Acute myeloid leukemia (AML) is a highly fatal blood cancer that is characterized by disruption of healthy differentiation of stem cells into functional blood cells in the bone marrow. Most patients with AML consequently die from infections due to the lack of immune cells. For decades, the standard method of remission induction for AML has been chemotherapy using an antineoplastic drug known as AraC. However, even after successful remission induction, aggressive, refractory relapse occurs in the majority of patients within 3 years with dismal survival rates. Here, we sought to develop a genome-wide screening approach to determine the causative genes in AML relapse. In the developed procedure, protein-coding genes of the human genome are screened using a leading-edge technology known as CRISPR (clustered regularly interspaced short palindromic repeats) activation screening. This involves usage of a pooled guide RNA library that upregulates a unique gene for each individual AML cell. By treating these cells with AraC in a mouse xenograft model, the bone marrow will gradually be enriched with cells that carry a guide RNA for a relapse-conducive gene. By harvesting and sequencing all enriched guide RNAs at relapse, the causative genes in AML relapse can be determined. All parameters of the in vivo CRISPR-activation screen have been optimized, and the workflow from preparation to the end of screening has been detailed. Follow-up studies that will validate the results of the screen have also been described. The long-term goal of this developed screen is to elucidate the mechanisms of AML relapse and find ways to clinically target these pathways in conjunction with standard the AraC-based chemotherapy. / Thesis / Master of Science (MSc)

Yes / Mycobacterium tuberculosis and Mycobacterium leprae have remained, for many years, the primary species of the genus Mycobacterium of clinical and microbiological interest. The other members of the genus, referred to as nontuberculous mycobacteria (NTM), have long been underinvestigated. In the last decades, however, the number of reports linking various NTM species with human diseases has steadily increased and treatment difficulties have emerged. Despite the availability of whole genome sequencing technologies, limited effort has been devoted to the genetic characterization of NTM species. As a consequence, the taxonomic and phylogenetic structure of the genus remains unsettled and genomic information is lacking to support the identification of these organisms in a clinical setting. In this work, we widen the knowledge of NTMs by reconstructing and analyzing the genomes of 41 previously uncharacterized NTM species. We provide the first comprehensive characterization of the genomic diversity of NTMs and open new venues for the clinical identification of opportunistic pathogens from this genus.

Influence de la micro-propagation par embryogenèse somatique sur la variation somaclonale chez le caféier (Coffea arabica): évaluation des changements au niveau phénotypique, cytologique, génétique et épigénique. La variation somaclonale (VS) est une préoccupation majeure de tous les systèmes de micropropagation. Elle est décrite comme un changement phénotypique présent chez les vitroplants et pourrait être générée par une large gamme de mécanismes génétique et épigénétiques. Des hybrides de Coffea arabica hautement productifs sont distribuées sous forme clonale par embryogenèse somatique (ES) en Meso-Amérique. L’objectif de ce travail chez le caféier est d’évaluer la conformité génétique des plants multipliés par ES et de comprendre les mécanismes impliqués dans les SV. Nous avons évalué les variations dans les plantes régénérées au niveau phénotypique, cytologique, génétique (mutations/AFLPs, transposition génétique/S-SAP) et épigénétique (méthylation/MSAP) en utilisant deux approches complémentaires. Tout d’abord, nous avons étudié chez deux hybrides des conditions de cultures industrielles supposées peu mutagènes i.e. une courte période de prolifération (6 mois) et faible apporte en auxine (0-1.4 µM 2,4-D). Deux systèmes de prolifération i.e. l’embryogenèse secondaire et les suspensions embryogènes seront comparés, le dernier étant plus productif et économique. Les analyses moléculaires AFLP et MSAP sur 145 somaplants montrent que les polymorphismes génétique et épigénétique entre plantes mères et somaplants sont extrêmement réduits, i.e. dans l’intervalle 0-0,003% et 0,07-0,18% respectivement, sans différence significative entre les systèmes de prolifération. Pour les deux hybrides testés, des observations phénotypiques massives en pépinière et au champ ont révélé de très faibles niveaux de VS (0,9% pour 800.000 plantes). Des analyses cytologiques ont mis en évidence des nombres de chromosomes anormaux (41-43, 45) chez la plupart des variants et des nombres normaux (44) chez les plants ayant un phénotype normal. Des conditions expérimentales a priori mutagènes ont également été appliquées en