Primary liver cancer is an aggressive, lethal malignancy that ranks as the fourth leading cause of cancer-related death worldwide. Its 5-year mortality rate is estimated to be more than 95%. This significant low survival rate is due to poor diagnosis, which can be referred to as the lack of sufficient and early-stage detection methods. Many liver cancer-associated non-coding RNAs (ncRNAs) have been extensively examined to serve as promising biomarkers for precise diagnostics, prognostics, and the evaluation of the therapeutic progress. For the simple, rapid, and selective ncRNA detection, various nanomaterial-enhanced biosensors have been developed based on electrochemical, optical, and electromechanical detection methods. This review presents ncRNAs as the potential biomarkers for the early-stage diagnosis of liver cancer. Moreover, a comprehensive overview of recent developments in nanobiosensors for liver cancer-related ncRNA detection is provided.

Not all transcribed DNA encodes protein, and some of these noncoding RNAs (ncRNAs), such as roX1 and roX2, may play important roles in the cell. The functional roles of the majority of these, however, remain largely unknown. In this thesis, I first used EST and mRNA evidence to define 2,788 lincRNA loci within the Drosophila melanogaster genome. I suggest that up to 1,652 of these are functional, as 1,411 show evidence for significant evolutionary constraint while 241 fast-evolving loci are enriched in short RNA species. A distinct set of 1,119 lincRNA loci were defined by RNA-seq, the vast majority of which show clear primary sequence constraint. Their expression profiles and enrichment in particular chromatin domains indicate that these lincRNAs are likely involved in developmental regulation. I also identified 42 potential analogous lincRNAs with shared genomic locations between Drosophila and mouse. Constrained, non-embryonic lincRNAs defined by ESTs are transcribed preferentially in the vicinity of protein-coding genes encoding transcription factors and I demonstrated that one of these, which I name dEvf-2, positively regulates the expression of its genomically adjacent transcription factor, Dll, in cell culture. Finally, I used a reverse genetics approach to search for lincRNA promoter mutations and examined the effect of these on lincRNA expression. My findings suggest that many, previously unknown, functional lincRNAs exist within the Drosophila genome and are worthy of further in-depth experimental investigation.

Pseudomonas brassicacearum a la particularité de générer une diversité intraclonale aussi bien in vitro qu'en conditions naturelles dans la rhizosphère de plantes. Ce phénomène de variation phénotypique commun chez les bactéries est un processus d'adaptation aux environnements changeants. Des données de transcriptomique issues de puces à ADN, contenant aussi bien des séquences codantes que non codantes, nous ont permis d'identifier les gènes dont l'expression est altérée et surtout de relier ce phénomène à l'expression d'ARN non codants régulateurs (ARNnc) de type Rsm qui sont sous le contrôle du système à deux composants GacS/GacA. Nous avons montré que des mutations ponctuelles dans les gènes gacS ou gacA sont à l'origine de cette variation phénotypique et que l'expression de l'un des trois gènes rsmX, rsmY ou rsmZ permet de restaurer le phénotype de la souche sauvage. L'importance de ces ARNnc dans la survie de la bactérie aux fluctuations de son environnement est dénotée par la duplication de rsmX en un gène que nous avons nommé rsmX-2, dont la fonction a été validée. Nos données suggèrent une activation exclusive des gènes rsmX-1 et rsmX-2 par GacA et l'intervention de régulateurs additionnels dans le cas de rsmY et rsmZ. Au vu de la redondance fonctionnelle de ces quatre ARNnc, nous avons investigué leur niveau d'expression et leur stabilité dans différentes conditions de culture et montré des différences pour les quatre ARNnc. En réponse à une carence en nutriments, l'expression des ARNnc Rsm est fortement activée et atteint son maximum quand le ppGpp est détecté dans le milieu, suggérant un lien entre le système Gac/Rsm et la réponse « stringente ». / The plant-beneficial bacterium Pseudomonas brassicacearum forms phenotypic variants in vitro as well as in planta during root colonisation under natural conditions. Transcriptome analysis of typical phenotypic variants using microarrays containing coding as well as non-coding DNA fragments showed differential expression of several genes relevant to secondary metabolism and of the small non-coding RNA (ncRNA) genes rsmX, rsmY and rsmZ, which was characterized by down-regulation. Naturally occurring mutations in the GacS/GacA two-component system accounted for phenotypic switching. The importance of these ncRNAs in the survival of the bacteria to changing environments is denoted by the duplication of rsmX gene, which we called rsmX-2 and whose function has been validated. Our data suggest an exclusive activation of rsmX-1 and rsmX-2 genes by GacA and the involvement of additional regulators in the case of rsmY and rsmZ. Given the functional redundancy of these ncRNAs, we investigated their expression level and stability in different culture conditions and showed differences for the four ncRNAs. In response to nutrient depletion, the four ncRNAs expression is strongly activated and reaches its maximum when the ppGpp is detected in bacterial cells, suggesting a link between the Gac/Rsm system and the "stringent" response. Determining the level of each Rsm ncRNA, which is defined by a balance between synthesis and degradation of each transcript, shows the maintenance of a very important pool of RsmZ compared to other ncRNAs.

