The postsynaptic density (PSD) is a complex, dynamic structure composed of ~2000 distinct proteins, found at the postsynaptic membrane. Interactions, of transient and non-transient nature, organise the PSD’s constituent parts into a protein complex, which functions as an intricately regulated molecular machine, orchestrating the mediation and regulation of synaptic transmission and synaptic plasticity. Furthermore, many of the proteins found in this complex have been linked to synaptic and behavioural plasticity, basic cognition or disease. Although, through proteomics we have accumulated a lot of information on the constituent parts of this machine as well smaller sub-networks representing pathways, not a lot is known about the organisational principles of the PSD. In this project our aim is to develop a standardised approach to reconstructing protein interaction networks from PSD proteomics data, providing a descriptive integrative model. Using these models we also performed an analysis elucidating parts of these organisational principles. We applied this method on two murine postsynaptic density proteomics datasets and found a persistent modular architecture of biological significance. Furthermore, given the lack of substantial evidence on the composition and architecture of postsynaptic density interaction networks of other model organisms, we decided to perform an affinity purification of Drosophila melanogaster postsynaptic density proteins and perform a similar analysis. The resulting model corroborated theoretical predictions of a lower complexity but similar functionality and also showed a modular architecture. As a final analysis we compared the two models from a structural and evolutionary perspective attempting to elucidate the mechanisms of evolution of this molecular machine. The results of this analysis implied that a whole component rather than just individual proteins of the fly protein interaction network have been conserved, highlighting the importance of the aforementioned organisational principles.

<p>On the Internet an enormous amount of information is available that is represented in an unstructured form. The purpose with a text mining tool is to collect this information and present it in a more structured form. In this report text mining is used to create an algorithm that searches abstracts available from PubMed and finds specific relationships between genes that can be used to create a network. The algorithm can also be used to find information about a specific gene. The network created by Mendoza et al. (1999) was verified in all the connections but one using the algorithm. This connection contained implicit information. The results suggest that the algorithm is better at extracting information about specific genes than finding connections between genes. One advantage with the algorithm is that it can also find connections between genes and proteins and genes and other chemical substances.</p>

No description available.

<i>Caulobacter crescentus</i> is an important model organism for studying regulation of cell growth, division and differentiation in prokaryotes. <i>C. crescentus</i> undergoes asymmetric division producing two progeny cells with identical genome but different developmental programs: the "swarmer" cell is flagellated and motile, and the "stalked" cell is sessile (attached to a surface by its stalk and holdfast). Only stalked cells undergo chromosome replication and cell division. A swarmer cell must shed its flagellum and grow a stalk before it can enter the replication-division cycle. Based on published experimental evidence, we propose a molecular mechanism controlling the cell division cycle in this bacterium. Our quantitative model of the mechanism illustrates detailed temporal dynamics of regulatory proteins and corresponding physiological changes during the process of cell cycle progression and differentiation of wild-type cells (both stalked cells and swarmer cells) and of a number of known and novel mutant strains. Our model presents a unified view of temporal regulation of protein activities during the asymmetric cell division cycle of <i>C. crescentus</i> and provides an opportunity to study and analyze the system dynamics of the <i>Caulobacter</i> cell cycle (as opposed to the dynamics of individual steps). The model can serve as a starting point for investigating molecular regulations of cell division and differentiation in other genera of alpha-proteobacteria, such as <i>Brucella</i> and <i>Rhizobium</i>, because recent experimental data suggest that these alpha-proteobacteria share similar genetic mechanisms for cell cycle control. / Ph. D.