utilisant des périodes de prolifération prolongées (4, 12 et 27 mois) chez trois lignées embryogènes indépendantes de la variété Caturra en présence de concentrations élevées en régulateurs de croissance (4.5 μM 2,4-D, 17.8 μM 6-BA), afin de comprendre les mécanismes liés vieillissement des cultures interviennent sur les VS. L’étude des 180 somaplants régénérés a montré que le temps de prolifération affecte fortement la fréquence de VS et d’une manière hautement similaire pour les 3 lignées embryogènes. Aucun variant n’a été trouvé après 4 mois de prolifération alors que 30% et 94% de variants phénotypiques ont été caractérisés chez les plants issus de cultures de 12 et 27 mois, respectivement. Quels que soient l’âge de culture et la lignée embryogène, aucun polymorphisme n’a été trouvé chez les 124 somaplants et un très faible nombre des changements des méthylation avec les MSAP (0,049-0,087%). Cependant, de façon similaire aux somaplants produits en conditions industrielles, les variants phénotypiques montrent systématiquement des nombres de chromosomes anormaux (41-43) et les plants normaux le nombre de chromosomes attendu. Ce travail montre que l’ES s’appuyant sur des suspensions embryogènes peut garantir une propagation conforme des variétés sélectionnées de C. arabica. Il démontre également l’importance de l’âge des cultures sur l’apparition de VS et donc le caractère non aléatoire du phénomène. Les changements génétiques et épigénétiques sont particulièrement limités durant l’ES. Le principal changement chez la plupart des variants est l’aneuploidie, ce qui montre que les aberrations mitotiques jouent un rôle majeur dans les VS chez le caféier. / Influence of micropropagation through somatic embryogenesis on somaclonal variation in coffee (Coffea arabica): assessment of variations at the phenotypical, cytological, genetic and epigenetic level Somaclonal variation (SV) is a major concern in all micropropagation systems. It is described as the phenotypic variation displayed in in vitro-derived regenerants and it is believed to be originated from a large array of genetic and epigenetic mechanisms. Highly productive Coffea arabica hybrids are clonally disseminated in Meso-American region through somatic embryogenesis (SE). The objective of the present work in coffee is to evaluate the trueness-to-type of SE and to understand the mechanisms involved in SV. We assessed the variations in the propagated plants at the phenotypic, cytogenetic, genetic (mutations/AFLP, genetic transposition/S-SAP) and epigenetic (methylation/MSAP) level by using two complementary approaches. First, with 2 hybrids we studied industrial culture conditions expected to be weakly mutagenic thanks to the combined use of short term proliferation period (6 months) and low auxin supply (0-1.4 µM 2,4-D). Two proliferation systems i.e. secondary embryogenesis and embryogenic suspensions were compared, the latter being more productive and economic. AFLP and MSAP molecular analyses on 145 somatic seedlings showed that genetic and epigenetic polymorphisms between mother plants and emblings were extremely low, i.e. ranges of 0–0.003% and 0.07–0.18% respectively, with no significant difference between the proliferation systems. For the two hybrids tested, massive phenotypic observations in nursery and field plots showed very low levels of SV (0.9% from 800,000 plants). Cytological analysis showed abnormal chromosome numbers (41-43, 45) in most of coffee somaclonal variants and normal numbers (44) in phenotypically normal plants. Stressful experimental conditions were also applied by using extended proliferation periods (4, 12 and 27 months) for three independent embryogenic lines established for the Caturra var. in presence of high growth regulator concentrations (4.5 μM 2,4-D, 17.8 μM 6-BA) to understand the mechanisms of culture ageing on SV. The proliferation time strongly affected the SV frequency among the 180 regenerated plants and in a highly similar way with the three embryogenic lines. No variant was found after 4 months proliferation although 30% and 94% phenotypic variants were observed in plants derived from 12 and 27 month-old cultures, respectively. Regardless the culture age and the embryogenic line, no polymorphisms were found in the 124 plants analyzed and very limited methylation changes with MSAP markers (0.049-0.087%). However, similarly to plants derived from industrial conditions, phenotypic variants systematically showed abnormal chromosome numbers and normal plants systematically showed normal numbers. This work showed that SE based on embryogenic suspensions is reliable for true-to-type propagation of selected C. arabica varieties. It also demonstrated the importance of culture age on SV and hence the non random nature of this phenomenon. The genetic and epigenetic alterations are particularly limited during SE. The main change in most of phenotypic variants was aneuploidy showing that mitotic aberrations play a major role in SV in coffee.