Le développement du séquençage ARN, ou RNAseq, a permis l'essor de la recherche intensive de biomarqueurs dans de nombreux domaines de la biologie. L’information complète du transcriptome contenue dans les données de sorties, permet à un bioinformaticien assidu de dépasser les connaissances actuelles et d’accéder, grâce à des pipelines informatiques avancés, à d’innombrables signatures d’intérêts inédites. Dans cette thèse nous mettons en avant que ces marqueurs potentiels, essentiellement explorés pour répondre à des problématiques clinique en conditions pathologiques, peuvent être utilisés pour affiner la caractérisation de types de cellules sans marqueurs strictement spécifiques. Nous nous sommes intéressés aux cellules souches mésenchymateuses (MSCs), un type de cellules souches adultes multipotentes, fortement utilisées en clinique mais ne possédant pas de marqueurs positifs strictement spécifiques.Notre étude se concentre sur la recherche des ARN longs non-codants non annotés. Ces ARNs, aussi nommés "lncRNA", constituent une classe émergente de transcrits encore peu explorée à ce jour. De plus, cette catégorie démontre une spécificité conditionnelle et tissulaire élevée. Nous avons élaboré un pipeline d’analyse RNAseq optimisé pour la reconstruction et la quantification de lncRNAs non annotés.En utilisant les données publiques de RNAseq, venant de différentes sources de MSCs et d'autres types de cellules, nous avons identifié de nouveaux lncRNA non annotés exprimés spécifiquement dans les MSCs.Nous avons développé pour ce projet Kmerator.jl, un outil qui permet de décomposer un transcrit en sous séquences spécifiques (k-mers) afin de chercher et quantifier plus rapidement la signature de nos candidats dans un grand nombre de données RNAseq. Kmerator a également été utilisé dans d'autres applications pour tester la qualité des données RNA-seq disponibles en accés public.Après validation de ces nouveaux biomarqueurs de MSCs par qPCR, nous avons eu recours à plusieurs outils informatiques pour prédire leurs fonctions potentielles. Enfin, nous avons analysé des données RNAseq « single-cell » pour aborder l’hétérogénéité d’expression au sein des populations MSCs. / The development of RNA sequencing, or RNAseq, have opened the path of intensive biomarkers research in many areas of biology. The complete information of the transcriptome contained in the output data, allows a bioinformatician to surpass the current knowledge and to access, thanks to advanced computer pipelines, to signatures of new interest. In this thesis, we are showing that these potential markers, classically used in clinical and pathological conditions, can be used to characterize cell types without extensive markers profile. We have studied mesenchymal stem cells, a type of adult multipotent stem cells, strongly used in clinics but without strickly specific positive markers. Our study mainly focuses on the search for non-annotated, long non-coding RNAs. These RNAs, also called "lncRNA", constitute an emerging class of transcripts and are still lightly explored.In addition, this category presents a highly tissue-related specificity. We have developed an optimized RNAseq pipeline for the reconstruction and quantification of non-annotated lncRNAs.Using public data from RNAseq, coming from different sources of MSC and other cell types, we have identified new non-annotated lncRNAs clearly and specifically expressed in MSCs. to complete this project, we developed Kmerator.jl, a bioinformatical tool that allows to decompose a transcript in k-mer, and select specific sub-sequences, in order to search and quantify at a faster rate the signature of our candidates in a large number of RNAseq dataset. After validation of these new biomarkers of MSCs by qPCR, we used several computer tools to predict their potential functions. Finally, we analyzed single-cell RNAseq data to address the heterogeneity of expression within MSC populations.

