Background: Obesity is a disease that is caused by genetic and environmental factors. However, epigenetic mechanisms of obesity are less well known. DNA methylation provides a mechanism whereby environmental factors can influence gene transcription. The aim of our study was to investigate skeletal muscle DNA methylation of sorbin and SH3 domain containing 3 (SORBS3) with weight loss induced by Roux-en-Y gastric bypass (RYGB). Results: Previously, we had shown increased methylation (5.0 to 24.4%) and decreased gene expression (fold change -1.9) of SORBS3 with obesity (BMI > 30 kg/m(2)) compared to lean controls. In the present study, basal muscle biopsies were obtained from seven morbidly obese (BMI > 40 kg/m(2)) female subjects pre-and3months post-RYGB surgery, in combination with euglycemic-hyperinsulinemic clamps to assess insulin sensitivity. We identified 30 significantly altered promoter and untranslated region methylation sites in SORBS3 using reduced representation bisulfite sequencing (RRBS). Twenty-nine of these sites were decreased (-5.6 to -24.2%) post-RYGB compared to pre-RYGB. We confirmed the methylation in 2 (Chr. 8: 22,423,690 and Chr. 8: 22,423,702) of the 29 decreased SORBS3 sites using pyrosequencing. This decreased methylation was associated with an increase in SORBS3 gene expression (fold change + 1.7) post-surgery. In addition, we demonstrated that SORBS3 promoter methylation in vitro significantly alters reporter gene expression (P < 0. 0001). Two of the SORBS3 methylation sites (Chr. 8: 22,423,111 and Chr. 8: 22,423,205) were strongly correlated with fasting plasma glucose levels (r = 0.9, P = 0.00009 and r = 0.8, P = 0.0010). Changes in SORBS3 gene expression post-surgery were correlated with obesity measures and fasting insulin levels (r = 0.5 to 0.8; P < 0.05). Conclusions: These results demonstrate that SORBS3 methylation and gene expression are altered in obesity and restored to normal levels through weight loss induced by RYGB surgery.

One of the most crucial steps towards nanopore-based nucleic acid analysis is extending the dwell time of DNA molecules within the sensing region of the nanopore. I address this issue by interfacing solid-state nanopores with gel media, which sterically hinders translocating DNA molecules, increasing dwell times. Specifically, my experimental results focus on two reptation regimes: when the DNA molecule is flexible on the length scale of the gel pore, and when the DNA molecule is inflexible on the length scale of the gel pore. The first regime is achieved through the use of agarose gel and 5 kbp dsDNA fragments, and produces a wide distribution of translocation times, spanning roughly three orders of magnitude. The second regime is achieved through the use of polyacrylamide gel and 100 bp dsDNA fragments, and displays a shift in translocation times by an order of magnitude while maintaining a tight distribution.

With the technological shift towards big data, internet of things (IoT), 5G applications and cloud computing, the demand for high capacity networks is dramatically increasing. To avoid congestion and saturation, content and service providers are re-designing their network (backbone, metro and data-centers interconnects) connectivity using gridless optical line systems along with programmable coherent transponders. The latter are expected to transmit data at different data rates up to 400 Gb/s. In 2008, the first coherent receiver was commercially available [1]. By means of high-speed analog to digital converters and adaptive digital signal processing (DSP) algorithms, such revolution in modern optical communication was possible. That allowed a better spectral efficiency using higher order modulation formats and further signal reach by means of compensating both linear and nonlinear impairments. Another key development was leveraging light polarization-diversity, that permits to double the data rate at the expense of receiver complexity. To further increase the capacity of fiber links, gridless DWDM networks are being developed for deployment in the next few years. The key idea is to allow variable bandwidth signals to be allocated on optical links and by performing the appropriate network layer optimization improved throughput can be achieved. These innovations are driving new types of challenges for routing and assignment methods, as well, DSP algorithms such as clock recovery and compensation of fiber non-linearity. This thesis is organized as a collection of contributions and composed of five major parts. The first part, consisting of chapters 2 and 3. Chapter 4 deals with tracking of fast state of polarization transient, i.e. dynamic aspect of optical channels, in presence of polarization dependent loss (PDL) and filtering effects due to reconfigurable optical add-drop multiplexers (ROADMs). Chapters 5 and 6 study the impact of filtering effects, quasi-static effects in optical links and transponders, represented by ROADMs in fixed-grid and Silicon Photonics (SiPh) modulators in flexible-grid networks, respectively. Chapters 7, 8 and 9, are related to clock recovery in digital coherent receivers. They cover mitigation of jitter in gridless applications, improving jitter when deploying phase interpolators (PI) and jitter injection as a test-mean to evaluate performance.