Les sarcomes pléomorphes sont des tumeurs mésenchymateuses rares caractérisées par de nombreux remaniements chromosomiques. Leur processus d’oncogenèse reste encore mal compris, aucune altération génétique motrice de l’initiation tumorale n’a pu être identifiée de façon récurrente et spécifique à ce jour. Les travaux que j’ai réalisés durant ma thèse avaient pour but de mieux comprendre la biologie de ces tumeurs, notamment les conséquences transcriptomiques de leur remodelage génomique.Nous avons caractérisé les transcrits de fusion exprimés dans ces tumeurs par séquençage haut-débit (RNA-seq). Ceci nous a amené à identifier l’expression de plusieurs transcrits chimériques impliquant le gène TRIO (5,1% des tumeurs). De plus, cette analyse ainsi que l’identification de variants exprimés nous ont permis d’identifier de fréquentes mutations de gènes suppresseurs de tumeurs tels qu’ATRX (16% des tumeurs) et plus généralement des membres du complexe SWI/SNF (47% des tumeurs). Les altérations de ce complexe majeur de remodelage de la chromatine sont associées à une plus grande instabilité génétique et à un phénotype plus agressif.Dans les sarcomes pléomorphes, l’instabilité génétique est liée à la progression tumorale via l’expression d’une signature transcriptomique pronostique. Cette signature, nommée CINSARC, est impliquée dans le contrôle de la mitose et de la ségrégation chromosomique. Nous avons voulu déterminer l’origine de cette expression via une étude intégrant la génomique et des mécanismes de régulation épigénétique, transcriptionnelle et post-transcriptionnelle. Si ces mécanismes ne semblent pas directement causals de l’expression de CINSARC, d’importantes modifications ont pu être mises en évidences. D’un point de vue clinique, nous avons démontré que l’expression de cette signature est un facteur pronostique universel de l’agressivité tumorale dans de nombreux types de cancers. De plus, CINSARC est un meilleur marqueur pronostique que le grade FNCLCC, actuel standard international d’évaluation du risque métastatique des sarcomes des tissus mous. Nous avons ainsi développé une méthode permettant une application clinique routinière de la signature CINSARC afin d’améliorer la prise en charge thérapeutique de ces tumeurs. / Pleomorphic sarcomas are rare mesenchymal tumors characterized by many chromosomal rearrangements. Their oncogenic process is still poorly understood, no recurrent and specific genetic alteration able to drive the tumor initiation has been identified yet. The work I did during my thesis had the objective to better understand the biology of these tumors, focusing on transcriptomic consequences of their genomic remodeling.We characterized fusion transcripts in these tumors by high-throughput sequencing (RNA-seq). This led us to identify the expression of several chimeric transcripts involving TRIO (5.1% of cases). Moreover, this analysis and the identification of expressed variants allowed us to identify frequent mutations of tumor suppressor genes such as ATRX (16% of cases) and more generally members of the SWI/SNF complex (47% of cases). Alterations of this major complex of chromatin remodeling are associated with higher genetic instability and more aggressive phenotype.In pleomorphic sarcomas, genetic instability is linked to tumor progression through the expression of a prognostic transcriptomic signature. This signature, termed CINSARC, is involved in mitosis control and chromosome segregation pathways. We wanted to determine the origin of such expression by integrating genomics and epigenetics, transcriptional and post-transcriptional regulation mechanisms. Though these mechanisms do not seem to directly regulate CINSARC expression, important changes have been highlighted. From a clinical standpoint, we demonstrated that the signature expression is a global prognostic factor of tumor aggressiveness in numerous cancer types. In addition, CINSARC is a better prognostic marker than the FNCLCC grading system, the current international standard to evaluate the metastatic risk in soft tissue sarcomas. We consequently developed a method allowing a daily clinical application of the CINSARC signature to improve the therapeutic management of these tumors.

