L’émergence et l’évolution des réseaux de nouvelles génération (NGN) a soulevé plusieurs défis surtout en termes d’hétérogénéité, de mobilité et de sécurité. En effet, l’utilisateur est capable, dans un tel environnement, d’avoir accès à plusieurs réseaux, à travers différents terminaux, avec un choix vaste de services fournis par différents fournisseurs. De plus, les utilisateurs finaux demandent à être constamment connectés n’importe où, n’importe quand et n’importe comment. Ils désirent également avoir un accès sécurisé à leurs services à travers une session dynamique, seamless et continue selon leurs préférences et la QoS demandée. Dans ce contexte, la sécurité représente une composante majeure. Face à cette session user-centric sécurisée, plusieurs défis se posent. L’environnement est de plus en plus ouvert, de multiples services ne sont pas connus d’avance et nous avons une diversité de communications entre les services et les utilisateurs. L’hétérogénéité des ressources (terminaux, réseaux et services) impliquées dans la session de l’utilisateur accentue la complexité des tâches de sécurité. Les différentes déclinaisons de mobilité (mobilité de l’utilisateur, mobilité du terminal, mobilité du réseau et mobilité du service) modifient la session user-centric que l’on veut unique, sécurisée et seamless avec la délivrance d’un service continu. / The emergence and evolution of Next Generation Networks (NGN) have raised several challenges mainly in terms of heterogeneity, mobility and security. In fact, the user is able, in such environment, to have access to many networks, via multiple devices, with a vast choice of services offered by different providers. Furthermore, end-users claim to be constantly connected anywhere, anytime and anyhow. Besides, they want to have a secure access to their services through a dynamic, seamless and continuous session according to their preferences and the desired QoS. In this context, security represents an important concern. In fact, this user-centric session should obviously be secured. However, many challenges arise. In such environment, system boundaries, which were well delimited, become increasingly open. Indeed, there are multiple services which are unknown in advance and multiple communications between services and with users. Besides, heterogeneity of involved resources (terminals, networks and services) in the user session increases the complexity of security tasks. In addition, the different types of mobility (user, terminal, network and service mobility) affect the user-centric session that should be unique, secure and seamless and ensure continuity of services.

Indiana University-Purdue University Indianapolis (IUPUI) / Several experimental methods has been developed for the study of the central dogma since late 20th century. Protein mass spectrometry and next generation sequencing (including DNA-Seq and RNA-Seq) forms a triangle of experimental methods, corresponding to the three vertices of the central dogma, i.e., DNA, RNA and protein. Numerous RNA sequencing and protein mass spectrometry experiments has been carried out in attempt to understand how the expression change of known genes affect biological functions in various of organisms, however, it has been once overlooked that the result data of these experiments are in fact holograms which also reveals other delicate biological mechanisms, such as RNA splicing and the expression of long non-coding RNAs. In this dissertation, we carried out five studies based on high-throughput sequencing data, in an attempt to understand how RNA splicing and differential expression of long non-coding RNAs is associated biological functions. In the first two studies, we identified and characterized 197 stimulant induced and 477 developmentally regulated alternative splicing events from RNA sequencing data. In the third study, we introduced a method for identifying novel alternative splicing events that were never documented. In the fourth study, we introduced a method for identifying known and novel RNA splicing junctions from protein mass spectrometry data. In the fifth study, we introduced a method for identifying long non-coding RNAs from poly-A selected RNA sequencing data. Taking advantage of these methods, we turned RNA sequencing and protein mass spectrometry data into an information gold mine of splicing and long non-coding RNA activities. / 2019-05-06

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第18122号 / 理博第4000号 / 新制||理||1577(附属図書館) / 30980 / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 村山 美穂, 教授 幸島 司郎, 教授 伊谷 原一 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

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Advances in next-generation sequencing (NGS) technologies have resulted in significant reduction of cost per sequenced base pair and increase in sequence data volume. On the other hand, most currently used NGS technologies produce relatively short sequence reads (50 - 150 bp) compared to Sanger sequencing (~700 bp). This represents an additional challenge in data analysis, because shorter reads are more difficult to assemble. At this point, production of sequencing data outpaces our capacity to analyze them. Newer NGS technologies capable of producing longer reads are emerging, which should simplify and speed up genome assembly. However, this will only increase the number of sequenced genomes without structural and functional annotation. In addition to multiple scientific initiatives to sequence thousands of genomes, personalized medicine centered on sequencing and analysis of individual human genomes will become more available. This poses a challenge for computer science and emphasizes the importance of developing new computational algorithms, methodology, tools, and pipelines. This dissertation focuses on development of these software tools, methodologies, and resources to help address the need for processing of volumes of data generated by new sequencing technologies. The research concentrated on genome structure analysis, individual variation, and comparative biology. This dissertation presents: (1) the Short Read Classification Pipeline (SRCP) for preliminary genome characterization of unsequenced genomes; (2) a novel methodology for phylogenetic analysis of closely related organisms or strains of the same organism without a sequenced genome; (3) a centralized online resource for standardized gene nomenclature. Utilizing the SRCP and the methodology for initial phylogenetic analysis developed in this dissertation enables positioning the organism in the evolutionary context. This should facilitate identification of orthologs between the species and paralogs within the species even in the initial stage of the analysis when only exome is sequenced and, thus, enable functional annotation by transferring gene nomenclature from well-annotated 1:1 orthologs, as required by the online standardized gene nomenclature resource developed in this dissertation. Thus, the tools, methodology, and resources presented here are tied together in following the initial analysis workflow for structural and functional annotation.

