Dissertations / Theses on the topic 'RNA signature'

Le stress oxydatif est l’une des voies menant à la sénescence cellulaire. Bien que les dommages causés par les espèces réactives de l'oxygène aux protéines et à l'ADN soient bien décrits, notre compréhension de la manière dont la transcription peut participer à l'apparition de la sénescence est encore limitée. Au niveau de la transcription, le stress oxydatif entraîne l’accumulation d’ARN promoteurs (ARNAu) et d’ARN amplificateur (ARNs), conséquence de la libération défectueuse du RNAPII de la chromatine, un phénomène connu sous le nom de RNAPII crawling. Nous avons observé que l'exploration de RNAPII était également détectée en aval d'une petite série de gènes connus pour être régulés par HP1Υ au niveau de leur terminaison. L'exploration de ce phénomène a donné un résultat inattendu, en ce sens qu'il a révélé un effet inhibiteur du peroxyde d'hydrogène sur le complexe exosome d'ARN impliqué dans la dégradation des ARN polyadénylés. Le RNAPII rampant a pour résultat la transcription de séquences d’ALU situées au voisinage des promoteurs et amplificateurs et en aval de gènes sans intron et de petites séries de gènes contenant un intron. Comme les séquences ALU contiennent des séquences A codées par le génome, elles doivent normalement être dégradées par l’exosome de l’ARN. Cependant, comme le stress oxydatif inhibe également cette activité d'ARNase, les ARNm contenant des séquences d'ALU transcrites par hasard se stabilisent et sont détectés dans le cytoplasme et même dans les fractions de polysomes. Ce phénomène peut participer à l'apparition de la réponse à l'interféron associée au stress oxydatif
Oxidative Stress is one of the routes leading to cellular senescence. While the damages that reactive oxygen species inflict on proteins and DNA are well described, our insight on how transcription may participate in the onset of senescence is still limited. At a transcriptional level, oxidative stress results in accumulation of promoter RNAs (uaRNAs) and enhancer RNAs (eRNAs) as a consequence of defective release of the RNAPII from the chromatin a phenomenon known as RNAPII crawling. We observed that RNAPII crawling was also detected downstream of a small series of genes known to be regulated by HP1Υ at the level of their termination. Exploring this phenomenon yielded an unexpected result in the sense that it revealed an inhibiting effect of hydrogen peroxide on the RNA exosome complex involved in degradation of polyadenylated RNAs. The crawling RNAPII results in the transcription of ALU sequences located in the neighborhood of promoters and enhancers and downstream of intron-less genes and of small series of intron-containing genes. As ALU sequences contain genome encoded A tracts, they should normally be degraded by the RNA exosome. Yet, as oxidative stress also inhibits this RNAse activity, mRNAs containing serendipitously transcribed ALU sequences get stabilized and are detected in the cytoplasm and even polysome fractions. This phenomenon may participate in the onset of the interferon response associated with oxidative stress