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The field of metagenomics has shown great promise in the ability to recover microbial DNA from communities whose members resist traditional cultivation techniques, although in most instances the recovered material comprises short anonymous genomic fragments rather than complete genome sequences. In order to effectively assess the microbial diversity and ecology represented in such samples, accurate methods for DNA classification capable of assigning metagenomic fragments into their most likely taxonomic unit are required. Existing DNA classification methods have shown high levels of accuracy in attempting to classify sequences derived from low-complexity communities, however genome distinguishability generally deteriorates for complex communities or those containing closely related organisms. The goal of this thesis was to identify factors both intrinsic or external to the genome that may lead to the improvement of existing DNA classification methods and to probe the fundamental limitations of composition-based genome distinguishability. To assess the suite of factors affecting the distinguishability of genomes, support vector machine classifiers were trained to discriminate between pairs of microbial genomes using the relative frequencies of oligonucleotide patterns calculated from orthologous genes or short genomic fragments, and the resulting classification accuracy scores used as the measure of genomic distinguishability. Models were generated in order to relate distinguishability to several measures of genomic and taxonomic similarity, and interesting outlier genome pairs were identified by large residuals to the fitted models. Examination of the outlier pairs identified numerous factors that influence genome distinguishability, including genome reduction, extreme G+C composition, lateral gene transfer, and habitat-induced genome convergence. Fragments containing multiple protein-coding and non-coding sequences showed an increased tendency for misclassification, except in cases where the genomes were very closely related. Analysis of the biological function annotations associated with each fragment demonstrated that certain functional role categories showed increased or decreased tendency for misclassification. The use of pre-processing steps including DNA recoding, unsupervised clustering, 'symmetrization' of oligonucleotide frequencies, and correction for G+C content did not improve distinguishability. Existing composition-based DNA classifiers will benefit from the results reported in this thesis. Sequence-segmentation approaches will improve genome distinguishability by decreasing fragment heterogeneity, while factors such as habitat, lifestyle, extreme G+C composition, genome reduction, and biological role annotations may be used to express confidence in the classification of individual fragments. Although genome distinguishability tends to be proportional to genomic and taxonomic relatedness, these trends can be violated for closely related genome pairs that have undergone rapid compositional divergence, or unrelated genome pairs that have converged in composition due to similar habitats or unusual selective pressures. Additionally, there are fundamental limits to the resolution of composition-based classifiers when applied to genomic fragments typical of current metagenomic studies.

It has long been evident that genome size is not an accurate measure of organismal complexity. This paradox was “solved” with the discovery of nonfunctional and selfish DNA in the 1970s. However, emerging from this explanation was an enigma of complexity. Neither neutral nor adaptive models can account for all genome size variation across the tree of life. An organism with intraspecific variation is needed to investigate the functional role of genome size differences. Here I use different populations of the seed beetle, Callosobruchus maculatus, with a known intraspecific genome size variation of ~4%. It has previously been shown that a larger genome is associated with higher scores in fitness-related traits for this species. In this study, genome size is regressed with phenotypic plasticity along three different environmental gradients. Genome size did not correlate with plasticity in mass and development time along environmental gradients of temperature and host types. However, the results show that larger genomes are consistent with higher canalization of fitness under different food regimes. This further supports the idea that natural selection acts on genome size variation in this species.

