La dystrophie myotonique de type 1 (DM1) est une maladie dégénérative impliquant des symptômes d’atrophie musculaire et de myotonie. Au niveau moléculaire, elle est caractérisée par une expansion aberrante de CUG dans la région 3’UTR de l’ARNm de DMPK (Dystrophia Myotonica protein kinase). Ces répétitions CUG forment des agrégats toxiques (appelés foci) majoritairement nucléaires dans les cellules de patients DM1 et causent la séquestration anormale de ribonucléoprotéines (RBP), tel que le facteur «Muscleblind-like 1» (MBNL1), qui lieraient normalement les motifs CUG d’autres ARN. Les fonctions normales de ces RBPs seraient alors perturbées. En plus de leur rôle dans l’épissage alternatif, MBNL a récemment été caractérisé pour son implication dans la localisation intracellulaire de ses ARN cibles. Ceci suggèrerait que la pathogénèse de la DM1 pourrait résulter de l’effet perturbateur des répétitions CUG sur la localisation d’ARN précis et de protéines RBPs. À cet effet, un criblage basé sur de la microscopie fluorescente de 322 RBPs dans des myoblastes de patients DM1 a permis d’identifier des nouveaux facteurs qui colocaliseraient avec les expansions pathogéniques CUG. De plus, ces myoblastes DM1 ont été fractionnés et un séquençage-ARN a par la suite permis l’identification de transcrits délocalisés. Les deux banques de données ainsi générées, tant par le criblage que par le fractionnement/séquençage-ARN, pourraient ouvrir des nouvelles avenues de recherches dans la compréhension des anomalies moléculaires associées à la DM1, et potentiellement d’autres maladies à expansions microsatellites. / Myotonic dystrophy of type 1 (DM1) is a degenerative disorder implicating symptoms of muscular atrophy and myotony. In a molecular level, it is caused by the aberrant expansion of CUG repeats in the 3’-UTR region of the DMPK mRNA (Dystrophia Myotonica protein kinase). Excessive CUG repeats then form toxic aggregates (foci) enriched within the nucleus of DM1 patient cells. These RNA foci cause the abnormal sequestration of RNA Binding Proteins (RBP), in particular members of the Muscleblind-like protein 1 (MBNL), that normally bind the CUG motif of other target RNAs, and will hence alter their normal functions. In addition to their role in alternative splicing, MBNL1 has recently been implicated in the intracellular localisation of its RNA targets. It remains elusive whether the pathogenesis of DM1 could result from the deregulating effect of CUG repeats on the localisation of specific RNAs and RBP proteins. In this thesis, a fluorescent imaging-based screening of 322 RBPs in DM1 patient’s myoblasts has been conducted and this had led to the identification of new factors that may colocalize with pathogenic CUG expansions. Moreover, these DM1 myoblasts have been fractionated and subsequent RNA-sequencing has permitted the identification of transcripts that are delocalised between subcellular compartments. From the two large datasets generated from the RBP imaging-based screening and fractionation/RNA-sequencing, new avenues of research can be initiated to further understand not only DM1, but perhaps also other disorders that implicate microsatellite expansions.

L'hypothyroïdie congénitale par dysgénésie thyroïdienne (HCDT, ectopie dans plus de 80 %) a une prévalence de 1 cas sur 4000 naissances vivantes. L’HCDT est la conséquence d'une défaillance de la thyroïde embryonnaire à se différencier, à se maintenir ou à migrer vers sa localisation anatomique (partie antérieure du cou), qui aboutit à une absence totale de la thyroïde (athyréose) ou à une ectopie thyroïdienne (linguale ou sublinguale). Les HCDT sont principalement non-syndromiques (soit 98% des cas sont non-familiale), ont un taux de discordance de 92% chez les jumeaux monozygotes, et ont une prédominance féminine et ethnique (i.e., Caucasienne). La majorité des cas d’HCDT n’a pas de cause connue, mais est associée à un déficit sévère en hormones thyroïdiennes (hypothyroïdie). Des mutations germinales dans les facteurs de transcription liés à la thyroïde (NKX2.1, FOXE1, PAX8, NKX2.5) ont été identifiées dans seulement 3% des patients atteints d’HCDT sporadiques et l’analyse de liaisons exclue ces gènes dans les rares familles multiplex avec HCDT. Nous supposons que le manque de transmission familiale claire d’HCDT peut résulter de la nécessité d’au moins deux « hits » génétiques différents dans des gènes importants pour le développement thyroïdien. Pour répondre au mieux nos questions de recherche, nous avons utilisé deux approches différentes: 1) une approche gène candidat, FOXE1, seul gène impliqué dans l’ectopie dans le modèle murin et 2) une approche en utilisant les techniques de séquençage de nouvelle génération (NGS) afin de trouver des variants génétiques pouvant expliquer cette pathologie au sein d’une cohorte de patients avec HCDT. Pour la première approche, une étude