Next generation sequencing, which allows concurrent parallel sequencing of many samples and makes it possible to distinguish the infection from multiple viral types in the sample, is well suited as a detection format for such assays described below. The aim of the thesis was to develop a method that could detect all known types of human adenoviruses, human enteroviruses, and bacteriophages selected for their presence in the intestine. Using the next- generation sequencing. The first step was to design primers capable of detecting all known types of viruses, covering the area that is capable of distinguishing these viruses. This method was tested on a set of 47 human adenovirus samples and 30 human enterovirus samples of known serotype. Samples with two serotypes in different proportions were also created. After amplification of the target genome, the samples were purified and sequenced on MiSeq, Illumina. The method was further used in the typing of adenoviruses, enteroviruses and bacteriophages in pre-diabetic cohorts of DIPP, MIDIA, and a cohort of diabetics from African and Asian countries. The tested sample was RNA / DNA isolated from the stool specimen. We have demonstrated that the method is capable to detect all tested virus types, including infections with two different types, even if the...

The use of short tandem repeats (STRs) for genotyping forensic case samples has long been an effective tool for human identification. However, interpretation of forensic STR mixture samples can be difficult and any additional information to aid in this process can be invaluable. Allele overlap and stutter during PCR can cause drop out of the minor contributor’s alleles and result in incorrect allele calling. The Scientific Working Group on DNA Analysis Methods (SWGDAM) provides a list of guidelines on how to interpret DNA typing results from forensic STRs and mixtures, but there is still a significant variation in the interpretation of mixture samples between analysts in the same laboratory and between laboratories. The Illumina MiSeq Forensic GenomicsTM system (Illumina Inc., San Diego, CA) is a massively parallel sequencing instrument that was developed specifically for the use in forensic DNA typing and which could provide sequence variations among on mixture samples. The ForenSeqTM DNA Signature Prep Kit is a kit that can be used with the MiSeq FGxTM platform. The DNA Primer Mix A (DPMA) included in the ForenSeqTM kit targets 27 autosomal STRs, 24 Y-STRs, 7 X-STRs and 94 identity single nucleotide polymorphisms (SNPs) on up to 32 or 96 samples, depending on the flow cell used. This study compares the STR performance on DNA mixtures of the MiSeq FGxTM and CE and evaluates its reliability and robustness. The MiSeq FGxTM provides data in read count and the CE in relative fluorescence units (RFU), so the two output data cannot be directly compared to one another. Instead, the ratio of two contributors was calculated at three mixture ratios (1:1, 1:4, and 1:9) to use as a mean of comparison. The mean contributor ratios calculated on the MiSeq FGxTM were 1.799, 7.595, and 13.524 for the 1:1, 1:4, and 1:9 mixtures, respectively. This was not significantly different from the CE mean contributor ratios of 1.818, 7.722, and 14.827, respectively. More allele dropouts occurred on the MiSeq FGxTM than the CE at both 1:4 and 1:9 mixture ratios, but sequencing provided the detection of six isoalleles based on sequence variants that could not be discerned by CE. Other studies have shown full profile generation at these ratios, indicating there could have been some issues during library preparation. Further studies should be performed to thoroughly validate the ForenSeqTM process and evaluate the sensitivity of the instrument. Until then, it is recommended that the ForenSeqTM kit and MiSeq FGxTM system be used at close to equal mixture ratios or in tandem with the CE to prevent genotypes miscalling.