Cereals contain naturally occurring biopolymers (for example proteins and starches) that can be used as renewable raw materials in a variety of speciality chemical applications. The separation of protein and starch biopolymers from wheat is well established and relies on a group of proteins called glutens that have a unique network-forming functionality. Oat and other cereals do not naturally contain these gluten proteins and typically rely on chemical-based separation techniques which alter the chemical and physical structures and damage the inherent natural functionality of the biopolymers. This research study investigated the separation of the protein and starch fractions from cereals using the Al-Hakkak Process, a new aqueous process. This process involves adding water and wheat gluten protein to cereals that do not contain gluten. The wheat gluten interacts with the cereal proteins, facilitating the separation of the starch and protein fractions whilst retaining their inherent natural functionality. The aim of this research project was to investigate and optimise the pilot scale separation performance of the Al-Hakkak Process using oat flour. As very little prior research had been carried out, the focus was to characterise the oat starch and protein separation performance and gain an understanding of the mechanisms involved. A variety of techniques were employed. Large scale deformation rheology was used to gain an understanding of the oat-gluten dough rheology and establish the relationship between the rheology and the separation performance. Confocal scanning laser microscopy was used to investigate the structure of the oat-gluten protein network. The molecular interactions between the oat and gluten proteins were studied using gel electrophoresis. The network-forming functionality of the new oat-gluten protein was explored. The influence of various processing parameters on the pilot scale separation performance was investigated and the results compared with other data collected through the study to identify key processing parameters. This research programme has resulted in interesting, encouraging and some unexpected outcomes and these are discussed in detail in the thesis. It was concluded that an insoluble protein network formed in the oat-gluten dough and both kneading and extraction processes were found to contribute to the formation of this. A key conclusion was that the changes that took place in the oat-gluten dough were similar to, but not identical to, the changes that occur in wheat dough. It was proposed that the mechanism for the development of a protein network in oat-gluten dough differed from wheat dough for two main reasons: a) the presence of the oat flour disrupted the normal wheat gluten behaviour, and b) components in the oat flour altered the activity of the gluten proteins. The research identified key processing parameters for the Al-Hakkak Process including kneading time, gluten content, and sodium chloride content of the oat-gluten dough as well as sodium chloride concentration, pH, and temperature of the extract liquor. An important discovery was that the oat and gluten proteins interacted at a molecular level through reducible, covalent, bonding (most likely disulphide linkages) to form the insoluble protein network in the oat-gluten dough. It was concluded that these reducible bonds coupled the individual protein subunits to form new hybrid oat-gluten protein molecules (a combination of oat proteins and gluten proteins). Both insoluble and soluble proteins in the oat and gluten flour were involved in the formation of the insoluble protein network in the oat-gluten dough. This outcome has applications beyond the Al-Hakkak Process, as this new knowledge can be applied to the wider dough processing industry. It was concluded that the wheat gluten was the source of the protein network-forming functionality of the hybrid oat-gluten protein and that the oat proteins had a diluting effect. It was proposed that oat-gluten protein flour from the Al-Hakkak Process could be reused to replace the commercial wheat gluten flour in subsequent production batches. During spray drying of the starch stream, the soluble biopolymers in the extract liquor were found to act as an adhesive and glued individual starch granules together to form spherical agglomerates. Acidification of the extract liquor was found to enhance this agglomeration. It was proposed the acidified starch granules were sticker during spray drying due to the partial acid hydrolysis of the starch granule suface which enhanced the agglomeration.

