deficiency on PEV of this transgene showed that HIRA was necessary for PEV in putative facultative heterochromatin and it appeared that, in this environment, it played a role both in the rate of establishment (during T cell development) and in the maintenance of PEV (in mature T cells). This was in contrast to its effect in constitutive (pericentromeric) heterochromatin where Hira knockout in vivo had no effect on the variegation of a transgene. On the other hand, the reduction in variegation usually induced by T cell activation was lessened by HIRA deficiency in another mouse strain where the transgene was located in close proximity to pericentromeric heterochromatin. The contribution of HIRA to the extent of expression changes in facultative heterochromatin was consistent with delays observed in the normal transcriptional changes of some genes during the differentiation of HIRA deficient murine ES cells. In common with S. pombe and chicken, cell cycle delay in G2/M was seen in HIRA deficient murine cell lines. This is believed to be the first observation of this effect in mammalian cells suggesting evolutionary conservation of this function.

The elucidation of molecular function of proteins encoded by genes is a major challenge in biology today. Genes regulate the amount of proteins (enzymes) needed to catalyse a metabolic reaction. There are several works on either the modelling of gene expression or metabolic network. However, an integrative model of both is not well understood and researched. The integration of both gene expression and metabolic network could increase our understanding of cellular functions and aid in analysing the effects of genes on metabolism. It is now possible to build genome-scale models of cellular processes due to the availability of high-throughput genomic, metabolic and fluxomic data along with thermodynamic information. Integrating biological information at various layers into metabolic models could also improve the robustness of models for in silico analysis. In this study, we provide a software tool for the in silico reconstruction of genome-scale integrative models of gene expression and metabolic network from relevant database(s) and previously existing stoichiometric models with automatic generation of kinetic equations of all reactions involved. To reduce computational complexity, compartmentalisation of the cell as well as enzyme inhibition is assumed to play a negligible role in metabolic function. Obtaining kinetic parameters needed to fully define and characterise kinetic models still remains a challenge in systems biology. Parameters are either not available in literature or unobtainable in the lab. Consequently, there have been numerous methods developed to predict biological behaviour that do not require the use of detailed kinetic parameters as well as techniques for estimation of parameter values based on experimental data. We present an algorithm for estimating kinetic parameters which uses fluxes and metabolites to constrain values. Our results show that our genetic algorithm is able to find parameters that fit a given data set and predict new biological states without having to re-estimate kinetic parameters.