Emerging sequencing approaches have revolutionized the way we can collect DNA sequence data for applications in bioforensics and biosurveillance. In this research, we present an approach to construct a database of known biological agents and use this database to develop a statistical framework to analyze raw reads from next-generation sequence data for species identification and strain attribution. Our method capitalizes on a Bayesian statistical framework that accommodates information on sequence quality, mapping quality and provides posterior probabilities of matches to a known database of target genomes. Importantly, our approach also incorporates the possibility that multiple species can be present in the sample or that the target strain is not even contained within the reference database. Furthermore, our approach can accurately discriminate between very closely related strains of the same species with very little coverage of the genome and without the need for genome assembly - a time consuming and labor intensive step. We demonstrate our approach using genomic data from a variety of known bacterial agents of bioterrorism and agents impacting human health.

Yes / Introduction: Mobile phones are used the world over, including in healthcare settings. This study aimed to investigate the viable microbial colonisation of mobile phones used by healthcare personnel. Methods: Swabs collected on the same day from 30 mobile phones belonging to healthcare workers from three separate paediatric wards of an Australian hospital were cultured on five types of agar plate, then colonies from each phone were pooled, extracted and sequenced by shotgun metagenomics. Questionnaires completed by staff whose phones were sampled assisted in the analysis and interpretation of results. Results and discussion: All phones sampled cultured viable bacteria. Overall, 399 bacterial operational taxonomic units were identified from 30 phones, with 1432 cumulative hits. Among these were 58 recognised human pathogenic and commensal bacteria (37 Gram-negative, 21 Gram-positive). The total number of virulence factor genes detected was 347, with 1258 cumulative hits. Antibiotic resistance genes (ARGs) were detected on all sampled phones and overall, 133 ARGs were detected with 520 cumulative hits. The most important classes of ARGs detected encoded resistance to beta-lactam, aminoglycoside and macrolide antibiotics and efflux pump mediated resistance mechanisms. Conclusion: Mobile phones carry viable bacterial pathogens and may act as fomites by contaminating the hands of their users and indirectly providing a transmission pathway for hospital-acquired infections and dissemination of antibiotic resistance. Further research is needed, but meanwhile adding touching mobile phones to the five moments of hand hygiene is a simple infection control strategy worth considering in hospital and community settings. Additionally, the implementation of practical and effective guidelines to decontaminate mobile phone devices would likely be beneficial to the hospital population and community at large.

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Introduction: Sepsis is a major source of morbidity and mortality in neonates; however, identification of the causative pathogens can be challenging. Next generation sequencing (NGS) is a high-throughput, parallel sequencing technique for DNA. Pathogen-targeted enrichment followed by NGS has the potential to be more sensitive and faster than current gold-standard blood culture. In this pilot study, we will test the feasibility and pathogen detection patterns of pathogen-targeted NGS in neonates with suspected sepsis. Additionally, the distribution and diagnostic accuracy of cell-free DNA and protein C levels at two time points will be explored. Methods: We will conduct a prospective, pilot observational study. Neonates over 1 kg with suspected sepsis from a single tertiary care children’s hospital will be recruited for the study. Recruitment will be censored at 200 events or 6 months duration. Two blood study samples will be taken: the first simultaneous to the blood culture (time = 0 hr, for NGS and biomarkers) via an exception to consent (deferred consent) and another 24 hours later after prospective consent (biomarkers only). Neonates will be adjudicated into those with clinical sepsis, culture-proven sepsis and without sepsis based on clinical criteria. Feasibility parameters (e.g. recruitment) and NGS process time will be reported. Analysis: NGS results will be described in aggregate, compared to the simultaneous blood culture (sensitivity and specificity) and reviewed via expert panel for plausibility. Pilot data for biomarker distribution and diagnostic accuracy (sensitivity and specificity) for distinguishing between septic and non-septic neonates will be reported. Study amendment and interim results: After obtaining ethics approval, study enrolment started October 15, 2020. Interim feasibility results showed successful deferred consent, but low enrolment. A study amendment was used to increase enrolment, create pre-packaged blood kits and implement a substitute decision maker Notification form. / Thesis / Master of Health Sciences (MSc)