La mise en place des circuits neuronaux est un processus complexe et précisément régulé. Une atteinte de ce processus est à l'origine de diverses pathologies neurodéveloppementales telles que la schizophrénie ou les troubles du spectre autistique, désordres psychiatriques partageant une altération des fonctions cognitives. Le récepteur 6 de la sérotonine (récepteur 5-HT6), notamment connu pour son implication dans la migration neuronale, s'est révélé être une cible thérapeutique de choix dans le traitement des symptômes cognitifs associés à la schizophrénie mais aussi à des pathologies neurodégénératives comme la maladie d'Alzheimer. Cependant la signalisation déclenchée par le récepteur 5-HT6 n'explique pas entièrement son implication dans les processus neurodéveloppementaux. Mon travail de thèse a donc visé à comprendre les mécanismes de signalisation engagés par le récepteur 5-HT6 au cours du développement neuronal. La réalisation d'un crible protéomique a permis de montrer que le récepteur 5-HT6 interagissait avec plusieurs protéines cruciales dans le développement neuronal comme la protéine Cdk5 et sa cible WAVE-1. J'ai ensuite pu démontrer qu'en plus de son rôle dans la migration, le récepteur 5-HT6 contrôlait de façon agoniste-indépendante l'élongation des neurites par un mécanisme impliquant la phosphorylation de son domaine C-terminal par la kinase Cdk5 et l'activation de la RhoGTPase Cdc42. La seconde partie de mon travail a visé à mettre en évidence le rôle du récepteur 5-HT6 dans la formation des épines dendritiques et à comprendre l'implication de la protéine WAVE-1, cible de Cdk5, dans ce processus. Les résultats obtenus au cours de ma thèse apportent de nouveaux éléments quant au contrôle des processus neurodéveloppementaux par le récepteur 5-HT6. Ce récepteur apparaît donc comme une cible thérapeutique de choix dans les atteintes neurodéveloppementales en contribuant au développement des circuits cognitifs en relation avec la physiopathologie des troubles du spectre autistique ou de la schizophrénie. / Brain circuitry patterning is a complex, highly regulated process. Alteration of this process is affected gives rise to various neurodevelopmental disorders such as schizophrenia or Autism Spectrum Disorders (ASD), which are both characterized by a wide spectrum of deficits. Serotonin 6 receptor (5-HT6 receptor), which is known for its implication in neuronal migration process, has been identified as a key therapeutic target for the treatment of cognitive deficits observed in schizophrenia, but also in neurodegenerative pathologies such as Alzheimer's disease. However, the signalling mechanisms knowned to be activated by the 5-HT6 receptor do not explain its involvement in neurodevelopmental processes. My thesis project therefore aimed at characterizing the signalling pathways engaged by 5-HT6 receptor during neural development. A proteomic approach allowed me to show that the 5-HT6 receptor was interacting with several proteins playing crucial roles in neurodevelopmental processes such as Cdk5 or WAVE-1. I then demonstrated that, besides its role in neuronal migration, the 5-HT6 receptor was also involved in neurite growth through constitutive phosphorylation of 5-HT6 receptor at Ser350 by associated Cdk5, a process leading to an increase in Cdc42 activity. The second part of my work aimed at understanding the role of 5-HT6 receptor in dendritic spines morphogenesis, and the involvement of WAVE-1 and Cdk5 in this process. These results provide new insights into the control of neurodevelopemental processes by 5-HT6 receptor. Thus, 5-HT6 receptor appears to be a key therapeutic target for neurodevelopmental disorders by contributing to the development of cognitive circuitry related to the pathophysiology of ASD or schizophrenia.

On the Internet an enormous amount of information is available that is represented in an unstructured form. The purpose with a text mining tool is to collect this information and present it in a more structured form. In this report text mining is used to create an algorithm that searches abstracts available from PubMed and finds specific relationships between genes that can be used to create a network. The algorithm can also be used to find information about a specific gene. The network created by Mendoza et al. (1999) was verified in all the connections but one using the algorithm. This connection contained implicit information. The results suggest that the algorithm is better at extracting information about specific genes than finding connections between genes. One advantage with the algorithm is that it can also find connections between genes and proteins and genes and other chemical substances.