cas-contrôles a été réalisée sur le promoteur de FOXE1. Il a récemment été découvert qu’une région du promoteur de FOXE1 est différentiellement méthylée au niveau de deux dinucléotides CpG consécutifs, définissant une zone cruciale de contrôle de l’expression de FOXE1. L’analyse d’association basée sur les haplotypes a révélé qu’un haplotype (Hap1: ACCCCCCdel1C) est associé avec le HCDT chez les Caucasiens (p = 5x10-03). Une réduction significative de l’activité luciférase est observée pour Hap1 (réduction de 68%, p<0.001) comparé au promoteur WT de FOXE1. Une réduction de 50% de l’expression de FOXE1 dans une lignée de cellules thyroïdienne humaine est suffisante pour réduire significativement la migration cellulaire (réduction de 55%, p<0.05). Un autre haplotype (Hap2: ACCCCCCC) est observé moins fréquemment chez les Afro-Américain comparés aux Caucasiens (p = 1.7x10-03) et Hap2 diminue l’activité luciférase (réduction de 26%, p<0.001). Deux haplotypes distincts sont trouvés fréquemment dans les contrôles Africains (Black-African descents). Le premier haplotype (Hap3: GTCCCAAC) est fréquent (30.2%) chez les contrôles Afro-Américains comparés aux contrôles Caucasiens (6.3%; p = 2.59 x 10-9) tandis que le second haplotype (Hap4: GTCCGCAC) est trouvé exclusivement chez les contrôles Afro-Américains (9.4%) et est absent chez les contrôles Caucasiens (P = 2.59 x 10-6). Pour la deuxième approche, le séquençage de l’exome de l’ADN leucocytaire entre les jumeaux MZ discordants n’a révélé aucune différence. D'où l'intérêt du projet de séquençage de l’ADN et l’ARN de thyroïdes ectopiques et orthotopiques dans lesquelles de l'expression monoallélique aléatoire dans a été observée, ce qui pourrait expliquer comment une mutation monoallélique peut avoir des conséquences pathogéniques. Finalement, le séquençage de l’exome d’une cohorte de 36 cas atteints d’HCDT a permis d’identifier de nouveaux variants probablement pathogéniques dans les gènes récurrents RYR3, SSPO, IKBKE et TNXB. Ces quatre gènes sont impliqués dans l’adhésion focale (jouant un rôle dans la migration cellulaire), suggérant un rôle direct dans les défauts de migration de la thyroïde. Les essais de migration montrent une forte diminution (au moins 60% à 5h) de la migration des cellules thyroïdiennes infectées par shRNA comparés au shCtrl dans 2 de ces gènes. Des zebrafish KO (-/- et +/-) pour ces nouveaux gènes seront réalisés afin d’évaluer leur impact sur l’embryologie de la thyroïde. / Congenital hypothyroidism by thyroid dysgenesis (CHTD) is a common disorder with prevalence at birth of 1 in 4000 live births. CHTD is the consequence of a failure of embryonic thyroid to differentiate or to migrate to its anatomical location (front of the neck), which leads to a total lack of thyroid (athyreosis) or an ectopic thyroid (lingual or sublingual). The most common category is ectopic thyroid diagnosis (up 85%). Most cases of CHTD have no known cause, but are associated with severe deficiency of thyroid hormones (hypothyroidism). The clinical diagnosis of hypothyroidism is usually possible only when permanent brain damage is already present. On the other hand, biochemical screening on the second day of life allows initiating replacement therapy from the second week of life, pre-empting severe intellectual deficit associated with the congenital hypothyroidism. Even with early treatment (an average of 9 days), loss of IQ, which is not exclusively due to the severity of hypothyroidism, can still be observed. Molecular markers may identify patients at risk for intellectual deficit (by e.g., genes involved in neuronal migration and the thyroid during development). These patients might benefit from early intervention to stimulate their neurocognitive development. Cases of CHTD are mainly non-syndromic and sporadic (in 98% of cases, there is no other affected in the family), have a discordant rate of 92% in monozygotic twins, and a female and ethnic (Caucasian) dominance. Germline mutations in thyroid-related transcription factors have been identified in only 3% of patients with sporadic CHTD, and linkage analysis has excluded these genes in rare multiplex families with CHTD. In addition, non-penetrating mutations among close relatives (for Nkx2.5 gene) suggest that modifying genes as germline variants de novo copy number (CNV) and / or somatic mutations are associated with CHTD. To respond to this research questions, we used two different approaches: 1) a candidate gene approach studying FOXE1, the only gene involved in ectopic thyroid in the mouse model and, 2) an approach using next generation sequencing techniques (NGS) to find genetic variants that could explain this pathology using a cohort of mostly sporadic CHTD. Variants and genes discovered by these two different approaches have been validated and their functional impact on the thyroid gland was evaluated by several experiments.