Η αποικοδόμηση των πρωτεϊνών είναι μια διαδικασία απαραίτητη για τη διατήρηση της ομοιόστασης του κυττάρου. Ένας από τους κύριους μηχανισμούς αποικοδόμησης των βραχύβιων πρωτεϊνών καθώς και όσων εμφανίζουν λανθασμένη αναδίπλωση, χωρεί μέσω του μονοπατιού ουβικιτίνης- πρωτεασώματος. Η ουβικιτινίωση είναι μια μετα-μεταφραστική διαδικασία, η οποία έγκειται στη σηματοδότηση των υποψήφιων για αποικοδόμηση πρωτεϊνών με ουβικιτίνη και περιλαμβάνει τρεις ενζυμικές ενεργότητες: Ε1 (εκκινητής ουβικιτίνης), Ε2(μεταφορέας ουβικιτίνης) και Ε3 (λιγάση ουβικιτίνης). Η πρωτεΐνη Arkadia (Rnf11) είναι μια Ε3 λιγάση ουβικιτίνης με συνολικό μήκος 994 αμινοξέα. Σε μοριακό επίπεδο, ενισχύει το TGF-β σηματοδοτικό μονοπάτι, διαμεσολαβώντας την εξαρτώμενη από το πρωτεάσωμα αποικοδόμηση των αρνητικών ρυθμιστών του, c-Ski και Sno-N. Η δραστικότητα Ε3 λιγάσης ουβικιτίνης εδράζεται στον C΄-τελικό RING-H2 τομέα, που σχηματίζεται από τα τελευταία 60 περίπου αμινοξέα της ακολουθίας. Η δομή και η σταθερότητα του RING τομέα εξαρτώνται από την πρόσδεση δύο ιόντων Zn μέσω ενός χαρακτηριστικού μοτίβου, που περιλαμβάνει 6 κυστεϊνικά και 2 ιστιδινικά κατάλοιπα. Στην προσπάθεια αποσαφήνισης της σχέσης δομής-δράσης της πρωτεΐνης Arkadia, ένα από τα κατάλοιπα που συναρμόζονται με Zn -συγκεκριμένα η His965- αντικαταστάθηκε από κυστεΐνη μέσω κατευθυνόμενης μεταλλαξιγένεσης. Η μετάλλαξη αυτή, με την οποία, ουσιαστικά, μετατρέψαμε τον RING-H2 σε RING-HC τομέα, μελετήθηκε με χρήση πολυπυρηνικής/πολυδιάστατης φασματοσκοπίας πυρηνικού μαγνητικού συντονισμού (NMR). H NMR δομή του RING-H2 τομέα της Η965C Arkadia επιλύθηκε σε υψηλή διακριτικότητα (tf=0.94±7.53*10-2, RMSD=0.75±0.20 και RMSD=1.45±0.24 για τα άτομα του πολυπεπτιδικού σκελετού και τα βαρέα άτομα αντίστοιχα) και αποκάλυψε μια ββαββ τοπολογία. Παράλληλα, πραγματοποιήθηκε μελέτη κινητικότητας, από την οποία προέκυψε ότι η εν λόγω μετάλλαξη υφίσταται ως μονομερές και διαθέτει έναν συμπαγή πυρήνα, που περικλείεται μεταξύ δύο ευκίνητων άκρων. / Protein degradation is necessary for the maintenance of cell homeostasis. A major mechanism for the degradation of short-lived as well as misfolded proteins involves the ubiquitin-proteasome pathway. Ubiquitination is a post translational modification, which targets the proteins to be degraded through the covalent attachment of a ubiquitin tag and consists of three enzyme activities: Ε1 (ubiquitin activator), E2 (ubiquitin carrier) and E3 (ubiquitin ligase). Arkadia (Rnf11) is a 994 amino acid protein, which acts as an E3 ubiquitin ligase. On a molecular level, Arkadia enhances TGF-β signaling by mediating the proteasome-dependent degradation of its negative regulators, c-Ski and Sno-N. Its E3 ubiquitin ligase activity lies on a C΄-terminal RING-H2 domain, formed by the last 60 residues. The structure as well as stability of the RING finger domain depend strongly on the binding of two zinc ions in a unique ΄΄cross-brace΄΄ arrangement through a defined motif of six cysteines and two histidines. Trying to elucidate the structure-activity relationship in the case of Arkadia, one of the amino acid ligands –specifically His965- was replaced by cysteine through site-directed mutagenesis. This particular mutation, which, in reality, transformed the RING-H2 to a RING-HC domain, was studied with the use of multinuclear/multidimensional nuclear magnetic resonance spectroscopy (NMR). The NMR solution structure of the H965 Arkadia RING-H2 domain was determined in high resolution (tf=0.94±7.53*10-2, RMSD=0.75±0.20 και RMSD=1.45±0.24 for backbone and heavy atoms respectively) and revealed a ββαββ topology. Furthermore, a mobility study was conducted with the following results: the mutated protein is not expected to form dimers and shows a compact core region including the four metal binding motifs flanked by two flexibly disordered termini.