Atrial fibrillation (AF) is the commonest disorder of cardiac rhythm. Following coronary artery surgery approximately 1 in 3 patients will develop de novo post-operative AF (POAF) leading not only to prolonged hospital stay but also to increased morbidity and mortality. The pathophysiology of this arrhythmia is highly complex, and combines factors such as ion channel dysfunction, inflammation, oxidative stress, fibrosis and autonomic dysfunction that through electrical and structural remodelling promote both triggering and maintenance of this arrhythmia. For many years POAF has been regarded as a reactive phenomenon, occurring in response to post surgical inflammatory stressors and electrolyte imbalance. However, it is also possible that in a proportion of patients, prior cardiomyocyte remodelling predisposes to atrial arrhythmogenesis when exposed to surgical stress. Understanding the genomic and metabolic mechanisms that underlie this substrate may not only provide novel diagnostic biomarkers to identify at risk patients, but also isolate previously unrecognised therapeutic targets for prevention and treatment. In this work, a clinical observational study was utilised to obtain microRNA, gene expression and metabolic profiles of patients developing POAF after coronary artery bypass graft (CABG) surgery. Based on these results, a network of genomic and subsequent downstream pathway interactions was established to characterise the atrial substrate of post-operative AF. Furthermore, analysis of pre-operative blood was performed in order to identify novel microRNA that may provide a platform for biomarker development. Finally, the metabolic signature of the atrial myocardium and its relationship with the surrounding epicardial adipose were investigated to complete a comprehensive overview of the central mechanisms thought to underlie POAF pathogenesis. This work demonstrates that prior to surgery and the onset of arrhythmia, several distinct genomic and post-translational characteristics are evident in the both the myocardium and circulating blood of patients going on to develop POAF. Analysis of right atrial biopsies highlights a characteristic microRNA profile associated with POAF, and identifies target genes regulating intracellular signalling pathways, leukocyte recruitment, and ion channel remodelling. Furthermore, selected gene expression analysis demonstrates a de-differentiated phenotype similar to that seen in chronic AF, whilst at the same time establishing that disordered cardiomyocyte calcium handling is apparent at the time of surgery. Finally, analysis of the pre-operative circulating blood serum highlights microRNA selectively upregulated in POAF and establishes the potential for future biomarker development. In summary, the results presented here support the presence of a pre-existing atrial substrate in POAF, suggesting the potential exists for high-risk patients to be identified prior to surgery and the onset of arrhythmia. Furthermore, for the first time a number of similarities have been made apparent between post-operative and chronic AF, implying a common mechanistic spectrum of structural and electrical remodelling. As a consequence, the results presented in this thesis have not only improved our understanding of the complex interplay of factors leading to the pathogenesis of AF, but also provide a platform for both the development of a unique clinical biomarker and the identification of novel therapeutic targets.

Les ARN circulaires désignent une classe d'ARN non codants qui se caractérisent par une structure en boucle fermée de manière covalente. Sur le plan fonctionnel, ils peuvent agir sur la physiologie cellulaire en inhibant les microARN et en régulant l'expression des gènes et des protéines. La fonction émergente de ces ARNc n'est pas entièrement comprise, mais des études initiales ont récemment montré que les ARNc sont impliqués dans la régulation de la sécrétion d'insuline. A travers ces travaux, nous avons cherché à identifier les ARNc dans le muscle squelettique au niveau des fibres glycolytiques et oxydatives de patients sains ou atteints de diabète de type 2. Nos résultats ont démontré une signature unique en ARN circulaire non seulement en fonction du statut (sain ou DT2) mais aussi en fonction du type de fibres musculaires (triceps ou soleus). Notre étude a pu mettre en évidence pour la première fois un nouveau moyen de régulation de l’expression des gènes et des protéines, indépendamment de ce qui est déjà connu au niveau du muscle squelettique. Ces résultats permettent l’identification de nouveaux acteurs impliqués dans le développement du diabète de type 2 avec l’identification potentielle de nouvelles cibles thérapeutiques
Circular RNAs are a class of non-coding RNAs characterized by a covalently closed loop structure. Functionally, they can act on cell physiology by inhibiting microRNAs and regulating gene and protein expression. The emerging function of circRNAs is not fully understood, but initial studies have recently shown that they are involved in the regulation of insulin secretion. In this work we tried to identify circRNAs in skeletal muscle at the level of glycolytic and oxidative fibers in healthy and type 2 diabetic patients. Our results showed a unique circular RNA signature not only as a function of status (healthy or T2DM) but also as a function of muscle fibre type (triceps or soleus). For the first time, our study has been able to identify a new way of regulating gene and protein expression independently of what is already known in skeletal muscle. These results allowed us to identify new key molecules involved in the development of type 2 diabetes, with the potential to identify new therapeutic targets