Afin d’approfondir les connaissances sur les liens entre la structure et les propriétés nutritionnelles de pâtes alimentaires, des pâtes de structures contrastées ont été produites en modifiant la formulation et/ou le procédé de fabrication. Leur structure a été caractérisée aux différentes échelles, puis les caractéristiques définies ont été reliées à la digestibilité des protéines et de l’amidon. Le remplacement partiel ou total de la semoule de blé dur par des farines de légumineuses dans la fabrication des pâtes a engendré des changements structuraux à plusieurs niveaux. Un affaiblissement marqué de la structure du réseau protéique proportionnel à la quantité de légumineuses incorporées a notamment été observé, conduisant à une amélioration de la digestibilité in-vitro des protéines. La digestibilité in-vitro de l’amidon était en revanche maintenue à son faible niveau voir même réduite. L’application de différentes températures de séchage a amplifié les modifications de la structure des pâtes enrichies en légumineuses. Ces différences s’atténuaient toutefois après cuisson, n’engendrant de fait aucune modification marquée de la digestibilité in-vitro des protéines entre une pâte cuite préalablement séchée à basse ou à très haute température. Les teneurs en facteurs antinutritionnels étaient réduites par le procédé de fabrication et de cuisson des pâtes. A quantité égale de protéines consommées, l’étude in-vivo a révélé une augmentation de la croissance et de l’efficacité azotée chez les rats nourris avec les pâtes mixtes blé/fèverole (65/35) par comparaison aux rats nourris aux pâtes 100% blé. L’assemblage blé/fèverole constituait un mélange protéique efficace pour la croissance des rats jeunes, probablement du fait de son profil équilibré en acides aminés, et de la structure affaiblie du réseau protéique. Par ailleurs, les différentes températures de séchage appliquées aux pâtes n’ont pas engendré de différence au niveau de la rétention azotée in-vivo. Les spécialités pastières développées au cours de ce travail de thèse sont jusqu’à trois fois plus riches en protéines et mieux équilibrées en acides aminés, comparativement à une pâte alimentaire classique. Ces spécialités pastières sont aussi appréciées que leurs homologues qui se vendent sur le marché. En outre, la recette mixte blé/légumineuses peut être adaptée à tout consommateur souhaitant diversifier ses apports en protéines, mais également à certaines populations spécifiques, notamment aux personnes âgées. La recette 100% légumineuses peut concerner les personnes intolérantes au gluten. Ces spécialités pastières pourraient de ce fait, trouver une large application à l’échelle industrielle. / In order to reinforce our knowledge about the links between structural and nutritional properties of pasta, pasta with contrasting structures were produced by modifying the formulation and/or the manufacturing process. Pasta structure was then characterized using a multiscale approach and linked to the digestibility of protein and starch fractions. The modification of pasta formulation by partial or total substitution of durum wheat semolina by legume flours changed pasta structure at various scales. A marked weakening in the structure of pasta protein network proportional to the level of legume protein incorporated was observed and related to an improvement of the in-vitro digestibility of pasta proteins. Interestingly the in-vitro starch digestibility was maintained at its low level or even reduced in mixed pasta. Changing pasta drying temperature amplified changes in the pasta structure. These differences, however, were reduced after cooking pasta resulting in unchanged in-vitro protein digestibility between cooked pasta dried at low or very high temperature. Anti-nutritional factors were also reduced by the manufacturing and cooking steps. In-vivo study revealed a better growth and nitrogen efficiency in rats consuming the same quantity of proteins from wheat/faba (65/35) mixed pasta in comparison with animals fed with 100% wheat pasta. This was probably related to the better amino acid balance, and to the weakened protein network in mixed pasta. The change in drying temperatures did not allow modifying protein retention. Pasta developed in this thesis are up to three fold richer in proteins than traditional wheat pasta, and more balanced in essential amino acids. Produced pasta are appreciated by the consumer as well as their counterparts found on the marked. Mixed wheat/legume may be adapted to the elderly people, whereas 100% legume pasta may be consumed by gluten intolerant subjects. Produced pasta may therefore find wide application at the industrial scale.

