Adaptations de la méthode de purification d’ARN par affinité avec l’étiquette ARiBo

Dans les dernières années, une explosion de la recherche sur les ARN a eu lieue à cause de nombreuses découvertes démontrant l’importance de l’ARN dans plusieurs processus biologiques. Ainsi, de grandes quantités d’ARN sont devenues indispensables au bon déroulement de plusieurs études, notamment pour la biologie structurale et la caractérisation fonctionnelle. Cependant, il existe encore peu de méthodes de purification simples, efficaces, fiables et produisant un ARN sous forme native. Dans les dernières années, le laboratoire Legault a mis au point une méthode de purification par affinité utilisant une étiquette ARiBo pour la purification d’ARN transcrits in vitro par la polymérase à ARN du phage T7. Cette méthode de purification d’ARN a été spécifiquement développée pour maximiser la pureté et le rendement. De plus, elle est très rapide et fonctionne avec plusieurs types d’ARN. Cependant, comme plusieurs autres méthodes de purification, cette méthode produit des ARN avec des extrémités 5′ hétérogènes. Dans ce mémoire, des solutions sont proposées pour remédier au problème d’hétérogénéité en 5ʹ′ des ARN transcrits avec la polymérase à ARN du phage T7 et purifiés par la méthode ARiBo. La première solution consiste à choisir la séquence en 5′ parmi celles des 32 séquences testées qui ne présentent pas d’hétérogénéité en 5ʹ′. La seconde solution est d’utiliser une étiquette clivable en 5ʹ′ de l’ARN d’intérêt, tel que le ribozyme hammerhead, déjà utilisée pour ce genre d’application, ou le système CRISPR/Cse3 que nous proposons dans l’article présenté dans ce mémoire. De plus, nous avons adapté la méthode ARiBo pour rendre possible la purification d’un long ARN de 614 nt, le polycistron miR-106b-25. Nous avons également démontré la possibilité d’utiliser la méthode ARiBo pour l’isolation de protéines qui se lient à un ARN donné, le précurseur de miRNA pre-miR-153-2. En conclusion, ce mémoire démontre la possibilité d’adapter la méthode ARiBo à plusieurs applications.

Purification par affinité et marquage isotopique spécifique pour études d’ARN fonctionnels

Il existe un lien étroit entre la structure tridimensionnelle et la fonction cellulaire de l’ARN. Il est donc essentiel d’effectuer des études structurales de molécules d’ARN telles que les riborégulateurs afin de mieux caractériser leurs mécanismes d’action. Une technique de choix, permettant d’obtenir de l’information structurale sur les molécules d’ARN est la spectroscopie RMN. Cette technique est toutefois limitée par deux difficultés majeures. Premièrement, la préparation d’une quantité d’ARN nécessaire à ce type d’étude est un processus long et ardu. Afin de résoudre ce problème, notre laboratoire a développé une technique rapide de purification des ARN par affinité, utilisant une étiquette ARiBo. La deuxième difficulté provient du grand recouvrement des signaux présents sur les spectres RMN de molécules d’ARN. Ce recouvrement est proportionnel à la taille de la molécule étudiée, rendant la détermination de structures d’ARN de plus de 15 kDa extrêmement complexe. La solution émergeante à ce problème est le marquage isotopique spécifique des ARN. Cependant, les protocoles élaborées jusqu’à maintenant sont très coûteux, requièrent plusieurs semaines de manipulation en laboratoire et procurent de faibles rendements. Ce mémoire présente une nouvelle stratégie de marquage isotopique spécifique d’ARN fonctionnels basée sur la purification par affinité ARiBo. Cette approche comprend la séparation et la purification de nucléotides marqués, une ligation enzymatique sur support solide, ainsi que la purification d’ARN par affinité sans restriction de séquence. La nouvelle stratégie développée permet un marquage isotopique rapide et efficace d’ARN fonctionnels et devrait faciliter la détermination de structures d’ARN de grandes tailles par spectroscopie RMN.

Tag based collaborative filtering for recommender systems

Collaborative tagging can help users organize, share and retrieve information in an easy and quick way. For the collaborative tagging information implies user’s important personal preference information, it can be used to recommend personalized items to users. This paper proposes a novel tag-based collaborative filtering approach for recommending personalized items to users of online communities that are equipped with tagging facilities. Based on the distinctive three dimensional relationships among users, tags and items, a new similarity measure method is proposed to generate the neighborhood of users with similar tagging behavior instead of similar implicit ratings. The promising experiment result shows that by using the tagging information the proposed approach outperforms the standard user and item based collaborative filtering approaches.

Collaborative filtering recommender systems using tag information

Recommender Systems is one of the effective tools to deal with information overload issue. Similar with the explicit rating and other implicit rating behaviours such as purchase behaviour, click streams, and browsing history etc., the tagging information implies user’s important personal interests and preferences information, which can be used to recommend personalized items to users. This paper is to explore how to utilize tagging information to do personalized recommendations. Based on the distinctive three dimensional relationships among users, tags and items, a new user profiling and similarity measure method is proposed. The experiments suggest that the proposed approach is better than the traditional collaborative filtering recommender systems using only rating data.