L'holoprosencéphalie (HPE) est une maladie rare qui affecte le développement de la ligne médiane du cerveau antérieur dès les premiers stades embryonnaires, rendant son diagnostic moléculaire complexe. Elle résulte principalement d’altérations génétiques entraînant une réduction de l'activité de la voie de signalisation Sonic Hedgehog (SHH). Cependant, un diagnostic moléculaire précis n’est possible que pour 30% des patients, ce qui souligne l’importance de développer des nouvelles approches diagnostiques. Le principal obstacle réside dans l'impossibilité d'accéder au tissu primaire affectée par la pathologie, soit le neuroectoderme antérieur. Pour surmonter cet obstacle, j’ai mis au point un modèle in vitro du développement du neuroectoderme antérieur en utilisant des cellules souches pluripotentes induites. Ce modèle m’a permis de produire des données transcriptomiques permettant d’évaluer les impacts moléculaires de la déficience en SHH et de définir des signatures transcriptomiques décrivant les variations de l'activité de la voie SHH pouvant être corrélées à la sévérité des phénotypes d’HPE. Ce travail a également révélé de nouveaux gènes co-exprimés et régulés par SHH, qui pourraient constituer de nouveaux marqueurs génétiques de l'HPE. Ces avancées ouvrent la voie à la création d’outils de diagnostic innovants, visant à améliorer la précision du diagnostic pour les patients atteints d'HPE
Abstract : Holoprosencephaly (HPE) is a rare disorder that affects the development of the midline of the forebrain during the earliest stages of embryogenesis, making molecular diagnosis challenging. It primarily results from genetic alterations that lead to a reduction in the activity of the Sonic Hedgehog (SHH) signaling pathway. However, a precise molecular diagnosis is only possible for 30% of patients, highlighting the importance of developing new diagnostic approaches. The main challenge is the inaccessibility of the primary tissue, specifically the anterior affected by HPE, namely the anterior neuroectoderm. To overcome this challenge, I established an in vitro model of anterior neuroectoderm using induced pluripotent stem cells. This model allowed me to generate transcriptomic data to assess the molecular impacts of SHH deficiency and define transcriptomic signatures that describe variations in SHH pathway activity, which may correlate with the severity of HPE phenotypes. This work also revealed new co-expressed and SHH-regulated genes, which could serve as new genetic markers for HPE. These advances pave the way for innovative diagnostic tools aimed at improving diagnostic accuracy for patients with HPE

Seul 1,2% du génome code des protéines :98,8% est non-codant,cependant 93% du génome est transcrit, principalement en longs ARN non-codants (lncRNA). Or ces lncRNA constituent une nouvelle classe de régulateurs génomique agissant à tous les niveaux d’expression des gènes et ils sont fortement spécifiques du tissu,modulés au cours du temps et en conditions physiopathologiques.Ainsi,nous proposons que chaque cellule spécifiée exprime son répertoire de lncRNA spécifique avec une carte des zones de chromatines ouvertes renseignant son identité cellulaire.Dans cette perspective,nous avons isolé par FACS 2types cellulaires impliqués dans des pathologies: i) des neurones dopaminergiques humains(nDA) différenciés à partir d’hiPS et ii) des neurones DA et sérotoninergiques (n5-HT)murins.Sur ces 2types neuraux isolés,nous avons identifié 1363 lncRNA exprimés dans les nDA (dont 989nouveaux) constituant le répertoire des neurones DA et 1257 lncRNA dans les n5-HT (719nouveaux) constituant le répertoire des n5-HT.Or leur comparaison a montré que seuls 194 lncRNA sont communs aux 2types cellulaires:la majorité des lncRNA est exprimée soit dans les nDA soit dans les n5-HT,attestant leur spécificité cellulaire.De plus,39%des zones de chromatines ouvertes/potentiellement régulatrices des nDA ne sont pas non plus retrouvées dans les n5-HT.Ainsi, nous avons généré un catalogue d’éléments non codants constituant des signatures moléculaires spécifiques des nDA et n5-HT,ouvrant de nouvelles pistes physiopathologiques:Dans cette optique,les signatures non codantes DA ont été comparées avec les SNP associés à la maladie de Parkinson et des études de fonction sur des lncRNA candidats ont été réalisées
Only 1.2% of the genome codes for proteins; 98.8% is thus non-coding, despite 93% of the human genome being actively transcribed, mostly in long non-coding RNA (lncRNA).These lncRNA constitute a new class of genomic regulator capable of acting at all levels of gene expression and their expression is highly tissue-specific,modulated during the time and under normal/pathological conditions.Thus, we propose that each specified cell expresses a specific repertoire of lncRNA correlated to open/active chromatin regions specifying its cellular identity.In this context, we isolated by FACS 2neural types involved in many pathologies: i) human dopaminergic neurons (nDA) differentiated from hiPS and ii) DA and serotoninergic (n5-HT) neurons. From these 2neural types, we identified 1,363 lncRNA in nDA (among which 989 new, whether 73%) constituting the repertoire of nDA, and 1,257 lncRNA (among which 719 new) constituting the repertoire of n5-HT. Moreover,their comparison has shown that only 194 lncRNA are common to both neural types:thus the majority of lncRNA is expressed either in nDA or in n5-HT, indicating a high degree of cell-specificity.In addition, 39% of open chromatin regions, potentially regulatory, were also not detected in the n5-HT.Thus, we have generated DA and 5-HT specific catalogues of non-coding elements of the genome, which constitute DA and 5-HT specific molecular signatures, that could participate in deepening our knowledge regarding nDA or n5-HT development and dysfunctions. With this in mind,these DA specific elements have been compared with the SNP described as Parkinson Disease risk variants and candidate lncRNA were selected to perform studies of function