Les organismes vivants doivent reproduire et transmettre ne variatur l’information contenue dans les chromosomes. Ainsi, la conservation de l’intégrité du génome est un processus biologique fondamental. La maintenance génomique constitue l’ensemble des processus biologiques impliqués dans la conservation, la duplication et la transmission de l’information génétique contenue dans les chromosomes. La machinerie réplicative des Archaea est décrite comme une version simplifiés de celle connue chez les Eucaryotes faisant des Archaea un excellent modèle d’étude de la réplication. Contrairement à la réplication, les processus Archaea de réparation de l’ADN sont encore mystérieux. En effet, plusieurs protéines essentielles de la réparation semblent absentes des génomes Archaea et ce même chez les espèces Hyperthermophiles (HA). Avec une température optimale de croissance proche de 100°C, ces Archaea doivent posséder des capacités considérables de réparation des dommages de l’ADN, catalysés à haute température. Ainsi les Archaea hyperthermophiles sont probablement dotées d’un système de réparation alternatif extrêmement efficace. Ce système et sa coordination avec la réplication sont inconnus. Un protocole de purification d’affinité couplée à la spectrométrie de masse des protéines a permis d’identifier les complexes protéiques impliqués dans la maintenance génomique de l’Archaea hyperthermophile P. abyssi. Les complexes identifiés sont compilées dans un réseau d’interaction. Soumis à une étude topologique le réseau révèle notamment de nouvelles interactions entre des protéines essentielles de la maintenance génomique, conservées avec les Eucaryotes. Plusieurs interactions sont vérifiées indépendamment où caractérisées fonctionnellement in vitro ou in vivo. Ces travaux mettent en lumière l’étroite collaboration entre la réplication et la recombinaison de l’ADN et révèlent de nouveaux aspects de la machinerie de transcription. / DNA replication, recombination and repair are central and essential mechanisms in all cells. Highly efficienthigh-fidelity chromosome replication is vital for maintaining the integrity of the genetic information and for theavoidance of genetic disease. Archaeal replisome is described as simplified version of the eukaryotic system.However, DNA repair is still enigmatic, as many essential repair proteins have not been identified in Archaealgenomes. The question of DNA repair is even more puzzling while many Archaea lives under extremetemperature that promotes DNA instability and catalyses nucleobase damages. Thus, HyperthermophilicArchaea (HA) must have solved a molecular problem (spontaneous loss of native DNA structure) at amagnitude that mesophilic organisms do not face. A highly adapted DNA maintenance system must operate inorder to maintain DNA integrity. Those mechanisms and their possible coordination with DNA replication arestill unknown. Here, I report the first protein-protein interaction network of genomic maintenance in HA. Using AP-MSapproach we identified new protein complexes potentially implicated in DNA replication, recombination andrepair of HA P. abyssi. Topological analysis of the network highlighted both known and unknown partners ofessential and conserved protein of genomic maintenance. From the network emerges multifunctional clustersintegrating both replication and recombination proteins and revealing new aspects of the transcriptionmachinery. I also provide experimental confirmation of some of the interactions we detected.I propose that the interactions we observe reflects the interplay between recombination and replicationmachineries that likely interfaces with regulatory elements involved in the control of the DNA damageresponse, as shown by the identification of a new factors, presumably involved in the coupling of DNArecombination and DNA synthesis at the replication fork.