The ability to precisely regulate gene expression patterns and to modify genome sequence in a site-specific manner holds much promise in determining gene function and linking genotype to phenotype. DNA-binding modules have been harnessed to generate customizable and programmable chimeric proteins capable of binding to site-specific DNA sequences and regulating the genome and epigenome. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like effectors (TALEs) are amenable to engineering to bind any DNA target sequence of interest. Deciphering the code of TALE repeat binding to DNA has helped to engineer customizable TALE proteins capable of binding to any sequence of interest. Therefore TALE repeats provide a rich resource for bioengineering applications. However, the TALE system is limited by the requirement to re-engineer one or two proteins for each new target sequence. Recently, the clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) has been used as a versatile genome editing tool. This machinery has been also repurposed for targeted transcriptional regulation. Due to the facile engineering, simplicity and precision, the CRISPR/Cas9 system is poised to revolutionize the functional genomics studies across diverse eukaryotic species. In this dissertation I employed transcription activator-like effectors and CRISPR/Cas9 systems for targeted genome regulation and editing and my achievements include: 1) I deciphered and extended the DNA-binding code of Ralstonia TAL effectors providing new opportunities for bioengineering of customizable proteins; 2) I repurposed the CRISPR/Cas9 system for site-specific regulation of genes in plant genome; 3) I harnessed the power of CRISPR/Cas9 gene editing tool to study the function of the serine/arginine-rich (SR) proteins.

Flavobacterium columnare is a Gram negative fish pathogen that causes columnaris disease, which infects populations of wild and cultured fish species. However, pathogenic mechanisms of F. columnare are largely unknown. The purpose of this research is to obtain the complete sequence of the F. columnare ATCC 49512 genome to advance pathogenesis research and increase our understanding of this pathogen. To accomplish this, genome sequencing by using Sanger and 454 sequencing was conducted. The sequences were assembled, gaps were filled, and the circular genome was autoannotated. The F. columnare genome size is 3.2 Mb and AT rich (68.5% AT). It contains 2,882 predicted proteins, 71 tRNA genes and five ribosomal RNA operons. More than half (57.1%) of the open reading frames have assigned function, which included chondroitin AC lyase, proteases, collagenases, and genes involved in biofilm formation, secretion systems, iron acquisition, and gliding motility.

High-throughput DNA sequencing technologies are currently revolutionizing the fields of biology and medicine by elucidating the structure and function of the components of life. Modern DNA sequencing machines typically produce relatively short reads of DNA which are then assembled by software in an attempt to produce a representation of the entire genome. Due to the complex structure of all but the smallest genomes, especially the abundant presence of exact or almost exact repeats, all genome assemblers introduce errors into the final sequence and output a relatively large set of contigs instead of full-length chromosomes (a contig is a DNA sequence built from the overlaps between many reads). These problems are dramatically worse when homologous copies of the same chromosome differ substantially. Currently such genomes are usually avoided as assembly targets and, when they are not avoided, they generally produce assemblies of relatively low quality. An improved algorithm for the assembly of such data would dramatically improve our understanding of the genetics of a large class of organisms. We present a unique algorithm for the assembly of diploid genomes which have a high degree of variation between homologous chromosomes. The approach uses coverage, graph patterns and machine-learning classification to identify haplotype-specific sequences in the input reads. It then uses these haplotype-specific markers to guide an improved assembly. We validate the approach with a large experiment that isolates and elucidates the effect of single nucleotide polymorphisms (SNPs) on genome assembly more clearly than any previous study. The experiment conclusively demonstrates that the Bioluminescence heterozygous genome assembler produces dramatically longer contigs with fewer haplotype-switch errors than competing algorithms under conditions of high heterozygosity.