Lors du développement, la coordination d’évènements moléculaires et cellulaires mène à la production du cortex qui orchestre les fonctions sensori-motrices et cognitives. Son développement s’effectue par étapes : les cellules gliales radiaires (RGs) – les cellules souches neurales (NSCs) du cerveau en développement – et les cellules progénitrices de la zone ventriculaire (VZ) et de la zone sous ventriculaire (SVZ) génèrent séquentiellement des vagues distinctes de nouveaux neurones qui formeront les différentes couches corticales. Autour de la naissance, les RGs changent de devenir et produisent des cellules gliales. Cependant, une fraction persiste tout au long de la vie dans la SVZ qui borde le ventricule, perdant au passage leur morphologie radiale. Ces NSCs produisent ensuite les différents sous types d’interneurones du bulbe olfactif ainsi que des cellules gliales en fonction de leur origine spatiale dans la SVZ. Ces observations soulèvent d’importantes questions non résolues sur 1) le codage transcriptionnel régulant la régionalisation de la SVZ, 2) le potentiel des NSCs postnatales dans la réparation cérébrale, et 3) le lignage et les spécificités transcriptionnelles entre les NSCs et leur descendants. Mon travail de doctorat repose sur une étude transcriptionnelle des domaines de la SVZ postnatale. Celle-ci soulignait le fort degré d’hétérogénéité des NSCs et progéniteurs et identifiait des régulateurs transcriptionnels clés soutenant la régionalisation. J’ai développé des approches bio-informatiques pour explorer ces données et connecter l’expression de facteurs de transcription (TFs) avec la genèse régionale de lignages neuraux distincts. J’ai ensuite développé un modèle d’ablation ciblée pour étudier le potentiel régénératif des progéniteurs postnataux dans divers contextes. Finalement, j’ai participé au développement d’une procédure pour explorer et comparer des progéniteurs pré et postnataux à l’échelle de la cellule unique. Objectif 1 : Des expériences de transcriptomique et de cartographie ont été réalisées pour étudier la relation entre l’expression régionale de TFs par les NSCs et l’acquisition de leur devenir. Nos résultats suggèrent un engagement précoce des NSCs à produire des types cellulaires définis selon leur localisation spatiale dans la SVZ et identifient HOPX comme un marqueur d’une sous population biaisé à générer des astrocytes. Objectif 2 : J’ai mis au point un modèle de lésion corticale qui permet l’ablation ciblée de neurones de couches corticales définies pour étudier la capacité régénérative et la spécification appropriée des progéniteurs postnataux. Une analyse quantitative des régions adjacentes, incluant la région dorsale de la SVZ, a révélé une réponse transitoire de progéniteurs définis. Objectif 3 : Nous avons développé la lignée de souris transgénique Neurog2CreERT2Ai14, qui permet le marquage de cohortes de progéniteurs glutamatergiques et de leurs descendants. Nous avons montré qu’une large fraction de ces progéniteurs persiste dans le cerveau postnatal après la fermeture de neurogénèse corticale. Ils ne s’accumulent pas pendant le développement embryonnaire mais sont produits par des RGs qui persistent après la naissance dans la SVZ et qui continuent de générer des neurones corticaux, bien que l’efficacité soit faible. Le séquençage d’ARN sur cellule unique a révélé une dérégulation transcriptionnelle qui corrèle avec le déclin progressif observé in vivo de la neurogénèse corticale. Ensemble, ces résultats soulignent le potentiel des études transcriptomiques à résoudre mais aussi à soulever des questions fondamentales comme les changements trancriptionnels intervenant dans une population de progéniteurs au cours du temps et participant aux changements de leur destinée. Cette connaissance sera la clé du développement d’approches novatrices pour recruter et promouvoir la génération de types cellulaires spécifiques, incluant les sous-types neuronaux dans un contexte pathologique. / During development, a remarkable coordination of molecular and cellular events leads to the generation of the cortex, which orchestrates most sensorimotor and cognitive functions. Cortex development occurs in a stepwise manner: radial glia cells (RGs) - the neural stem cells (NSCs) of the developing brain - and progenitor cells from the ventricular zone (VZ) and the subventricular zone (SVZ) sequentially give rise to distinct waves of nascent neurons that form cortical layers in an inside-out manner. Around birth, RGs switch fate to produce glial cells. A fraction of neurogenic RGs that lose their radial morphology however persists throughout postnatal life in the subventricular zone that lines the lateral ventricles. These NSCs give rise to different subtypes of olfactory bulb interneurons and glial cells, according to their spatial origin and location within the postnatal SVZ. These observations raise important unresolved questions on 1) the transcriptional coding of postnatal SVZ regionalization, 2) the potential of postnatal NSCs for cellular regeneration and forebrain repair, and 3) the lineage relationship and transcriptional specificities of postnatal NSCs and of their progenies. My PhD work built upon a previously published comparative transcriptional study of defined microdomains of the postnatal SVZ. This study highlighted a high degree of transcriptional heterogeneity within NSCs and progenitors and revealed transcriptional regulators as major hallmarks sustaining postnatal SVZ regionalization. I developed bioinformatics approaches to explore these datasets further