Contexte: La maladie de Waldenstrom (MW) est un syndrome lymphoprolifératif B caractérisé par une infiltration de la moelle osseuse par des lymphoplasmocytes et un pic monoclonal de type IgM. La mutation MYD88L265P est considérée comme un événement fondateur dans cette hémopathie. Les études par séquençage complet du génome ont identifié des mutations du gène CXCR4 (CXCR4 Muté) dans la MW. CXCR4 est un récepteur couplé aux protéines G qui participe aux processus de migration cellulaire et d’activation de différentes cascades de signalisation parmi lesquelles figurent RAS, Akt et NFKB. Les mutations de CXCR4 joueraient un rôle important dans la physiopathologie de la MW et dans les mécanismes de chimiorésistance de certaines thérapeutiques ciblées. Notre objectif a été d’étudier le profil génomique des MW avec mutations CXCR4 par séquençage à haut débit ciblé et par puces à SNP (single nucleotide polymorphism) et d’évaluer leur impact clinique. Matériel et Méthodes. Les échantillons de moelle osseuse de 98 MW ont été analysés. L’ADN tumoral a été extrait après sélection des cellules B. Des échantillons appariés (tumeur/ lymphocytes T) ont été utilisés comme référence intra-individuelle. Les mutations de CXCR4 ont été étudiées en séquençage ciblé à haut débit (NGS) ce qui a permis d’évaluer l’architecture clonale dans les cellules tumorales de MW et /ou par séquençage Sanger (exon 2). Le spectre mutationnel de 8 gènes candidats impliqués dans les voies des Toll Like Receptor, RAS et du BCR a été étudié : MYD88L265P, CD79A (domaine ITAM), CD79B (domaine ITAM), CARD11 (exon 5-9), N RAS (exon 2-3), K RAS (exons 2-3), BRAF6 (exon 15), PTEN (exon 5-7). L’analyse des profils génomiques a été réalisée en puces à SNP (Genome-Wide Human SNP Array 6.0 (Affymetrix chips) dans une cohorte de 53 patients. L’expression de CXCR4 et du CD49d (VLA4) ont été étudiées en cytométrie de flux (n=53). Résultats. Nous avons identifié une mutation de CXCR4 dans 24.5% des MW. Toutes mutations sont hétérozygotes, acquises et localisées dans le domaine C terminal de la protéine. Parmi les 17 variants observés, nous identifions 12 nouvelles mutations dans la MW. Les mutations les plus fréquentes sont le variant C1013G (S338X) (5/98) et la mutation C1013A (S338X) (3/98). Les mutations de CXCR4 muté sont associées à une plus forte expression de la protéine CXCR4 en cytométrie de flux, indépendamment du type de mutations de CXCR4 (n=53) (p=0.003). Aucun impact en terme de niveau d’expression de l’intégrine VLA4 (CD49d), qui interagit directement avec CXCR4, n’est observé. Des mutations sous clonales de CXCR4 sont identifiées en NGS dans 4/14 cas. Les mutations de CXCR4 sont présentes dans le même clone que MYD88 L265P, mais sont mutuellement exclusives des mutations de la voie du BCR (CD79A / CD79B). Nous avons ensuite identifié une signature génomique plus complexe dans le groupe des MW CXCR4 muté. Les mutations de CXCR4 sont associées aux gains du chromosome 4 (p=0.002), aux gains en Xq (p=0.002), aux délétions 6q (p=0.038) et présentent un nombre d’anomalies plus élevées (5.8 versus 2.8 per patient, p=0.046). Nous avons ensuite cherché à caractériser le profil clinico biologique des patients MW avec mutation CXCR4 muté. Les mutations de CXCR4 sont associées à un pic monoclonal IgM plus élevé (p=0.006) et à une thrombopénie (p=0.018). Aucun impact en terme de survie globale n’a été observé dans notre cohorte selon le statut mutationnel de CXCR4. Conclusion. Notre étude a permis de décrire de nouvelles mutations de CXCR4 dans la MW et d’identifier une signature génomique plus complexe associée dans les MW CXCR4 muté parmi les MW MYD88L265P. Cette analyse des mutations de CXCR4 a d’autre part montré l’existence d’une hétérogénéité intra-clonal (dans le clone muté MYD88 et CXCR4) et interclonal (mutations du BCR et de CXCR4 dans le groupe des WM mutés MYD88L265P). Nos résultats suggèrent donc l’existence de différents sous groupes génomiques dans la MW. / Purpose. Waldenstrom macroglobulinemia (WM) is a B-cell malignancy characterized by bone marrow infiltration of clonal lymphoplasmacytic cells, which produce a monoclonal immunoglobulin M (IgM). MYD88L265P mutation may be considered as a founder event because of it high frequency in WM. Whole-genome sequencing has revealed CXCR4 mutations (CXCR4Mut) in WM. CXCR4 is a G-protein-coupled receptor that promotes migration and activation of several pathways including RAS, Akt and NFKB. CXCR4 mutation has proved to be of critical importance in WM, in part due to its role as a mechanism of resistance to several agents of targeted therapy. We have therefore sought to unravel the different aspects of CXCR4 mutations in WM using next generation sequencing, and SNP (single nucleotide polymorphism) arrays and to study the clinical impact.Experimental Design. Bone marrow samples of 98 patients with WM (mean age: 67 years) were analyzed. Tumoral DNA was extracted following CD19 B cell selection. Paired samples (tumor/T lymphocytes) were used as an intra-individual reference. CXCR4 mutation was analyzed by ultra deep targeted NGS (next generation sequencing) (all exons) allowing study of the clonal architecture in WM cells and/or sanger sequencing (SaS) (exon 2). Mutational spectrum of 8 candidate genes involved in Toll Like Receptor, RAS and B Cell Receptor (BCR) pathway along with MYD88L265P, CD79A (ITAM domain), CD79B( ITAM domain), CARD11 (exon 5-9), N RAS (exon 2-3), K RAS (exons 2-3), BRAF6 (exon 15), PTEN( exon 5-7), was also analysed in an integrative study. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was performed in 53 patients to decipher genomic signature of CXCR4Mut. Flow cytometry was performed to assess CXCR4 and CD49d (VLA4) expression (n=53).Results. We found all mutations to be heterozygous, somatic and located in the C-terminal domain of CXCR4 in 24.5% of the WM. CXCR4 mutations led to a truncated receptor protein. Among the 17 variants, 12 new variants were identified in WM. The most frequent mutation was the CXCR4 C1013G (S338X) mutation (5/98) followed by CXCR4C1013A (S338X) (3/98). Interestingly, CXCR4Mut was associated to higher expression of CXCR4 protein by flow cytometry, independently of the type of CXCR4 mutation (n=53) (p=0.003). No impact on the expression profile of the integrin VLA4 (CD49d) which directly interacts with CXCR4 was observed. Sub clonal CXCR4 mutations identified using NGS were identified in 4/14 cases. CXCR4 mutations pertain to the same clone as to MYD88 L265P mutations, but were mutually exclusive to CD79A/ CD79B mutations (BCR pathway). We identified a genomic signature in CXCR4Mut WM traducing a more complex genome. CXCR4 mutations were also associated with gain of chromosome 4 (p=0.002), gain of Xq (p=0.002) and deletion 6q (p=0.038) and a higher number of genomic abnormalities (5.8 versus 2.8 per patient, p=0.046). We thought to identify clinical-biologic characteristics of WM with CXCR4Mut features. CXCR4 mutations were associated with higher IgM monoclonal component (p=0.006) and thrombocytopenia (p=0.018). However, no impact in overall survival was observed according to CXCR4 mutational status. Conclusions. Our study panned out new CXCR4 mutations in WM, and identified a specific signature associated to CXCR4Mut, characterized with complex genomic aberrations among MYD88L265P WM. The study of CXCR4 mutations showed existence of intraclonal (variation in co-expression of MYD88 and CXCR4 mutations) and interclonal (BCR and CXCR4 mutations in MYD88L265P WM) heterogeneity. Our results suggest the existence of various genomic subgroups in WM.