Mon travail de thèse a consisté à caractériser une sous-population de lymphocytes T régulateurs naturels de phénotype CD8+CD25+.

Nous avons purifié les CD8+CD25+ nTregs et vérifié par cytométrie de flux leur expression en FOXP3 et CTLA-4. Puis nous avons pu montrer que ces cellules possèdent des propriétés suppressives dans un test d’inhibition de la prolifération de lymphocytes T activés allogéniquement. Les lymphocytes CD8+CD25+ nTregs expriment les gènes FOXP3, CTLA-4, GARP et CCL-4 et les cytokines IL-10 et TGF-β. Par contre, les gènes CD28, ICOS, FOXO1 et Helios sont sous-exprimés dans les nTregs CD8+CD25+ par rapport aux lymphocytes T CD8+CD25-.

Nous avons établi une signature micro-ARN qui comprend 10 micro-ARNs différentiellement exprimés :7 micro-ARNs sous-exprimés "miR-9, -24, -31, -155, -210, -335 et -449 " et 3 micro-ARNs surexprimés " miR-214, -205 et -509". De plus, nous avons pu explorer la relevance biologique de cette signature micro-ARN en montrant dans un premier temps que les miRs "-31, -24, -210, -335" ciblent spécifiquement la région 3'UTR de FOXP3, de même les miR-9 et miR-155 ciblent la région 3'UTR de CTLA-4, et les miR-24, et -335 ciblent la région 3'UTR de GARP. Ceci a été fait par des expériences de co-transfections suivies d'une mesure de l'activité rapportrice luciférase. De plus, nous avons pu démontrer par des expériences de transduction lentivirale ex vivo, de cellules T primaires, que des micro-ARNs de la signature régulent l’expression de FOXP3, CTLA-4 et GARP dans les Tregs naturels CD8+CD25+ humains.

Cette étude montre l'importance des micro-ARNs dans la régulation post-transcriptionnelle des gènes impliqués dans la fonction régulatrice des lymphocytes T régulateurs.