and relate expression of defined transcription factors (TFs) to the regional generation of distinct neural lineages. I then developed a model of targeted ablation that can be used to investigate the regenerative potential of postnatal progenitors in various contexts. Finally, I participated to the development of a pipeline for exploring and comparing select populations of pre- and postnatal progenitors at the single cell level. Objective 1: Transcriptomic as well as fate mapping were used to investigate the relationship between regional expression of TFs by NSCs and their acquisition of distinct neural lineage fates. Our results supported an early priming of NSCs to produce defined cell types depending of their spatial location in the SVZ and identified HOPX as a marker of a subpopulation biased to generate astrocytes. Objective 2: I established a cortical lesion model, which allowed the targeted ablation of neurons of defined cortical layers to investigate the regenerative capacity and appropriate specification of postnatal cortical progenitors. Quantitative assessment of surrounding brain regions, including the dorsal SVZ, revealed a transient response of defined progenitor populations. Objective 3: We developed a transgenic mouse line, i.e. Neurog2CreERT2Ai14, which allowed the conditional labeling of birth-dated cohorts of glutamatergic progenitors and their progeny. We used fate-mapping approaches to show that a large fraction of Glu progenitors persist in the postnatal forebrain after closure of the cortical neurogenesis period. Postnatal Glu progenitors do not accumulate during embryonal development but are produced by embryonal RGs that persist after birth in the dorsal SVZ and continue to give rise to cortical neurons, although with low efficiency. Single-cell RNA sequencing revealed a dysregulation of transcriptional programs, which correlates with the gradual decline in cortical neurogenesis observed in vivo. Altogether, these data highlight the potential of transcriptomic studies to unravel but also to approach fundamental questions such as transcriptional changes occurring in a population of progenitors over time and participating to changes in their fate potential. This knowledge will be key in developing innovative approaches to recruit and promote the generation of selected cell types, including neuronal subtypes in pathologies.

Les carcinomes spinocellulaires cutanés (cSCCs) sont le deuxième type de cancer par ordre de fréquence et sont responsables de 25% des décès dus aux cancers de la peau. Il est donc essentiel de caractériser les mécanismes responsables de la cancérisation de l'épiderme afin de développer de nouveaux traitements. Dans ce contexte, les miRNAs apparaissent comme des cibles de choix pour le développement de futures thérapies anti-tumorales. Toutefois, leur implication dans la physiopathologie des cSCCs est encore peu documentée. Au cours de cette étude, j’ai identifié, par séquençage à haut débit, 112 miRNAs dont l’expression est altérée au cours du développement tumoral dans un modèle murin de carcinogénèse chimique cutanée. J’ai ensuite focalisé mon attention sur le cluster miR-193b/365a et sur miR-708 dont les niveaux diminuent au cours de la progression tumorale, suggérant des fonctions de suppresseurs de tumeur. En accord avec cette hypothèse, l’expression ectopique de ces miRNAs inhibe la prolifération, la survie et la migration de cellules tumorales, alors que le blocage de leur action par des anti-sens stimule ces fonctions cellulaires dans des kératinocytes normaux. L’association d’approches in silico et d’analyses du transcriptome de cellules de cSCC sur-exprimant ces miRNAs m’a permis d’identifier leurs gènes cibles potentiels. J’ai validé KRAS et MAX comme cibles communes de miR-193b et miR-365a, et montré par l’utilisation de siRNAs que la répression de ces cibles mime les effets de ces miRNAs. Ces résultats suggérent que le ciblage de ces gènes pourrait médier en partie les effets suppresseurs de tumeur de miR-193b et de miR-365a dans les cSCCs. / Cutaneous squamous cell carcinomas (cSCCs) are the second most common cancer and are responsible for up to 25% of all skin cancer deaths. It is therefore essential to characterize the mechanisms responsible for epidermis carcinogenesis to develop new treatments. In this context, miRNAs appear to be prime targets for the development of future anti-tumor therapies. However, their involvement in the pathophysiology of cSCCs is still poorly documented. In this study, I identified using Small RNA sequencing, 112 miRNAs whose expression is altered during tumor development in a mouse model of cutaneous two-stage chemical carcinogenesis. Then, I focused my attention on the miR-193b/365a cluster and on miR-708, that are down-regulated during tumorigenesis, suggesting tumor suppressor functions. Consistent with this hypothesis, the ectopic expression of these miRNAs inhibit the proliferation, survival and migration of tumor cells, while blocking their action with antisense oligonucleotides stimulates these cellular functions in normal keratinocytes. Combining in silico target-prediction approaches and transcriptome analyzes of cSCC cells over-expressing these miRNAs, I identified their potential target genes. I validated KRAS and MAX as direct targets of miR-193b and miR-365a, and I showed that repression of these genes using siRNAs mimics the effects of these miRNAs. These results suggest that targeting these genes might mediate, at least in part, the tumor suppressor action of miR-193b and miR-365a in cSCCs.