HapMap tag-SNP analysis confirms a role for COMT in schizophrenia risk and reveals a novel association

Catechol-O-methyl transferase (COMT) encodes an enzyme involved in the metabolism of dopamine and maps to a commonly deleted region that increases schizophrenia risk. A non-synonymous polymorphism (rs4680) in COMT has been previously found to be associated with schizophrenia and results in altered activity levels of COMT. Using a haplotype block-based gene-tagging approach we conducted an association study of seven COMT single nucleotide polymorphisms (SNPs) in 160 patients with a DSM-IV diagnosis of schizophrenia and 250 controls in an Australian population. Two polymorphisms including rs4680 and rs165774 were found to be significantly associated with schizophrenia. The rs4680 results in a Val/Met substitution but the strongest association was shown by the novel SNP, rs165774, which may still be functional even though it is located in intron five. Individuals with schizophrenia were more than twice as likely to carry the GG genotype compared to the AA genotype for both the rs165774 and rs4680 SNPs. This association was slightly improved when males were analysed separately possibly indicating a degree of sexual dimorphism. Our results confirm that COMT is a good candidate for schizophrenia risk, by replicating the association with rs4680 and identifying a novel SNP association.

Analysis of HapMap tag-SNPs in dysbindin (DTNBP1) reveals evidence of consistent association with schizophrenia

Dystrobrevin binding protein 1 (DTNBP1), or dysbindin, is thought to be critical in regulating the glutamatergic system. While the dopamine pathway is known to be important in the aetiology of schizophrenia, it seems likely that glutamatergic dysfunction can lead to the development of schizophrenia. DTNBP1 is widely expressed in brain, levels are reduced in brains of schizophrenia patients and a DTNBP1 polymorphism has been associated with reduced brain expression. Despite numerous genetic studies no DTNBP1 polymorphism has been strongly implicated in schizophrenia aetiology. Using a haplotype block-based gene-tagging approach we genotyped 13 SNPs in DTNBP1 to investigate possible associations with DTNBP1 and schizophrenia. Four polymorphisms were found to be significantly associated with schizophrenia. The strongest association was found with an A/C SNP in intron 7 (rs9370822). Homozygotes for the C allele of rs9370822 were more than two and a half times as likely to have schizophrenia compared to controls. The other polymorphisms showed much weaker association and are less likely to be biologically significant. These results suggest that DTNBP1 is a good candidate for schizophrenia risk and rs9370822 is either functionally important or in disequilibrium with a functional SNP, although our observations should be viewed with caution until they are independently replicated.

Website customization : exploring a tag-based approach in the Australian banking context

Website customization can help to better fulfill the needs and wants of individual customers. It is an important aspect of customer satisfaction of online banking, especially among the younger generation. This dimension, however, is poorly addressed particularly in the Australian context. The proposed research aims to fulfill this gap by exploring the use of a popular Web 2.0 technology known as tags or user assigned metadata to facilitate customization at the interaction level. A prototype is proposed to demonstrate the various interaction-based customization types, evaluated through a series of experiments to assess the impact on customer satisfaction. The expected research outcome is a set of guidelines akin to interaction design patterns for aiding the design and implementation of the proposed tag-based approach.

Online banking customization via tag-based interaction

In this paper, we describe ongoing work on online banking customization with a particular focus on interaction. The scope of the study is confined to the Australian banking context where the lack of customization is evident. This paper puts forward the notion of using tags to facilitate personalized interactions in online banking. We argue that tags can afford simple and intuitive interactions unique to every individual in both online and mobile environments. Firstly, through a review of related literature, we frame our work in the customization domain. Secondly, we define a range of taggable resources in online banking. Thirdly, we describe our preliminary prototype implementation with respect to interaction customization types. Lastly, we conclude with a discussion on future work.

Learning domain ontology for tag recommendation

Recently, user tagging systems have grown in popularity on the web. The tagging process is quite simple for ordinary users, which contributes to its popularity. However, free vocabulary has lack of standardization and semantic ambiguity. It is possible to capture the semantics from user tagging and represent those in a form of ontology, but the application of the learned ontology for recommendation making has not been that flourishing. In this paper we discuss our approach to learn domain ontology from user tagging information and apply the extracted tag ontology in a pilot tag recommendation experiment. The initial result shows that by using the tag ontology to re-rank the recommended tags, the accuracy of the tag recommendation can be improved.