Melanoma is a heterogeneous neoplasm at the histomorphologic, immunophenotypic, and molecular levels. Melanoma with extreme histomorphologic heterogeneity can pose a diagnostic challenge in which the diagnosis may predominantly rely on its immunophenotypic profile. However, tumor survival and response to therapy are linked to tumor genetic heterogeneity rather than tumor morphology. Therefore, understating the molecular characteristics of such melanomas become indispensable. In this study, DNA was extracted from 11 morphologically distinct regions in eight formalin-fixed, paraffin-embedded melanomas. In each region, mutations in 50 cancer-related genes were tested using next-generation sequencing (NGS). A tumor was considered genetically heterogeneous if at least one non-overlapping mutation was identified either between the histologically distinct regions of the same tumor (intratumor heterogeneity) or among the histologically distinct regions of the paired primary and metastatic tumors within the same patient (intertumor heterogeneity). Our results revealed that genetic heterogeneity existed in all tumors as non-overlapping mutations were detected in every tested tumor (n = 5, 100%; intratumor: n = 2, 40%; intertumor: n = 3, 60%). Conversely, overlapping mutations were also detected in all the tested regions (n = 11, 100%). Melanomas exhibiting histomorphologic heterogeneity are often associated with genetic heterogeneity, which might contribute to tumor survival and poor response to therapy.

Introduction: The surprising presence of P. vivax in West Africa and their ability to infect a Duffy negative population is one more threat to public health. In order to contribute to malaria elimination efforts, there is a need to investigate the origin and characteristics of P. vivax population isolates in Northern Mali. Next Generation Sequence Analysis (NGSA) can help us understand parasite genetic characteristics although low parasite density is a challenge for whole genome sequencing (WGS). In the present work, we investigated if selective whole genome amplification (sWGA) can enrich P. vivax DNA extracted from Rapid Diagnostic Tests (RDTs) for Whole Genome Sequencing. We also investigated the origin and the susceptibility to antimalarial drugs of the strains isolated in Northern Mali. Methods: Parasite DNA was extracted from 267 RDTs using the QIAamp DNA mini kit, then nested PCR and 7 samples were positive for P. vivax. After sWGA, the whole genomes were sequenced using the Illumina platform. Next Generation Sequences Analysis was done followed by population differentiation analyses. Twenty-two additional P. vivax whole genomes from other parts of the World were downloaded from the European Nucleotide Archive for further Neighbour Joining analysis. Results: The sequences extracted from RDTs showed high contamination with human DNA (80%). From the parasite DNA, in total 69529 SNPs were found in the seven P. vivax strains of Northern Mali. The most significant p-values per SNP were carried by the chromosomes 2, 3, 4, 5, 12, 13 and 14. With regard to variant effects, the Transition/Transversion ratio was 1.1. The density of variants with a high effect was 1.62%. There was no mutation associated with antimalarial drugs resistance on pvcrt-o or pvmdr-1 genes. Pairwise differentiation suggests a high degree of relatedness between P. vivax strains isolated in Northern Mali. The NeighboursJoining analysis shows clearly that strains from Mali cluster together and are genetically distinct from those from Mauritania, which shares a border with Mali. The strains isolated in Northern Mali are genetically closer to those from Madagascar, India and Latina America. Conclusion: We did not identify mutations associated to the resistance to antimalarial drugs in pvcrt-o and pvmdr-1 genes. This study confirms that P. vivax strains genetically distinct from those of Mauritania are circulating in Mali. Finally, we conclude that sWGA is a feasible approach for P. vivax DNA enrichment for WGS despite the high proportion of human contamination.

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