La principale contribution de cette thèse est le développement d'une méthode fiable, robuste, et rapide pour la prédiction des pré-miARNs. Deux objectifs avaient été assignés : efficacité et flexibilité. L'efficacité a été rendue possible au moyen d'un algorithme quadratique. La flexibilité repose sur deux aspects, la nature des données expérimentales et la position taxonomique de l'organisme (en particulier plantes ou animaux). Mirinho accepte en entrée des séquences de génomes complets mais aussi les très nombreuses séquences résultant d'un séquençage massif de type NGS de “RNAseq”. “L'universalité” taxonomique est obtenu par la possibilité de modifier les contraintes sur les tailles de la tige (double hélice) et de la boule terminale. Dans le cas de la prédiction des miARN de plantes la plus grande longueur de leur pré-miARN conduit à des méthodes d'extraction de la structure secondaire en tige-boule moins précises. Mirinho prend en compte ce problème lui permettant de fournir des structures secondaires de pré-miARN plus semblables à celles de miRBase que les autres méthodes disponibles. Mirinho a été utilisé dans le cadre de deux questions biologiques précises l'une concernant des RNAseq l'autre de l'ADN génomique. La première question a conduit au traitement et l'analyse des données RNAseq de Acyrthosiphon pisum, le puceron du pois. L'objectif était d'identifier les miARN qui sont différentiellement exprimés au cours des quatre stades de développement de cette espèce et sont donc des candidats à la régulation des gènes au cours du développement. Pour cette analyse, nous avons développé un pipeline, appelé MirinhoPipe. La deuxieme question a permis d'aborder les problèmes liés à la prévision et l'analyse des ARN non-codants (ARNnc) dans la bactérie Mycoplasma hyopneumoniae. Alvinho a été développé pour la prédiction de cibles des miRNA autour d'une segmentation d'une séquence numérique et de la détection de la conservation des séquences entre ncRNA utilisant un graphe k-partite. Nous avons finalement abordé un problème lié à la recherche de motifs conservés dans un ensemble de séquences et pouvant ainsi correspondre à des éléments fonctionnels / The main contribution of this thesis is the development of a reliable, robust, and much faster method for the prediction of pre-miRNAs. With this method, we aimed mainly at two goals: efficiency and flexibility. Efficiency was made possible by means of a quadratic algorithm. Flexibility relies on two aspects, the input type and the organism clade. Mirinho can receive as input both a genome sequence and small RNA sequencing (sRNA-seq) data of both animal and plant species. To change from one clade to another, it suffices to change the lengths of the stem-arms and of the terminal loop. Concerning the prediction of plant miRNAs, because their pre-miRNAs are longer, the methods for extracting the hairpin secondary structure are not as accurate as for shorter sequences. With Mirinho, we also addressed this problem, which enabled to provide pre-miRNA secondary structures more similar to the ones in miRBase than the other available methods. Mirinho served as the basis to two other issues we addressed. The first issue led to the treatment and analysis of sRNA-seq data of Acyrthosiphon pisum, the pea aphid. The goal was to identify the miRNAs that are expressed during the four developmental stages of this species, allowing further biological conclusions concerning the regulatory system of such an organism. For this analysis, we developed a whole pipeline, called MirinhoPipe, at the end of which Mirinho was aggregated. We then moved on to the second issue, that involved problems related to the prediction and analysis of non-coding RNAs (ncRNAs) in the bacterium Mycoplasma hyopneumoniae. A method, called Alvinho, was thus developed for the prediction of targets in this bacterium, together with a pipeline for the segmentation of a numerical sequence and detection of conservation among ncRNA sequences using a kpartite graph. We finally addressed a problem related to motifs, that is to patterns, that may be composed of one or more parts, that appear conserved in a set of sequences and may correspond to functional elements.