Doctorat en Sciences
info:eu-repo/semantics/nonPublished

Angiogênese é o processo de formação de novos vasos sanguíneos a partir dos vasos existentes. É um processo vital, mas muitas doenças também dependem deste mecanismo para obter nutrientes e progredir. Estas \"doenças dependentes de angiogênese\" incluem cânceres, retinopatias e degeneração macular. Alguns inibidores da angiogênese foram desenvolvidos na última década, com o objetivo de auxiliar no manejo dessas doenças e melhorar a qualidade de vida dos pacientes. A maioria destes compostos funciona inibindo a ligação de VEGFA/VEGFR2, que também é um elemento importante para a sobrevivência de células endoteliais quiescentes; e isso pode explicar parcialmente eventos adversos observados em alguns ensaios clínicos. Nossa hipótese é que a melhoria das terapias anti-angiogênicas depende de uma compreensão melhor e mais ampla desse processo, especialmente quando relacionada à progressão das doenças. Utilizando RNA-Seq e um modelo animal bem aceito de angiogênese, o modelo murino de Retinopatia Induzida por Oxigênio, exploramos o transcritoma e identificamos 153 genes diferencialmente expressos durante a angiogênese. Uma extensiva validação de vários genes realizada por qRT-PCR e hibridização in-situ confirmou a superexpressão de Esm1 em células endoteliais de tecidos com angiogênese ativa. A análise de enriquecimento desta lista de genes confirmou a ligação da angiogênese com genes frequentemente mutados em tumores, consistente com a conhecida ligação entre câncer e angiogênese, e forneceu sugestões de fármacos já aprovados que podem ser reutilizados para controlar a angiogênese em circunstâncias patológicas. Finalmente, com base neste panorama amplo da angiogênese, fomos capazes de criar um biomarcador molecular com poder prognóstico para a predição da recidiva de câncer de mama, com aplicações clínicas promissoras. Em resumo, este trabalho revelou com sucesso genes relacionados à angiogênese e forneceu novas alternativas terapêuticas, incluindo potenciais fármacos para reposicionamento. Esse conjunto de genes diferencialmente expressos é também um recurso valioso para investigações futuras.
Angiogenesis is the process of formation of new blood vessels based on existing vessels. It is a vital process but many diseases also rely on this mechanism to get nourishment and progress. These so called angiogenesis-dependent diseases include cancers, retinopathies and macular degeneration. Some angiogenesis inhibitors were developed in the past decade, aiming to help the management of such diseases and improve patients quality of life. Most of these compounds work by inhibiting VEGFA/VEGFR2 binding, which is also a key element to the survival of quiescent endothelial cells; this may partly explain unanticipated adverse events observed in some clinical trials. We hypothesize that the improvement of anti-angiogenesis therapies hinges on a better and broader understanding of the process, especially when related to diseases\' progression. Using RNA-seq and a well accepted animal model of angiogenesis, the murine model of Oxygen Induced Retinopathy, we have explored the transcriptome landscape and identified 153 genes differentially expressed in angiogenesis. An extensive validation of several genes carried out by qRT-PCR and in-situ hybridization confirmed Esm1 overexpression in endothelial cells of tissues with active angiogenesis, providing confidence on the results obtained. Enrichment analysis of this gene list endorsed a narrow link of angiogenesis and frequently mutated genes in tumours, consistent with the known connection between cancer and angiogenesis, and provided suggestions of already approved drugs that may be repurposed to control angiogenesis under pathological circumstances. Finally, based on this comprehensive landscape of angiogenesis, we were able to create a prognostic molecular biomarker for prediction of breast cancer relapse, with promising clinical applications. In summary, this work successfully unveiled angiogenesis-related genes, providing novel therapeutic alternatives, including potential drugs for repositioning. The set of differentially expressed genes is also a valuable resource for further investigations.

Náplní této práce je teoretický úvod a následné praktické zpracování tématu Molekulární signatura jako optimální multi-objektivní funkce s aplikací v predikci v onkogenomice. Úvodní kapitoly jsou zaměřeny na téma rakovina, zejména pak rakovina prsu a její podtyp triple negativní rakovinu prsu. Následuje literární přehled z oblasti optimalizačních metod, zejména se zaměřením na metaheuristické metody a problematiku strojového učení. Část se odkazuje na onkogenomiku a principy microarray a také na statistiku a s důrazem na výpočet p-hodnoty a bimodálního indexu. Praktická část je pak zaměřena na konkrétní průběh výzkumu a nalezené závěry, vedoucí k dalším krokům výzkumu. Implementace vybraných metod byla provedena v programech Matlab a R, s využitím dalších programovacích jazyků a to konkrétně programů Java a Python.

Thesis (Ph. D.)--Ohio State University, 2004.
Title from first page of PDF file. Document formatted into pages; contains x, 117 p.; also includes graphics (some col.) Includes bibliographical references (p. 99-117). