In this project, two tests were performed. On the first test, Mininet-WiFi was used to simulate a Software Defined Network to demonstrate Mininet-WiFi’ s ability to be used as the Software Defined Network emulator which can also be integrated to the existing network using a Network Virtualized Function (NVF). A typical organization’s computer network was simulated which consisted of a website hosted on the LAMP (Linux, Apache, MySQL, PHP) virtual machine, and an F5 application delivery controller (ADC) which provided load balancing of requests sent to the web applications. A website page request was sent from the virtual stations inside Mininet-WiFi. The request was received by the application delivery controller, which then used round robin technique to send the request to one of the web servers on the LAMP virtual machine. The web server then returned the requested website to the requesting virtual stations using the simulated virtual network. The significance of these results is that it presents Mininet-WiFi as an emulator, which can be integrated into a real programmable networking environment offering a portable, cost effective and easily deployable testing network, which can be run on a single computer. These results are also beneficial to modern network deployments as the live network devices can also communicate with the testing environment for the data center, cloud and mobile provides. On the second test, a Software Defined Network was created in Mininet using python script. An external interface was added to enable communication with the network outside of Mininet. The amazon web services elastic computing cloud was used to host an OpenDaylight controller. This controller is used as a control plane device for the virtual switch within Mininet. In order to test the network, a webserver hosted on the Emulated Virtual Environment – Next Generation (EVENG) software is connected to Mininet. EVE-NG is the Emulated Virtual Environment for networking. It provides tools to be able to model virtual devices and interconnect them with other virtual or physical devices. The OpenDaylight controller was able to create the flows to facilitate communication between the hosts in Mininet and the webserver in the real-life network. / Electrical and Mining Engineering / M. Tech. (Electrical Engineering)

MSc (Microbiology) / Department of Microbiology / BACKGROUND: Human papillomavirus (HPV) is a common sexually transmitted virus known to be a causative agent of cervical cancer (CC), one of the most frequent cancers in women worldwide. HPV is a double stranded DNA virus of approximately 7,900 bp; belonging to Papillomaviridae family. To date, about 202 low risk (LR) and high risk (HR) HPV genotypes have been identified. However, available vaccines against HPV infection are designed based on the most common known genotypes. Therefore, it is critical to understand the scope and diversity of HPV genotypes in all geographical locations which can help to inform the design and development of future vaccines. OBJECTIVE: The objective of this study was to describe the burden and diversity of HPV genotypes in a cohort of women in routine care in northern South Africa. METHODS: Eighty seven women consented to participate in the study and each provided a specimen for analysis. With the help of qualified health care practitioners, Aptima Cervical Specimen Collection and Transport Kit (Hologic, San Diego, CA) was used to collect cervical specimens from each study participant following the manufacturer’s procedure. Total DNA was purified from the cervical pellet using QIAamp DNA mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. The purified DNA was then subjected to a single round conventional PCR in a reaction volume of 100 μl to amplify HPV L1 gene comprising of approximately 450 bp. A portion of each PCR amplicon from each participant was denatured, hybridized and genotyped using the Linear Array HPV genotyping Test Kit (Roche Molecular Systems, Inc. Branchburg, NJ USA). The kit is designed to detect 37 HPV genotypes (genotypes 6, 11, 16, 18, 26, 31, 33, 35, 39, 40, 42, 45, 51, 52, 53, 54, 55, 56, 58, 59, 61, 62, 64, 66, 67, 68, 69, 70, 71, 72, 73, 81, 82, 83, 84, IS39 and CP6108). To detect the HPV genotypes, the Linear Array (LA) reference guide was used for results interpretation following the manufacturer’s instructions. The other portion of each of the amplicons was subjected to next generation sequencing (NGS) using the Illumina MiniSeq platform. Using the Nextera XT DNA Library preparation kit, an initial input of 1ng genomic DNA was tagmented, cleaned up, normalized and pooled. The pooled library was then denatured with 0.1 N NaOH and diluted into a final volume of 500 μl at 1.8 pM then sequenced using the Local Run Manager option following the manufacturer’s instructions. The generated sequence data was downloaded into fastaQ format and analysed using Genious 11.0.5 software. RESULTS: Of the 87 participants, the overall proportion of women harbouring HPV DNA by linear array (LA) PCR was 23% (n=20). Of the 20, 16 (80%) were living with HIV. However, this difference was not significant (p=0.077). Genotyping data generated by Roche LA method was successful for all the 20 positive amplicons. In this study, 27 (73%) of the 37 HPV genotypes incorporated in the Roche Linear Array method were detected. The detected genotypes include: types 84, 83, 81, 73, 72, 71, 70, 69, 68, 66, 62, 61, 59, 54, 53, 52, 51, 45, 42, 39, 35, 26, 18, 16, 6, IS39 and CP6108. Most women (15/20;75%) harboured multiple infections compared to single infection. In terms of genotypes distribution, the most frequent genotypes detected LR HPV types in increasing order of frequency included HPV type 61 and 83 (12%), 62 (36%) and 81 (43%). On the other hand, HPV type 66, 53, 52, 51, 18 and 16 were the most common genotypes detected HR HPV types. In contrast, although genotyping data was successfully generated from 15 of 20 women (75%), NGS technology was seen to be more sensitive compared to Roche LA method. Nearly all the detected genotypes identified by the commercial kit were detected by NGS. In addition, NGS detected 10 namely: HPV types 11, 31, 33, 40, 55, 56, 58, 64, 67, and 82 that were not detected by the LA yet incorporated in the kit. Moreover, it was observed that NGS identified additional 6 HPV types including HPV types 2, 27, 30, 35, 85 and 102 not incorporated in the Roche LA kit. A similar distribution of HPV multiple infections was observed in the study population, however, high frequency of 93% (14 of 15) was detected by NGS. The proportion of women harbouring one or more of the 22 LR HPV types was 100% (n=15).The most frequent LR genotypes in increasing order of frequency was HPV type 62 and 70 (27%), 6 (40%) and 11 (47%). HPV types 40, 42, 54, 72, 64, and 81 were the least detected genotypes with n=1 (7%) each. Furthermore, the common combination observed among the participants was type 6 and 11. In contrast, the most frequent detected genotypes in the study population by NGS under the HR HPV types in increasing order of frequency include type 35 (21%), 39, 56 and 82 (29%), 68 (36%) and 51 (50%). In addition, HPV types 26, 31, 45, 53, 56, 58 and 66 were the least detected genotypes n=1 (7%) in the study population. HPV 39 and 68 were observed as the common combination detected under HR HPV types. Following genotyping by LA and NGS, the demographic and clinical data of all the 20 positive subjects by PCR were subjected to statistical analysis to determine the association between HPV positive DNA status and associated risk factors. Smoking status (p=0.000), age at first sexual intercourse (p=0.011), vaccination status (p=0.000), gender of sexual partner (p=0.000), highest level of education (p=0.004), marital status (p=0.008) and number of sexual partners (p=0.000) were found to be having a positive statistical association. CONCLUSION: Amplification of targeted HPV DNA from cervical specimens demonstrated the presence of HPV infection in the study cohort, with a proportion of 23%. The findings illustrate that there is a diversity of HPV genotypes prevalent in the study population as shown by Roche LA and NGS methods. However, the NGS method was observed to be more sensitive than Roche LA in detecting HPV genotypes. Furthermore, NGS identified 6 additional HPV types not incorporated in the Roche LA. Thus, there are genotypes that may be present in the study population that the Roche commercial kit may fail to detect. Therefore, is it imperative to use both genotyping methods to confirm HPV genotypes. / NRF

Autosomal recessive polycystic kidney disease (ARPKD) is a rare severe inherited disease manifested by cystic renal disease, congenital hepatic fibrosis and dilatatation of bile ducts. The spectrum of clinical manifestations is very wide and variable, depends on the age at which the disease was manifested. In severe forms of the disease, it is possible to detect the first symptoms prenatally around the 20th week of pregnancy due to increased echogenic kidneys and the presence of oligohydramnios. The causal gene of this disease is thePKHD1 gene with protein product fibrocystin that is most likely contributing on maintaining the intracellular concentration of Ca2+ cations. The exact phatophysiology mechanism of ARPKD remains unknown. Phenotypic manifestations of this disease may overlap with mutations associated with other genes. One of the genes mimicking the ARPKD phenotype is the HNF1B gene. Mutations associated with HNF1B gene are the most common monogenic cause of developmental kidney abnormalities. HNF1B is a tissue-specific transcription factor that regulates the expression of PKHD1. In experimental part I worked on genetic analysis of the HNF1B gene in 28 patients who have not been confirmed ARPKD diagnosis by detection of 2 PKHD1 mutations. For the purposes of mutational screening, I used...

Le microbiote intestinal équin joue un rôle important dans le maintien de la santé de l'hôte. Le microbiote intestinal est composé de nombreux micro-organismes tels que les bactéries, les virus, les champignons et les archées. Cependant, la majorité de ces cellules microbiennes sont bactériennes, et par conséquent, de nombreuses études, y compris la présente, se concentrent sur l'exploration des communautés bactériennes dans l'intestin. Un déséquilibre du microbiote intestinal, appelé dysbiose, a été observé dans plusieurs conditions, telles que la colite, après l’administration d'antibiotiques ou la modification du régime alimentaire. La restauration du microbiote peut être effectuée par la transplantation de microbiote fécal (FMT). Des études utilisant les recommandations actuelles pour la FMT ont montré une récupération clinique chez les chevaux souffrant de diarrhée, mais le microbiote reste largement inchangé après la FMT et aucune étude randomisée avec contrôle placébo n'a été réalisée. Les hypothèses de ce projet étaient que le traitement avec une FMT concentrée corrigera la dysbiose plus rapidement qu’une FMT conventionnelle et le véhicule, et que le microbiote intestinal des chevaux traités avec une FMT concentrée ressemblera au microbiote intestinal du cheval donneur. L'objectif de ce projet était de développer un protocole pour améliorer la FMT chez les chevaux, en augmentant la concentration de bactéries présentes dans les selles du donneur par centrifugation, et de le tester chez les chevaux atteints de dysbiose intestinale induite par les antibiotiques. L'antibiotique triméthoprime sulfadiazine (TMS) a été administré à neuf chevaux pour induire une dysbiose intestinale. Les chevaux ont été séparés en trois groupes: les chevaux recevant une FMT concentrée (cFMT, n = 3); les chevaux recevant la FMT fraîche (fFMT), selon les recommandations actuelles (n = 3); et les chevaux recevant un véhicule (VEH) avec 10% de glycérol dans une solution saline à 0,9% (n=3). Des échantillons fécaux ont été prélevés avant et après l'administration du TMS, ainsi qu'avant, pendant et après la transplantation. Le séquençage a été réalisé à l'aide de la plateforme Illumina MiSeq et les données analysées à l'aide du logiciel Mothur. Tel qu’attendu, l'antibiotique TMS a significativement diminué la richesse microbienne chez tous les chevaux. De manière inattendue, la composition des suspensions fécales des donneurs cFMT et fFMT était significativement différente de la composition de base des receveurs cFMT et fFMT, respectivement. La composition du microbiote des chevaux ayant reçu une transplantation fécale (concentrée ou non) était significativement différente après la transplantation, alors que ce n’était pas le cas chez les chevaux ayant reçu le véhicule. En outre, l’abondance relative de Escherichia était significativement plus élevée dans les suspensions fécales du donneur cFMT par rapport aux suspensions fécales du donneur fFMT. Les principales limites de ce projet sont la petite taille des groupes et l'exposition des selles des donneurs à l'oxygène et à la congélation-décongélation. En outre, le modèle de dysbiose peut ne pas être optimal pour tester l'efficacité de la FMT, et des études réalisant la FMT chez les chevaux souffrant de diarrhée sont nécessaire. Cette étude a contribué à la recherche de nouvelles approches pour améliorer la FMT chez les chevaux. Le faible effet mesuré avec les deux protocoles de FMT et l’augmentation de Escherichia démontre que les protocoles actuels doivent être optimisés avant de pouvoir recommander la FMT pour traiter et prévenir la dysbiose chez les chevaux. / The equine gut microbiota plays an important role in maintaining the health of the host. The gut microbiota is composed of many microorganisms such as bacteria, viruses, fungi, and archaea. However, the majority of these microbial cells are bacterial cells, and consequently, many studies, including the present one, focus on exploring bacterial communities in the gut. An imbalance of the gut microbiota, termed dysbiosis, has been observed in several conditions such as colitis, colic, after antibiotic administration, or diet modification. Restoration of the gut to a healthy state can be performed through fecal microbiota transplantation (FMT). Studies using current recommendations for FMT have shown clinical recovery in horses with diarrhea, but the microbiota remains largely unchanged after FMT and no controlled studies have been performed. The hypotheses of this project were that treatment with concentrated FMT will correct dysbiosis faster than conventional FMT and the vehicle, and that the gut microbiota of horses treated with concentrated FMT will resemble the gut microbiota of the donor. The objective of this project was to develop an improved protocol for FMT in horses, by increasing the concentration of bacteria found in the donor stool using centrifugation, and to test it in horses with antibiotic-induced intestinal dysbiosis. The antibiotic trimethoprim sulfadiazine (TMS) was administered to nine horses to induce intestinal dysbiosis. Horses were separated into three groups: horses receiving concentrated FMT (cFMT) (n=3); horses receiving fresh FMT (fFMT), as per current recommendations (n=3); horses receiving a vehicle (VEH) with 10% glycerol in 0.9% saline (n=3). Fecal samples were collected before and after antibiotic administration, as well as before, during, and after transplantation. Sequencing was performed using the Illumina MiSeq platform and data analysed using the software Mothur. As expected, the antibiotic TMS significantly decreased the richness in all horses (P < 0.05). Unexpectedly, the membership of the cFMT and fFMT donor fecal suspensions was significantly different from cFMT and fFMT recipients’ baseline membership, respectively. The membership of the cFMT and fFMT recipient horses was significantly different after transplantation, while the vehicle recipients were not. In addition, the Escherichia genus was found in significantly higher relative abundances in the cFMT donor fecal suspensions when compared to the fFMT donor fecal suspensions. The main limitations of this study are the small sample size and exposure of cFMT donor stool to oxygen and freeze-thawing. In addition, the dysbiosis model may not be optimal to test the efficacy of FMT, and studies performing FMT in horses with diarrhea are warranted. This study contributed to the search for novel approaches to improve FMT in horses. The weak effect of both FMT protocols on the gut microbiota and the increase in Escherichia suggest that further clinical studies are needed before FMT can be recommended to treat and prevent dysbiosis in horses.

La Sindrome da Affaticamento Cronico/Encefalomielite Mialgica (CFS/ME), è una grave malattia multisistemica caratterizzata da anomalie immunologiche e disfunzioni del metabolismo energetico. Recenti evidenze suggeriscono l’esistenza di una forte correlazione tra disbiosi e condizione patologica. La presente ricerca ha analizzato la composizione del microbiota intestinale ed orale in pazienti con CFS/ME rispetto a controlli sani e ha determinato se eventuali differenze osservate potrebbero essere utili in futuro per l'identificazione di biomarcatori diagnostici. La composizione batterica fecale e salivare dei pazienti con CFS/ME è stata studiata mediante sequenziamento Illumina degli ampliconi del gene 16S rRNA. Il microbiota fecale dei pazienti con CFS/ME ha mostrato una significativa riduzione di Lachnospiraceae, in particolare di Anaerostipes, rispetto ai gruppi di soggetti senza CFS/ME e un incremento di Phascolarctobacterium faecium e unclassified Ruminococcus. Bacteroides vulgatus, unclassified Bacteroides, Bacteroides uniformis e unclassified Barnesiella sono risultati significativamente più abbondanti nei pazienti con CFS/ME. Il microbiota orale dei pazienti con CFS/ME ha mostrato un aumento significativo di Rothia dentocariosa. Il profilo metabolico fecale di un sottogruppo di pazienti con CFS/ME ha mostrato un aumento complessivo di SCFA e di derivati dell'indolo rispetto ai gruppi non CFS/ME, suggerendo un aumento dei processi di fermentazione. I nostri risultati supportano l'ipotesi autoimmune per la CFS/ME e se saranno confermati da studi più ampi, le differenze rilevate nei profili microbici dei pazienti CFS/ME potrebbero essere utilizzate come markers per una diagnosi più accurata e per lo sviluppo di strategie terapeutiche specifiche. / The Chronic Fatigue Syndrome/Myalgic Encephalomyelitis (CFS/ME), is a severe multisystemic disease characterized by immunological abnormalities and dysfunction of energy metabolism. Recent evidence suggest that there is a strong correlation between dysbiosis and pathological condition. The present research investigated the composition of the intestinal and oral microbiota in CFS/ME patients in comparison to healthy controls and determined whether any observed differences could be useful for the identification of diagnostic biomarkers. The fecal and salivary bacterial composition in CFS/ME patients was investigated by Illumina sequencing of 16S rRNA gene amplicons. The fecal microbiota of CFS/ME patients showed a significant reduction of Lachnospiraceae, particularly Anaerostipes, compared to the non-CFS/ME groups, and an increase of Phascolarctobacterium faecium and unclassified Ruminococcus. Bacteroides vulgatus, unclassified Bacteroides, Bacteroides uniformis and unclassified Barnesiella resulted significantly more abundant in CFS/ME patients. The oral microbiota of CFS/ME patients showed a significant increase of Rothia dentocariosa. The fecal metabolic profile of a subgroup of CFS/ME patients revealed an overall increase of SCFAs and indole derivatives compared to the non-CFS/ME groups, suggesting an increase in the fermentation processes. Our results support the autoimmune hypothesis for CFS/ME condition and if confirmed by larger studies, the differences detected in the microbial profiles of CFS/ME patients may be used as markers for a more accurate diagnosis and for the development of specific therapeutic strategies.

Ubiquitous Sensor Network (USN) est un réseau conceptuel construit sur des réseaux physiques existantes. Il se sert des données détectées et fournit des services de connaissances à quiconque, n'importe où et à tout moment, et où l'information est générée en utilisant la sensibilité au contexte. Dispositifs et USN portables intelligents émergent rapidement en offrant de nombreux services fiables facilitant la vie des gens. Ces petits terminaux et terminaux très utiles besoin d'un substrat de communication globale pour fournir un service complet de l'utilisateur final global. En 2010, ITU -T a fourni les exigences pour supporter des applications et services USN dans le Next Generation Network (NGN) de l'environnement d'exploiter les avantages du réseau de base. L'un des principaux marchés prometteurs pour l'application et les services USN est la e- santé. Il fournit le suivi des patients en continu et permet une grande amélioration dans les services médicaux. D'autre part, des Véhicules Ad-hoc NETwork (VANET) est une technologie émergente qui permet une communication intelligente entre les véhicules mobiles. Intégrer VANET avec USN a un grand potentiel pour améliorer la sécurité routière et la fluidité du trafic. La plupart des applications VANET sont appliqués en temps réel et ils sont sensibles à retarder, en particulier ceux liés à la sécurité et à la santé. Dans ce travail, nous proposons d'utiliser l'IP Multimédia Subsystem (IMS) comme une sous- couche de contrôle de service dans l'environnement USN fournir un substrat mondiale pour un service complet de bout en bout. De plus, nous vous proposons d'intégrer VANETs avec USN pour des applications et des installations riches plus, ce qui facilitera la vie des humains. Nous avons commencé à étudier les défis sur la route pour atteindre cet objectif / Ubiquités Sensor Network (USN) is a conceptual network built over existing physical networks. It makes use of sensed data and provides knowledge services to anyone, anywhere and at anytime, and where the information is generated by using context awareness. Smart wearable devices and USNs are emerging rapidly providing many reliable services facilitating people life. Those very useful small end terminals and devices require a global communication substrate to provide a comprehensive global end user service. In 2010, the ITU-T provided the requirements to support USN applications and services in the Next Génération Network (NGN) environment to exploit the advantages of the core network. One of the main promising markets for the USN application and services is the e-Health. It provides continuous patients’ monitoring and enables a great improvement in medical services. On the other hand, Vehicular Ad-Hoc NETwork (VANET) is an emerging technology, which provides intelligent communication between mobile vehicles. Integrating VANET with USN has a great potential to improve road safety and traffic efficiency. Most VANET applications are applied in real time and they are sensitive to delay, especially those related to safety and health. In this work, we propose to use IP Multimedia Subsystem (IMS) as a service controller sub-layer in the USN environment providing a global substrate for a comprehensive end-to-end service. Moreover, we propose to integrate VANETs with USN for more rich applications and facilities, which will ease the life of humans. We started studying the challenges on the road to achieve this goal

Indiana University-Purdue University Indianapolis (IUPUI) / Phormia regina (Meigen), commonly known as the black blow fly is a dipteran that belongs to the family Calliphoridae. Calliphorids play an important role in various research fields including ecology, medical studies, veterinary and forensic sciences. P. regina, a non-model organism, is one of the most common forensically relevant insects in North America and is typically used to assist in estimating postmortem intervals (PMI). To better understand the roles P. regina plays in the numerous research fields, we re-constructed its genome using next generation sequencing technologies. The focus was on generating a reference genome through de novo assembly of high-throughput short read sequences. Following assembly, genetic markers were identified in the form of microsatellites and single nucleotide polymorphisms (SNPs) to aid in future population genetic surveys of P. regina. A total 530 million 100 bp paired-end reads were obtained from five pooled male and female P. regina flies using the Illumina HiSeq2000 sequencing platform. A 524 Mbp draft genome was assembled using both sexes with 11,037 predicted genes. The draft reference genome assembled from this study provides an important resource for investigating the genetic diversity that exists between and among blow fly species; and empowers the understanding of their genetic basis in terms of adaptations, population structure and evolution. The genomic tools will facilitate the analysis of genome-wide studies using modern genomic techniques to boost a refined understanding of the evolutionary processes underlying genomic evolution between blow flies and other insect species.

[pt] Nesta tese estudamos o potencial que terão a próxima geração de detectores de neutrinos (JUNO, Hyper-Kamiokande e DUNE) para a detecção dos efeitos da matéria da Terra através da identificação das modulações no espectro de energia dos neutrinos de supernovas de colapso de núcleo em nossa galáxia, assumindo a possibilidade do decaimiento invisível de v2 após os neutrinos terem deixado a estrela, caminho da Terra. Simulações recentes do colapso gravitacional (e subsequente explosão) de estrelas com massa maior do que ~ 8Mo mostram que durante a fase de esfriamento as energias médias (Eve) e (Evx) tornam-se muito semelhantes e os fluxos tendem a se igualar, tornando difícil observar os efeitos da matéria da Terra usando um único detector. Neste trabalho mostramos que a inclusão do decaimiento dos neutrinos também cria a possibilidade de observar os efeitos em consideração no canal de detecção de neutrinos se o ordenamento de massa for normal e no canal anti-neutrino se o ordenamento for invertido, o que não é esperado na ausência de decaimento. Em particular, se a taxa de decaimento for maior do que ~ 70%, descobrimos que o decaimento invisível de v2 pode aumentar as possibilidades de observação dos efeitos da matéria da Terra, mesmo para supernovas a uma distância de 10 kpc de nós. / [en] In this thesis we studied the potential that the next-generation neutrino detectors (JUNO, Hyper-Kamiokande and DUNE) will have to the detection of the Earth matter effects through the identification of the modulations in the energy spectrum of neutrinos from core-collapse supernovae in our galaxy, assuming the possibility of the invisible decay of v2 after the neutrinos have left the star, on their way to Earth. Recent simulations of gravitational collapse (and subsequent explosion) of stars more massive than ~ 8Mo show that during the cooling phase the average energies (EVe) and (Evx) become very similar and the fluxes tend to equalize, making it difficult to observe the Earth matter effects using a single detector. In this work we show that the inclusion of neutrino decay creates also the possibility of observing the effects under consideration in the neutrino detection channel if the mass ordering is normal and in the anti-neutrino channel if the ordering is inverted, which is not expected in the absence of neutrino decay. In particular, if the decay rate is more than ~ 70%, we find that the invisible neutrino decay of v2 can enhance the observation possibilities of Earth matter effects even for supernovae at a distance of 10 kpc from us.

Les leucémies myéloïdes aiguës (LMA) consistent en un groupe de cancers agressifs causés par une accumulation de mutations génétiques et épigénétiques survenant dans les cellules souches ou progénitrices de la moelle osseuse. Il s’agit d’un groupe de maladies très hétérogène, caractérisé par un grand nombre de combinaisons d’altérations qui perturbent à la fois les voies de signalisation qui y sont exprimées, leur sensibilité aux différents traitements et le pronostic des patients. Le déploiement des technologies de séquençage de nouvelle génération au courant de la dernière décennie a permis l’exploration à une échelle sans précédent du paysage mutationnel et transcriptomique de différents cancers, incluant les LMA. Dans le cadre de nos travaux, nous avons voulu tester l'hypothèse selon laquelle les LMA se déclinent en plusieurs sous-groupes génétiques caractérisés chacun par des mutations distinctes et une expression génique dérégulée, ainsi qu’une réponse différentielle à des molécules qui pourraient représenter de nouvelles stratégies thérapeutiques. Nous avons testé cette hypothèse au sein de la cohorte Leucegene, qui comprend un grand nombre de LMA primaires analysées par le séquençage du transcriptome, et nous avons analysé les différences entre les différents sous-groupes en les analysant un à la fois. Cette étude des différents sous-groupes nous a permis de disséquer le profil génomique, transcriptomique et les sensibilités aux petites molécules de sept sous-groupes génétiques, représentant environ la moitié des cas de LMA de l’adulte. Notre approche a permis de découvrir plusieurs nouvelles mutations spécifiques aux différents sous-groupes, dont certaines ont été validées dans des cohortes indépendantes. Nous avons également confirmé que les gènes différentiellement exprimés dans les sous-groupes sont plus informatifs que les signatures d'expression non supervisées pour identifier les biomarqueurs de la maladie. Nous avons ainsi identifié dans la majorité des sous-groupes des gènes représentant un biomarqueur d'intérêt, ayant une pertinence fonctionnelle ou pronostique. Ces données ont également mené à des criblages chimiques ciblés qui ont identifié de nouvelles vulnérabilités dépendant du contexte génétique. Au-delà de ces observations, nos travaux pourraient avoir une portée translationnelle tandis que le séquençage de nouvelle génération est de plus en plus utilisé en clinique. La combinaison avec d’autres modalités de séquençage et l’incorporation de technologies émergentes aideront à poursuivre la dissection génomique, transcriptomique et chimique de la LMA et l’approche utilisée pourra même éventuellement s’appliquer à d’autres types de cancers. / Acute myeloid leukemias (AML) are a group of cancers caused by an accumulation of genetic and epigenetic mutations occurring in the stem or progenitor cells of the bone marrow. They represent a very heterogeneous group of diseases, characterized by a large number of combinations of alterations which disrupt to varying degrees key networks in these cells, their sensitivity to treatments and the prognosis of the patients. The deployment of next-generation sequencing technologies over the past decade has enabled exploration on an unprecedented scale of the mutational and transcriptomic landscape of various cancers, including AML. As part of our work, we tested the hypothesis according to which AMLs comprise several genetic subgroups, each characterized by distinct mutations and deregulated gene expression profiles, as well as a differential response to molecules that could represent novel therapies. We tested this hypothesis in the Leucegene cohort, which includes a large number of primary AMLs analyzed by transcriptome sequencing, which we explored one subgroup after the other, dissecting the genomic, transcriptomic or small molecule sensitivities profile of seven AML subgroups representing approximately half of adult AML cases. Our approach has allowed us to discover several new mutations specific to different subgroups, some of which have been validated in independent cohorts. We also confirmed that genes differentially expressed in subgroups are more informative than unsupervised expression signatures, and we identified genes representing potential biomarkers, or having a functional or prognostic relevance in the majority of subgroups. Generated data also led to targeted chemical screens performed on primary AML cells, which identified new context-dependent vulnerabilities. Beyond these observations, our work could have a translational scope while next-generation sequencing is paving its way in the clinic. The combination with other Omics and the incorporation of emerging technologies will help to further the multi-dimensional dissection of these groups and additional ones, as the presented approach could be applied to additional disease subsets and cancer types.

Background: Coronary Artery Disease (CAD) represents the leading cause of mortality and morbidity worldwide despite declines in the prevalence of environmental risk factors. This trend has drawn attention to the risk conferred by genetic variation. Twin and linkage studies demonstrate a profound hereditary risk for CAD, especially in young individuals. Rare genetic variants conferring high risk for extreme disease phenotypes can provide invaluable insight into novel mechanisms underlying CAD development. Methods: Whole exome sequencing was performed to characterize rare protein-altering variants in 52 early-onset CAD (EOCAD) patients encompassing the DECODE study. The enrichment of Mendelian dyslipidemias in EOCAD was assessed through interrogation of pathogenic mutations among known lipid genes. The identification of novel genetic CAD associations was conducted through case-only and case-control approaches across all protein-coding genes using rare variant burden and variance component tests. Lastly, beta coefficients for significant risk genes from the European population in the Early-onset Myocardial Infarction (EOMI) cohort (N=552) were used to construct calibrated, single-sample rare variant gene scores (RVGS) in DECODE Europeans (N=39) and a local European CAD-free cohort (N=77). Results: A 20-fold enrichment of Familial hypercholesterolemia mutation carriers was detected in EOCAD cases compared to CAD-free controls (P=0.005). Association analysis using EOMI Europeans revealed exome-wide and nominal significance for two known CAD/MI genes: CELSR2 (P=1.1x10-17) and APOA5 (P=0.001). DECODE association revealed exome-wide and nominal significance for genes involved in endothelial integrity and immune cell activity. RVGS based upon beta coefficients of significant CAD/MI risk genes were significantly increased in DECODE (z-score=1.84; p=0.03) and insignificantly decreased among CAD-free individuals (z-score=-1.61; p=0.053). Conclusion: Rare variants play a pivotal role in the development early CAD through Mendelian and polygenic mechanisms. Construction of RVGS that are calibrated against population and technical biases can facilitate discovery of single-sample and cohort-based associations beyond what is detectable using standard methods. / Thesis / Master of Science (MSc)