Massive parallel sequencing (MPS) data analysis tasks are often computationally demanding and their execution time would take too long using standard computing machines. Thus there is a need for parallelization of this tasks and ability to execute them on a sufficiently powerful computing machines. In the first chapter we describe a newly created platform for resequencing analysis of MPS data - MOLDIMED and novel annotation tool, which is ready to deploy on HPC infrastructure. The second chapter describes MPS approaches in Diamond-Blackfan anaemia (DBA), which is predominantly underlined by mutations in genes encoding ribosomal proteins (RP); however, its etiology remains unexplained in approximately 25% of patients. We performed panel sequencing of all ribosomal genes in DBA patient without previously known molecular pathology. A novel heterozygous RPS7 mutation coding RPS7 p.V134F was found in one female patient and subsequently confirmed in two asymptomatic family members, in whom mild anemia were detected on further examination. Subsequently, we performed whole transcriptome analysis in all family members and patient with RPS7 mutation in comparison with healthy control group and with DBA patients with known mutation in RPS19. We observed dysregulation mainly in signal pathways of translation,...

Cellular function and physiology are largely established through regulated gene expression. The first step in gene expression, transcription of the genomic DNA into RNA, is a process that is highly aligned at the levels of initiation, elongation and termination. In eukaryotes, protein-coding genes are exclusively transcribed by RNA polymerase II (Pol II). Upon transcription of the first 15-20 nucleotides (nt), the emerging nascent RNA 5’ end is modified with a 7-methylguanosyl cap. This is one of several RNA modifications and processing steps that take place during transcription, i.e. co-transcriptionally. For example, protein-coding sequences (exons) are often disrupted by non-coding sequences (introns) that are removed by RNA splicing. The two transesterification reactions required for RNA splicing are catalyzed through the action of a large macromolecular machine, the spliceosome. Several non-coding small nuclear RNAs (snRNAs) and proteins form functional spliceosomal subcomplexes, termed snRNPs. Sequentially with intron synthesis different snRNPs recognize sequence elements within introns, first the 5’ splice site (5‘ SS) at the intron start, then the branchpoint and at the end the 3’ splice site (3‘ SS). Multiple conformational changes and concerted assembly steps lead to formation of the active spliceosome, cleavage of the exon-intron junction, intron lariat formation and finally exon-exon ligation with cleavage of the 3’ intron-exon junction. Estimates on pre-mRNA splicing duration range from 15 sec to several minutes or, in terms of distance relative to the 3‘ SS, the earliest detected splicing events were 500 nt downstream of the 3‘ SS. However, the use of indirect assays, model genes and transcription induction/blocking leave the question of when pre-mRNA splicing of endogenous transcripts occurs unanswered. In recent years, global studies concluded that the majority of introns are removed during the course of transcription. In principal, co-transcriptional splicing reduces the need for post-transcriptional processing of the pre-mRNA. This could allow for quicker transcriptional responses to stimuli and optimal coordination between the different steps. In order to gain insight into how pre-mRNA splicing might be functionally linked to transcription, I wanted to determine when co-transcriptional splicing occurs, how transcripts with multiple introns are spliced and if and how the transcription termination process is influenced by pre-mRNA splicing. I chose two yeast species, S. cerevisiae and S. pombe, to study co-transcriptional splicing. Small genomes, short genes and introns, but very different number of intron-containing genes and multi-intron genes in S. pombe, made the combination of both model organisms a promising system to study by next-generation sequencing and to learn about co-transcriptional splicing in a broad context with applicability to other species. I used nascent RNA-Seq to characterize co-transcriptional splicing in S. pombe and developed two strategies to obtain single-molecule information on co-transcriptional splicing of endogenous genes: (1) with paired-end short read sequencing, I obtained the 3’ nascent transcript ends, which reflect the position of Pol II molecules during transcription, and the splicing status of the nascent RNAs. This is detected by sequencing the exon-intron or exon-exon junctions of the transcripts. Thus, this strategy links Pol II position with intron splicing of nascent RNA. The increase in the fraction of spliced transcripts with further distance from the intron end provides valuable information on when co-transcriptional splicing occurs. (2) with Pacific Biosciences sequencing (PacBio) of full-length nascent RNA, it is possible to determine the splicing pattern of transcripts with multiple introns, e.g. sequentially with transcription or also non-sequentially. Part of transcription termination is cleavage