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.

Background: Palliative care can be given to patients with progressive, incurable disease. In palliative care the patient is seen in a holistic perspective, the goal is to prevent and relieve physical, psychological, social and existential suffering. The four cornerstones of palliative care are symptom relief, teamwork, communication/relation and support to next of kin. The nurse in palliative care builds a relationship with the patient and next of kin. The nurse also works with symptom relief and informing patient and next of kin. Next of kin to patients in palliative care often put themselves aside to care for the patient and wish to be with the patient until the end of the patients’ life. Aim: The aim of the study was to describe next of kins’ need for support from the nurse in end-of-life care in hospitals. Method: A literature study based on nine qualitative articles retrieved through systematic search in database Cinahl. The articles were analysed with a five-step method. Results: Two categories emerged in the results. The first was A respectful nurse, with the subcategories Emotional support, To be together and The patient is well taken care of. The second was To be involved as next of kin with the subcategories To be informed and Participation. Conclusion: Next of kin to patients in end-of-life care need a nurse who cares for them, enables them to be with the patient and participate in the patients’ care, guides and informs them in the end-of-life process and takes good care of the patient.

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.

Indiana University-Purdue University Indianapolis (IUPUI) / Machine learning models are rising every day. Most of the Computer Vision oriented machine learning models arise from Convolutional Neural Network’s(CNN) basic structure. Machine learning developers use CNNs extensively in Image classification, Object Recognition, and Image segmentation. Although CNN produces highly compatible models with superior accuracy, they have their disadvantages. Estimating pose and transformation for computer vision applications is a difficult task for CNN. The CNN’s functions are capable of learning only shift-invariant features of an image. These limitations give machine learning developers motivation towards generating more complex algorithms. Search for new machine learning models led to Capsule Networks. This Capsule Network was able to estimate objects’ pose in an image and recognize transformations to these objects. Handwritten digit classification is the task for which capsule networks are to solve at the initial stages. Capsule Networks outperforms all models for the MNIST dataset for handwritten digits, but to use Capsule networks for image classification is not a straightforward multiplication of parameters. By replacing the Capsule Network’s initial layer, a simple Convolutional Layer, with complex architectures in CNNs, authors of Residual Capsule Network achieved a tremendous change in capsule network applications without a high number of parameters. This thesis focuses on improving this recent Residual Capsule Network (RCN) to an extent where accuracy and model size is optimal for the Image classification task with a benchmark of the CIFAR-10 dataset. Our search for an exemplary capsule network led to the invention of RCN2: Residual Capsule Network 2 and RCNX: Residual Capsule NeXt. RCNX, as the next generation of RCN. They outperform existing architectures in the domain of Capsule networks, focusing on image classification such as 3-level RCN, DCNet, DC Net++, Capsule Network, and even outperforms compact CNNs like MobileNet V3. RCN2 achieved an accuracy of 85.12% with 1.95 Million parameters, and RCNX achieved 89.31% accuracy with 1.58 Million parameters on the CIFAR-10 benchmark.

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.