When conspecific individuals are crossed, the ensuing hybridization creates a spectrum of phenotypes in the resulting offspring. Many of hybrid traits will be additive, similar to the parental phenotypes. In some cases however, transgressive phenotypes are formed, outside the range of that of the parental phenotypes. Transgressive phenotypes can either be restricted to the F1 generation or be heritable throughout the hybrid lineage. While the mechanism behind heritable transgressive phenotyped is yet to be determined, transgressive gene expression is thought to be the root cause of their formation. Epigenetics modifications, heritable variation separate to the DNA code, can alter gene expression, persist through generations, and vary between individuals and over time. This makes them ideal candidates to be involved in the formation of transgressive phenotypes. RNA silencing is an epigenetic mechanism of gene regulation relying on 20Q24nt single stranded small RNAs (sRNAs). Small RNAs, due to their ability to set up persistent epigenetic marks at a locus, have the potential to create heritable transgressive gene expression. For example, when genetic variation from one parental genome presents novel targets to the sRNAs of the other parental genome, new epigenetic marks such as DNA methylation or secondary sRNAs can be created at target sites. In order to understand the potential of small RNAs to influence hybrid phenotype, I designed crossing experiments with Chlamydomonas reinhardtii, choosing this unicellular alga due to the genetic tools available and the haploid nature of its vegetative cells. The specific aim of the experiment was to identify transgressively expressed sRNA populations. Crossing two geographically distinct strains of C. reinhardtii, and sequencing both the genomes and sRNAomes of parents and recombinants, I was able catalogue both genetic and epigenetic variation in the parental strains providing unique insight into the inheritance of small RNAs in this alga. In this thesis, I first compare the genomes of the parental strains, identifying polymorphisms and assessing genetic variation in RNA silencing pathway components. I then describe the sRNA profiles of the parental strains, identifying differentially expressed sRNA loci. I then describe my approach to identifying transgressively expressed sRNA loci in the hybrids. While many sRNA loci in the recombinants exhibit additive sRNA expression, I found multiple transgressively expressed sRNA loci. Using the available bioinformatics tools, I identified potential miRNAs and phased secondary sRNAs within the list of transgressively expressed loci. Target analysis of one of the transgressively expressed miRNAs linked it with the transgressive expression of certain phased loci, suggesting a potential for sRNAs to be able to set up heritable epigenetic marks in recombinant C. reinhardtii cells.

Le cancer du rein est un problème majeur de santé publique. Un diagnostic précoce améliore les chances de survie. Le diagnostic repose essentiellement sur les examens d’imagerie comme l’échographie, la tomodensitométrie et l’IRM. Ces examens sont parfois associés à la biopsie et sont couteux et parfois invasifs. De plus, l’imagerie n’est pas capable de faire la distinction entre les tumeurs bénignes et les tumeurs malignes et entre les sous-types histologiques de carcinome à cellules claires qui est le plus fréquent. Il n’existe pas dans le cancer rénal de marqueur comme la PSA dans le cancer de la prostate ou la Foetoprotéine et l’HCG dans le cancer du testicule. Le but de cette étude est concentré sur la recherche des marqueurs mARNs ou miARNs dans les liquides biologiques (sérum, plasma, urine) pour le cancer du rein à cellules claires. Nous avons montré que les petits ARNs dans le sérum et les urines et l’intégrité des ARNs dans les urines étaient des outils diagnostiques dans le cancer du rein à cellules claires. Si ces petits ARNs circulants sont validés, on peut éventuellement imaginer l’intérêt pratique en clinique comme la détection des petits ARNs circulants dans l’urine pour prédire le cancer, la classification de la tumeur pour aider le clinicien, la prédiction d’une récidive ou d’une progression soit après néphrectomie soit au cours d’un traitement médical / Kidney cancer is a major public health problem. Early diagnosis improves the chances of survival. The diagnosis is mainly