of the nascent transcript at the polyA site. The splicing status of cleaved and non-cleaved transcripts can provide insights into links between splicing and transcription termination and can be obtained from PacBio data. I found that co-transcriptional splicing in S. pombe is similarly prevalent to other species and that most introns are removed co-transcriptionally. Co-transcriptional splicing levels are dependent on intron position, adjacent exon length, and GC-content, but not splice site sequence. A high level of co-transcriptional splicing is correlated with high gene expression. In addition, I identified low abundance circular RNAs in intron-containing, as well as intronless genes, which could be side-products of RNA transcription and splicing. The analysis of co-transcriptional splicing patterns of 88 endogenous S. cerevisiae genes showed that the majority of intron splicing occurs within 100 nt downstream of the 3‘ SS. Saturation levels vary, and confirm results of a previous study. The onset of splicing is very close to the transcribing polymerase (within 27 nt) and implies that spliceosome assembly and conformational rearrangements must be completed immediately upon synthesis of the 3‘ SS. For S. pombe genes with multiple introns, most detected transcripts were completely spliced or completely unspliced. A smaller fraction showed partial splicing with the first intron being most often not spliced. Close to the polyA site, most transcripts were spliced, however uncleaved transcripts were often completely unspliced. This suggests a beneficial influence of pre-mRNA splicing for efficient transcript termination. Overall, sequencing of nascent RNA with the two strategies developed in this work offers significant potential for the analysis of co-transcriptional splicing, transcription termination and also RNA polymerase pausing by profiling nascent 3’ ends. I could define the position of pre-mRNA splicing during the process of transcription and provide evidence for fast and efficient co-transcriptional splicing in S. cerevisiae and S. pombe, which is associated with highly expressed genes in both organisms. Differences in S. pombe co-transcriptional splicing could be linked to gene architecture features, like intron position, GC-content and exon length.

The mortality rate is high for cancer patients and treatments are only efficient in a fraction of patients. To be able to cure more patients, new treatments need to be invented. Immunotherapy activates the immune system to fight against cancer and one treatment targets immune checkpoints. If more targets are found, more patients can be treated successfully. In this project, interactions between immune and cancer cells that drive tumour growth were investigated in an attempt to find new potential targets. This was achieved by creating a machine learning model that finds genes expressed in cells involved in tumour-driving interactions. Single-cell RNA sequencing and spatial transcriptomic data from breast cancer patients were utilised as well as single-cell RNA sequencing data from healthy patients. The tumour rate was based on the cumulative expression of G2/M genes. The G2/M related genes were excluded from the analysis since these were assumed to be cell cycle genes. The machine learning model was based on a supervised variational autoencoder architecture. By using this kind of architecture, it was possible to compress the input into a low dimensional space of genes, called a latent space, which was able to explain the tumour rate. Optuna hyperparameter optimizer framework was utilised to find the best combination of hyperparameters for the model. The model had a R2 score of 0.93, which indicated that the latent space was able to explain the growth rate 93% accurately. The latent space consisted of 20 variables. To find out which genes that were in this latent space, the correlation between each latent variable and each gene was calculated. The genes that were positively correlated or negatively correlated were assumed to be in the latent space and therefore involved in explaining tumour growth. Furthermore, the correlation between each latent variable and the growth rate was calculated. The up- and downregulated genes in each latent variable were kept and used for finding out the pathways for the different latent variables. Five of these latent variables were involved in immune responses and therefore these were further investigated. The genes in these five latent variables were mapped to cell types. One of these latent variables had upregulated immune response for positively correlated growth, indicating that immune cells were involved in promoting cancer progression. Another latent variable had downregulated immune response for negatively correlated growth. This indicated that if these genes would be upregulated instead, the tumour would be thriving. The genes found in these latent variables were analysed further. CD80, CSF1, CSF1R, IL26, IL7, IL34 and the protein NF-kappa-B were interesting finds and are known immune-modulators. These could possibly be used as markers for pro-tumour immunity. Furthermore, CSF1, CSF1R, IL26, IL34 and the protein NF-kappa-B could potentially be targeted in immunotherapy.