La dysfonction endothéliale vasculaire, processus majeur initiant l’athérosclérose, est étroitement liée à l’altération de la synthèse et/ou biodisponibilité du monoxyde d’azote (NO), dont l’arginine est le précurseur. Elle apparait aussi dès la phase postprandiale après un repas gras et sucré. Chez des sujets avec des facteurs de risque cardiométabolique, une supplémentation orale en arginine a un effet bénéfique sur des fonctions associées au NO. Aucune donnée ne permet cependant de lier la mise à disposition de l’arginine et la synthèse de NO, en situation normale ou de risque cardiométabolique. De plus, peu d’études ont étudié l’effet d’une supplémentation en arginine, dans un contexte nutritionnel (faible dose et libération lente) chez des sujets avec des facteurs de risque cardiométabolique. Ce travail vise donc à évaluer l’effet d’une supplémentation nutritionnelle en arginine, sur le métabolisme de l’arginine et la fonction endothéliale (FE), chez des sujets sains présentant des facteurs de risque cardiométabolique. Dans une première étude clinique, nous avons ainsi comparé la biodisponibilité de l’arginine ingérée et son utilisation pour la synthèse de NO, selon la présence de facteurs de risque cardiométabolique, et selon qu’elle était consommée sous une forme à libération immédiate (LI) ou sous une forme à libération prolongée (LP), mimant la mise à disposition naturelle de l’arginine alimentaire. Puis, dans une deuxième étude clinique, nous avons étudié l’effet de la supplémentation en arginine LP sur la FE à jeun et sur son altération postprandiale, chez des sujets sains présentant des facteurs de risque cardiométabolique; et si cet effet pourrait varier selon leur argininémie basale. Ce travail de thèse a ainsi mis en évidence une utilisation de l’arginine ingérée plus élevée pour la synthèse de NO chez les sujets avec des facteurs de risque cardiométabolique, et plus élevée avec la forme LP qu’avec la forme LI, en particulier chez ces sujets à risque. La deuxième étude, quant à elle, a révélé que les effets de la supplémentation en arginine-LP variaient selon l’argininémie basale des sujets présentant des facteurs de risque cardiométabolique. Chez les sujets avec une argininémie basale relativement plus faible, l’arginine-LP a atténué la diminution postprandiale de la FE et a conduit à une FE significativement meilleure à la fin de la période postprandiale. / Vascular endothelial dysfunction, the hallmark of early atherosclerosis, results from an impairment of the synthesis and/or bioavailability of nitric oxide (NO), the precursor of which is arginine. Endothelial dysfunction is also known to be induced transiently by a high-fat meal. In subject with cardiometabolic risk factors, oral arginine supplementation has a beneficial effect on NO-related physiological functions. However, no data relates the availability of arginine to the synthesis of NO in normal or cardiometabolic risk condition. In addition, few studies only have investigated the effect of arginine supplementation in a nutritional context (low dose and slow release) in subjects with cardiometabolic risk factors. This work aims to evaluate the effect of a nutritional arginine supplementation, on the arginine metabolism and endothelial function in healthy subjects with cardiometabolic risk factors. In a first clinical study, we have compared the bioavailability of oral arginine and its utilization for NO synthesis, as a function of the presence of cardiometabolic risk factors, and as a function of the form of release (immediate release, IR, as free arginine, or sustained release, SR, which mimics the slow release of dietary arginine). Then, in a second clinical study, we studied the effect of SR-arginine supplementation on fasting endothelial function and its postprandial alteration in healthy subjects with cardiometabolic factors. A further aim was to investigate whether this effect may vary according to the baseline arginine status of subjects. This thesis work has demonstrated a higher utilization of oral arginine for NO synthesis in subjects with cardiometabolic risk factors, and a higher utilization with the SR form, particularly in these subjects at risk. As to the second study, it showed that the SRarginine supplementation effects largely varied with baseline fasting arginine concentration of subjects with cardiometabolic risk factors. In subjects with a relatively lower baseline arginine concentration, SR-arginine attenuated the decrease in postprandial endothelial function and led to a significantly higher endothelial function at the end of the postprandial period.