based on the imaging tests such as ultrasound, CT and MRI. These tests are sometimes associated with biopsy and are expensive and sometimes invasive. In addition, the imaging test is not able to distinguish between benign and malignant tumors and between histological subtypes of renal cell carcinoma. There don’t exist tumor marker in renal cancer such as PSA in prostate cancer or Fetoprotein and HCG in testicular cancer. For this purpose, the study has focused on finding markers mRNAs and miRNAs in biological fluids (serum, plasma, urine) for clear cell renal cell carcinoma. We have shown that the small cell-free RNAs in serum and urine and the integrity of cell-free RNA in urine were diagnostic tools for clear cell renal cell carcinoma. If these circulating small RNAs are validated, we can possibly imagine the interest in clinical practice as the detection of circulating small RNAs in urine to predict cancer, the classification of the tumor to help the clinician, the prediction of recurrence and progression after nephrectomy or during medical treatment

Les petits ARN régulateurs et les systèmes à deux composants sont des régulateurs très répandus de l’expression des gènes chez les bactéries. Dans la plupart des cas, les systèmes à deux composants agissent comme des régulateurs transcriptionnels. Un grand nombre de petits ARN agissent quant à eux au niveau post-transcriptionnel en modulant la traduction et/ou la stabilité de leur(s) ARN messager(s)-cible. Des connexions entre ces deux systèmes ont récemment pu mettre en lumière des circuits de régulations complexes aux propriétés encore peu connues.Mon travail a tout d’abord porté sur la connexion entre le système à deux composants EnvZ-OmpR et les petits ARN OmrA et OmrB chez Escherichia coli. Dans un premier temps, nous avons montré qu’OmpR activait directement la transcription d’omrA et d’omrB en se fixant à leur promoteur. Cette activation permet la production des petits ARN OmrA et OmrB qui, via leur extrémité 5’ conservée, ciblent à leur tour plusieurs ARN messagers-cibles et notamment le messager ompR-envZ. En accord avec des études précédentes, le contrôle d’ompR-envZ par les Omr n’affecte pas le niveau de forme phosphorylée d’OmpR. Ce phénomène posait donc la question de l’intérêt d’une telle régulation. Nous avons ensuite pu montrer que la régulation d’omrA et d’omrB est assez unique car leurs promoteurs répondent non seulement à la forme phosphorylée mais aussi à la forme non phosphorylée d’OmpR. Ce phénomène permet à ces ARN régulateurs de limiter leur propre synthèse en ayant un effet limité sur l’expression des autres cibles d’OmpR comme les porines OmpC et OmpF.Ce travail nous a conduits à chercher à caractériser d’autres exemples de modulation des systèmes à deux composants par des petits ARN régulateurs. Nous avons notamment étudié la régulation du système NarQ-NarP. En effet, nos travaux ont montré que la synthèse de narP était contrôlée par le petit ARN RprA. Cette régulation semble affecter les cibles de NarP et en particulier l’opéron napFDAGHBC. De plus, RprA répondrait au même stimulus que le système NarQ-NarP créant ainsi un lien physiologique entre le petit ARN et sa cible.Pour finir, un autre aspect de ce travail de thèse a été de s’intéresser à la régulation d’OmrA/B dans un contexte d’infection des macrophages par une souche d’Escherichia coli pathogène, la souche LF82. En effet, des données suggéraient que ces petits ARN étaient induits au cours de l’infection. J’ai pu valider ces données et montrer que cette induction était dépendante de la présence du système EnvZ/OmpR.En conclusion, j’ai pu montrer par diverses approches que les circuits de régulation intégrant des systèmes à deux composants et des petits ARN régulateurs possédaient des propriétés assez inédites permettant à la bactérie de s’adapter à divers stress. / Small regulatory RNA (sRNAs) and two component systems (TCS) are both widespread regulators of gene expression in bacteria. While TCS are mostly transcriptional regulators, a large class of sRNAs acts as post-transcriptional regulators of gene expression by modulating translation and/or stability of target-mRNAs. Many connections have recently been unraveled between these two types of regulators, resulting in mixed regulatory circuits with poorly characterized properties.First, we have investigated in details the negative feedback circuit that