« Myopathie héréditaire » est un terme générique pour les maladies génétiques rares caractérisées par une faiblesse musculaire et une hypotonie avec ou sans atrophie musculaire. Les personnes atteintes d'une forme légère peuvent présenter des contractures, une scoliose, une hyporéflexie ou des caractéristiques dysmorphiques, et les plus sévères peuvent être accompagnées de symptômes cardiaques ou respiratoires pouvant s'avérer mortel. Alors que les méthodes de séquençage de nouvelle génération basées sur l'ADN ont considérablement accéléré la découverte de gènes responsables de maladies rares, de nombreux patients demeurent sans diagnostiques génétiques. L'une des principales raisons de ce problème est le grand nombre de variants de signification inconnue identifiés, où l’impact biologique est peu ou pas connu. Ce mémoire de maîtrise contient trois projets distincts dont l'objectif global est d'augmenter le rendement diagnostique pour les patients atteints de myopathies héréditaires rares. La première étude porte sur trois frères et soeurs atteints d'une dystrophie musculaire non diagnostiquée. Une combinaison de techniques « omic » a été utilisée pour identifier un variant faux-sens dans le gène IARS accompagné d’un déséquilibre allélique spécifique aux tissus musculaires. L’inhibition de iars-1 chez le C. elegans a entraîné une désorganisation progressive du muscle de la paroi corporelle, mais sans perte significative de la motilité. Ainsi, nous avons conclu que iars-1 joue clairement un rôle dans l'organisation des myotubes. La pathogénicité du variant, cependant, nécessite une enquête plus approfondie. La deuxième étude porte sur une femme présentant une myopathie statique congénitale se manifestant par une faiblesse proximale et distale. En utilisant le séquençage de l’ARN, nous avons identifié pour la première fois un profil d'expression génique compatible avec une prédominance des fibres musculaires de type I, focussant l’intérêt sur un variant dans le gène RYR1. La troisième étude englobe une cohorte de vingt-huit patients porteurs de la même mutation RYR1, mais présentant une hétérogénéité clinique significative. Des modèles « knock-in » de C. elegans pour les études deux et trois ont démontrés des changements en transmission synaptique, la durée de vie, la taille corporelle et la locomotion. Ainsi, nous avons conclu que les deux variants identifiés dans RYR1 ont probablement également des conséquences cliniques chez les porteurs humains. En fin de compte, ces études mettent en évidence l'utilité du séquençage de l’ARN en tant qu'outil de diagnostic complémentaire, capable de restreindre la liste de candidats potentiellement pathogéniques, ainsi que le pouvoir du C. elegans en tant que modèles pour des tests rapides et coordonnés de variants candidats. / “Hereditary myopathies” is an umbrella term for rare inherited diseases characterized by muscle weakness and hypotonia with or without muscle atrophy. Individuals with a mild affliction may present with contractures, scoliosis, hyporeflexia or dysmorphic features, while those more severely affected may present cardiac or respiratory involvement that could prove deadly. While traditional DNA-based next-generation sequencing techniques have greatly accelerated discovery of genes causing rare diseases, many patients remain without a known genetic cause. The main reason for this diagnostic shortfall is the vast number of variants of unknown significance identified whose biological functions are unknown. This master’s thesis contains three separate projects with an overarching goal to increase the diagnostic yield of patients with rare hereditary myopathies. The first study focuses on three siblings with an undiagnosed muscular dystrophy. A combination of “omic” techniques were used to identify a missense variant as well as a muscle-specific allelic imbalance in the gene IARS leading to the exclusive expression of the mutant allele. Iars-1 knock-down in C. elegans resulted in progressive disorganization of the body wall muscle but with no significant loss of motility. Thus, we concluded that iars-1 likely plays a role in the organization of myotubes. The pathogenicity of the variant, however, requires further investigation. The second study involves a woman with a congenital static myopathy exhibited as proximal and distal weakness. Using RNA-sequencing, we identified for the first time a gene expression profile consistent with type I fiber predominance in the proband which guided the search for the causative RYR1 variant. The third study encompasses a cohort of twenty-eight patients who carry the same RYR1 mutation but display significant clinical heterogeneity. Knock-in models of C. elegans for both studies demonstrated altered synaptic transmission, lifespan, body size and locomotion. Thus, we concluded that both variants identified in RYR1 likely have consequences for human carriers as well. Ultimately, these studies highlight the utility of RNA-seq as a complimentary diagnostic tool capable of narrowing the search for novel pathogenic mutations as well as the value of C. elegans as models for rapid and coordinated testing of candidate variants.

<p>Across the tree of life, novel genes are thought to be a source of much of the unique behaviors and adaptions between the different taxa. This is especially true in the social insects where novel genes are proposed to contribute to novel social behaviors. In the honey bee (Apis mellifera L.), a group of novel genes called the major royal jelly proteins (MRJPs) are proposed to be important to the expression of novel social behaviors, particularly those related to nursing versus foraging tasks. Unfortunately, identifying the functional role of novel genes is often not possible due to a lack of functional genomic tools in non-model species such as the honey bee. Here I have developed a novel ectopic expression system in Drosophila melanogaster and used it to elucidate how the MRJPs contribute to behavioral and transcriptional changes in the insect brain. I found that the MRJPs regulated the expression of hundreds of genes in Drosophila, and these overlap with genes regulated differentially between nursing and foraging honey bees. Furthermore, I found that MRJP expression impairs or negatively regulated phototaxis. My results demonstrate the MRJPs play a role in behavioral plasticity and highlight that the MRJPs may have a much larger role in the nurse-forager transition than previously thought.</p>