Constructing tag ontology from folksonomy based on WordNet

With the emergence of Web 2.0, Web users can classify Web items of their interest by using tags. Tags reflect users’ understanding to the items collected in each tag. Exploring user tagging behavior provides a promising way to understand users’ information needs. However, free and relatively uncontrolled vocabulary has its drawback in terms of lack of standardization and semantic ambiguity. Moreover, the relationships among tags have not been explored even there exist rich relationships among tags which could provide valuable information for us to better understand users. In this paper, we propose a novel approach to construct tag ontology based on the widely used general ontology WordNet to capture the semantics and the structural relationships of tags. Ambiguity of tags is a challenging problem to deal with in order to construct high quality tag ontology. We propose strategies to find the semantic meanings of tags and a strategy to disambiguate the semantics of tags based on the opinion of WordNet lexicographers. In order to evaluate the usefulness of the constructed tag ontology, in this paper we apply the extracted tag ontology in a tag recommendation experiment. We believe this is the first application of tag ontology for recommendation making. The initial result shows that by using the tag ontology to re-rank the recommended tags, the accuracy of the tag recommendation can be improved.

An expressed sequence tag (EST) library for Drosophila serrata, a model system for sexual selection and climatic adaptation studies

The native Australian fly Drosophila serrata belongs to the highly speciose montium subgroup of the melanogaster species group. It has recently emerged as an excellent model system with which to address a number of important questions, including the evolution of traits under sexual selection and traits involved in climatic adaptation along latitudinal gradients. Understanding the molecular genetic basis of such traits has been limited by a lack of genomic resources for this species. Here, we present the first expressed sequence tag (EST) collection for D. serrata that will enable the identification of genes underlying sexually-selected phenotypes and physiological responses to environmental change and may help resolve controversial phylogenetic relationships within the montium subgroup.

A novel DRD2 single-nucleotide polymorphism associated with schizophrenia predicts age of onset : HapMap tag-single-nucleotide polymorphism analysis

Background: Dopamine D2 receptor (DRD2) is thought to be critical in regulating the dopaminergic pathway in the brain which is known to be important in the aetiology of schizophrenia. It is therefore not surprising that most antipsychotic medication acts on the Dopamine D2 receptor. DRD2 is widely expressed in brain, levels are reduced in brains of schizophrenia patients and DRD2 polymorphisms have been associated with reduced brain expression. We have previously identified a genetic variant in DRD2, rs6277 to be strongly implicated in schizophrenia susceptibility. Methods: To identity new associations in the DRD2 gene with disease status and clinical severity, we genotyped seven single nucleotide polymorphisms (SNPs) in DRD2 using a multiplex mass spectrometry method. SNPs were chosen using a haplotype block-based gene-tagging approach so the entire DRD2 gene was represented. Results: One polymorphism rs2734839 was found to be significantly associated with schizophrenia as well as late onset age. Individuals carrying the genetic variation were more than twice as likely to have schizophrenia compared to controls. Conclusions: Our results suggest that DRD2 genetic variation is a good indicator for schizophrenia risk and may also be used as a predictor age of onset.

Learning personalized tag ontology from user tagging information

The cross-sections of the Social Web and the Semantic Web has put folksonomy in the spot light for its potential in overcoming knowledge acquisition bottleneck and providing insight for "wisdom of the crowds". Folksonomy which comes as the results of collaborative tagging activities has provided insight into user's understanding about Web resources which might be useful for searching and organizing purposes. However, collaborative tagging vocabulary poses some challenges since tags are freely chosen by users and may exhibit synonymy and polysemy problem. In order to overcome these challenges and boost the potential of folksonomy as emergence semantics we propose to consolidate the diverse vocabulary into a consolidated entities and concepts. We propose to extract a tag ontology by ontology learning process to represent the semantics of a tagging community. This paper presents a novel approach to learn the ontology based on the widely used lexical database WordNet. We present personalization strategies to disambiguate the semantics of tags by combining the opinion of WordNet lexicographers and users’ tagging behavior together. We provide empirical evaluations by using the semantic information contained in the ontology in a tag recommendation experiment. The results show that by using the semantic relationships on the ontology the accuracy of the tag recommender has been improved.

Personalization in tag ontology learning for recommendation making

Due to the explosive growth of the Web, the domain of Web personalization has gained great momentum both in the research and commercial areas. One of the most popular web personalization systems is recommender systems. In recommender systems choosing user information that can be used to profile users is very crucial for user profiling. In Web 2.0, one facility that can help users organize Web resources of their interest is user tagging systems. Exploring user tagging behavior provides a promising way for understanding users’ information needs since tags are given directly by users. However, free and relatively uncontrolled vocabulary makes the user self-defined tags lack of standardization and semantic ambiguity. Also, the relationships among tags need to be explored since there are rich relationships among tags which could provide valuable information for us to better understand users. In this paper, we propose a novel approach for learning tag ontology based on the widely used lexical database WordNet for capturing the semantics and the structural relationships of tags. We present personalization strategies to disambiguate the semantics of tags by combining the opinion of WordNet lexicographers and users’ tagging behavior together. To personalize further, clustering of users is performed to generate a more accurate ontology for a particular group of users. In order to evaluate the usefulness of the tag ontology, we use the tag ontology in a pilot tag recommendation experiment for improving the recommendation performance by exploiting the semantic information in the tag ontology. The initial result shows that the personalized information has improved the accuracy of the tag recommendation.