Με την αλληλούχιση του ανθρώπινου γονιδιώματος αλλά και άλλων οργανισμών, μια νέα εποχή άρχισε για την επιστήμη της βιολογίας, γνωστή ως "μεταγονιδιωματική εποχή". Ο όρος σε καμιά περίπτωση δεν σηματοδοτεί το τέλος της αλληλούχισης ή της ανάλυσης των ήδη αλληλουχημένων γονιδιωμάτων, απλά δηλώνει πως οι τεχνικοί περιορισμοί για την ανίχνευση γονιδιωματικής πληροφορίας έχουν σε μεγάλο βαθμό αντιμετωπισθεί . Η επόμενη μεγάλη πρόκληση είναι η κατανόηση του πρωτεόματος, δηλαδή του συνόλου των πρωτεϊνών που εκφράζονται σε ένα κύτταρο. Εξαιρετικά σημαντικό βήμα προς την κατεύθυνση αυτή αποτελεί η μελέτη του δικτύου των πρωτεϊνικών αλληλεπιδράσεων. Οι πρωτεϊνικές αλληλεπιδράσεις αποτελούν ένα ιδιαίτερα σημαντικό πεδίο έρευνας καθώς ρυθμίζουν ένα τεράστιο αριθμό διεργασιών οι οποίες είναι βασικές για τη δομή και τη λειτουργία του. Εκτιμάται πως το δίκτυο των πρωτεϊνικών αλληλεπιδράσεων στον άνθρωπο αφορά περίπου 130,000 ζεύγη αλληλεπιδράσεων, ενώ ο αριθμός των αλληλεπιδράσεων που έχουν ταυτοποιηθεί αλλά παραμένει ως ζητούμενο η επιβεβαίωση των αντίστοιχων πειραματικών αποτελεσμάτων, αποτελεί μόνο το 8%, περίπου, του πραγματικού δικτύου. Ο κύριος όγκος πληροφορίας στο πεδίο αυτό δίνεται μέσα από βάσεις δεδομένων και έρχεται από μεγάλης κλίμακας πειράματα υψηλής απόδοσης τα οποία όμως, έχει βρεθεί πως δίνουν σε μεγάλο ποσοστό ανακριβή αποτελέσματα εξ αιτίας μιας σειράς εγγενών προβλημάτων των μεθόδων και της φύσεως των πρωτεϊνών. Η εκτίμηση του πραγματικού μεγέθους του δικτύου των πρωτεϊνικών αλληλεπιδράσεων υποδεικνύει και τον όγκο των δεδομένων που θα παραχθεί στο εγγύς μέλλον αλλά και την ανάγκη για βελτίωση της αξιοπιστίας της πληροφορίας που διατίθεται στις σχετικές βάσεις δεδομένων. Σκοπός αυτής της εργασίας είναι η ανάπτυξη μιας αναλυτικής βάσης δεδομένων γνώσης για την ταυτοποίηση δικτύων πρωτεϊνικών αλληλεπιδράσεων αντλώντας πληροφορία από επιλεγμένες μετά από αξιολόγηση, δημόσιες βάσεις πρωτεϊνικών αλληλεπιδράσεων. Η συλλογή αυτή της πληροφορίας θα οδηγήσει μεσοπρόθεσμα στη δημιουργία μιας βάσης δεδομένων για τη διαχείριση της συλλογής, της οργάνωσης και της ανάκτησης δεδομένων πρωτεϊνικών αλληλεπιδράσεων όπου κατάλληλα υπολογιστικά εργαλεία θα αναπτυχθούν ώστε να αξιολογηθεί ο βαθμός αξιοπιστίας της καταχωρημένης σχετικής πληροφορίας σε μια μεγάλη ομάδα δημόσιων γονιδιωματικών βάσεων δεδομένων. Επίσης θα υπάρξει η δυνατότητα χρήσης εργαλείων για την ανάλυση των αξιολογημένων δεδομένων πρωτεϊνικών αλληλεπιδράσεων, την μελέτη πρωτεϊνών με άγνωστη λειτουργία αλλά και τον προσδιορισμό μη καταγεγραμμένων έως τώρα πρωτεϊνικών συμπλεγμάτων. / The elucidation of protein-protein interaction (PPI) networks (protein interactomes) is a major objective of systems biology. This task is crucial for furthering our understanding of the cellular machinery dynamics, in light of the fundamental role of proteins in cellular function. The development of high-throughput methods for PPI identification, including the widely used yeast two hybrid and the mass spectrometry of co-immunoprecipitated complexes, drastically increased the PPI data in model organisms and the human. However, these methods suffer from intrinsic detection biases and >70% of false positives. Moreover, independent public databases (dbs) of experimentally derived PPIs exhibit a remarkably limited overlap, mainly due to uncoordinated data validation and curation criteria. These limitations scale up in highly complex systems like the human for which only 8-10% of the estimated PPIs have been determined. Furthermore, new PPI prediction is affected by the current data quality and quantity along with predictive algorithm limitations to incorporate the available protein information. Thus, there is initially a need for a systematic curation of multiple datasets into one common db. We have integrated high- and low- throughput experimental data, ortholog protein interactions, protein domain interactions, and protein structure/function and expression data from twelve highly informative and updated dbs into one local db using the Microsoft_SQL_Server. The db selection was based on their unique gene content, PPIs recorded, annotation depth, curation methodology and relational scheme. This data will be enriched with PPI information mined from the literature. The final dataset will be appropriately scored to rank and validate the PPIs with increased confidence, forming the basis for a human interactome knowledge base.