exists between the EnvZ-OmpR TCS and the OmrA/B sRNAs in Escherichia coli. We have found that OmpR directly activates transcription from omrA and omrB promoters, allowing production of OmrA/B sRNAs that target multiple mRNAs through their conserved 5’ end, including the ompR-envZ mRNA. In agreement with previous reports, we have found that this control of ompR-envZ by OmrA/B sRNAs does not affect the amount of OmpR-P i.e. the presumably active form of the regulator. This phenomenon therefore raised the question of the possible interest of such a regulation. Thereafter, we found that OmrA/B regulation is really unique because they respond to the phosphorylated form but also to the unphosphorylated form of OmpR. As a result, OmrA/B limit their own synthesis while they have only a limited effect on others targets of OmpR, such as the OmpC or OmpF porins.This work led us to try to characterize other examples of modulation of two-component systems synthesis by small regulatory RNAs. In particular, we studied the regulation of the NarQ-NarP system. Indeed, our work showed that the synthesis of narP is controlled by the RprA sRNA. This regulation appears to affect NarP targets and in particular the napFDAGHBC operon. Moreover, RprA would respond to the same stimulus as the NarQ-NarP system, thus creating a physiological link between the small RNA and its target.Finally, another aspect of this work was to study the regulation of OmrA/B in a context of infection of macrophages by an Escherichia coli pathogenic strain, LF82. Indeed, data suggested that these small RNAs were induced during infection. I was able to validate these data and showed that this induction was dependent on the presence of the EnvZ-OmpR system.

Proteins and RNAs are two of the most versatile macromolecules that carry out almost all functions within living organisms. In this thesis I have explored evolutionary and regulatory aspects of proteins and RNAs by studying their structures, modifications and interactions. In the first chapter of my thesis I investigate domain atrophy, a term I coined to describe large-scale deletions of core structural elements within protein domains. By looking into truncated domain boundaries across several domain families using Pfam, I was able to identify rare cases of domains that showed atrophy. Given that even point mutations can be deleterious, it is surprising that proteins can tolerate such large-scale deletions. Some of the structures of atrophied domains show novel protein-protein interaction interfaces that appear to compensate and stabilise their folds. Protein-protein interactions are largely influenced by the surface and charge complementarity, while RNA-RNA interactions are governed by base-pair complementarity; both interaction types are inherently different and these differences might be observed in their interaction networks. Based on this hypothesis I have explored the protein-protein, RNA-protein and the RNA-RNA interaction networks of yeast in the second chapter. By analysing the three networks I found no major differences in their network properties, which indicates an underlying uniformity in their interactomes despite their individual differences. In the third chapter I focus on RNA-protein interactions by investigating post-translational modifications (PTMs) in RNA-binding proteins (RBPs). By comparing occurrences of PTMs, I observe that RBPs significantly undergo more PTMs than non-RBPs. I also found that within RBPs, PTMs are more frequently targeted at regions that directly interact with RNA compared to regions that do not. Moreover disorderedness and amino acid composition were not observed to significantly influence the differential PTMs observed between RBPs and nonRBPs. The results point to a direct regulatory role of PTMs in RNA-protein interactions of RBPs. In the last chapter, I explore regulatory RNA-RNA interactions. Using differential expression data of mRNAs and lncRNAs from mouse models of hereditary hemochromatosis, I investigated competing regulatory interactions between mRNA, lncRNA and miRNA. A mutual interaction network was created from the predicted miRNA interaction sites on mRNAs and lncRNAs to identify regulatory RNAs in the disease. I also observed interesting relations between the sense-antisense mRNA-lncRNA pairs that indicate mutual regulation of expression levels through a yet unknown mechanism.

The discovery of RNA in 1868 by Friedrich Miescher was meant to be the prologue to an exciting new era in Biology full of scientific breakthroughs and accomplishments. Since then, RNAs have been proven to play an indispensable role in biological processes such as coding, decoding, regulation and expression of genes. In particular, the discovery of small non-coding RNAs and especially miRNAs, in C. elegans first and thereafter to almost all animals and plants, started to fill in the puzzle of a complex gene regulatory network present within cells. The aim of this thesis is to shed more light on the features and functionality of small RNAs. In particular, we will focus on the function and biogenesis of miRNAs and piRNAs, across multiple species, by employing advanced computational methods and machine learning. We first introduce a novel method (Chimira) for the identification of miRNAs from sets of animal and plant hairpin precursors along with post-transcriptional terminal modifications that are not encoded by the genome. This method allows the characterisation of the prevalence of miRNA isoforms within different cell types and/or conditions. We have applied Chimira within a larger study that examines the effect of terminal uridylation in RNA degradation in oocytes and cells in either embryonic or adult stage. This study showed that uridylation is the predominant transcriptional regulation mechanism in oocytes while it does not retain the same functionality on mRNAs and miRNAs, both in embryonic and adult cells. We then move on to a large-scale analysis of small RNA-Seq datasets in order to identify potential modification signatures across specific conditions and cell types or tissues in Human and Mouse. We extracted the full modification profiles across 461 samples, unveiling the high prevalence of modification signatures of mainly 1 to 4 nucleotides. Additionally, samples of the same cell type and/or condition tend to cluster together based on their miRNA modification profiles while miRNA gene precursors with close genomic proximity showed a significant degree of co-expression. Finally, we elucidate the determinant factors in strand selection during miRNA biogenesis as well as update the miRBase annotation with corrected miRNA isoform sequences. Next, we introduce a novel computational method (mirnovo) for miRNA prediction from RNA-Seq data with or without a reference genome using machine learning. We demonstrate its efficiency by applying it to multiple datasets, including single cells and RNaseIII deficient samples, supporting previous studies for the existence of non-canonical miRNA biogenesis pathways. Following this, we explore and justify a novel piRNA biogenesis pathway in Mouse which is independent of the MILI enzyme. Finally, we explore the efficiency of CRISPR/Cas9 induced editing of miRNA targets based on the computationally predicted accessibility of the targeted regions in the genome. We have publicly released two web-based novel computational methods and one on-line resource with results regarding miRNA biogenesis and function. All findings presented in this study comprise another step forward within the journey of elucidation of RNA functionality and we believe they will be of benefit to the scientific community.

DNA methylation entails the addition of a methyl group to the 5-carbon of the cytosine base of the DNA. This modification is important during many biological processes such as imprinting, X-chromosome inactivation, cell differentiation as well as silencing of transposable elements (TEs). DNA methylation is dynamic during early mammalian development, despite being a more static mark in somatic cells. Global hypomethylation is a hallmark of epigenetic reprogramming in mammalian primordial germ cells (PGCs), the early embryo and in naïve embryonic stem cells (ESCs). Genome integrity is crucial during early development, as the germline DNA needs to be protected for future generations. Therefore, epigenetic reprogramming presents a critical phase for TE defence since presumably alternative silencing pathways need to be employed to limit their activity. In this thesis, I investigate the role of small RNAs to control TEs during global waves of DNA demethylation in cellular reprogramming, naïve pluripotency as well as early mammalian development. Following an introduction to the research questions, in chapter 3 I investigate the mechanism of TE regulation in an in vitro model of Dnmt1 deletion in mouse ES cells to recapitulate in vivo epigenetic reprogramming. I find that certain classes of TEs become transcriptionally upregulated and subsequently resilenced by a mechanism independent of DNA methylation. I identify ARGONAUTE 2 (AGO2) bound siRNAs as the prominent mechanism to control certain classes of TEs, while others appear to be regulated by redistribution of repressive histone modifications. In chapter 4, I construct Dicer constitutive and conditional KO ESCs in the background of the Dnmt1f l/f l ESCs using CRISPR-Cas9. I dissect the role of DNA methylation and of DICER dependent small RNAs on transcriptional changes of ESCs. Additionally, I find that DICER dependent small interfering RNAs (siRNAs) re-silence transcriptionally active TE classes. Finally, in chapter 5, I examine the role of small RNAs in TE silencing in different models of global hypomethylation in vivo and in vitro PGCs, during iPSC reprogramming and in a transition from serum to 2i culturing of mouse ESCs.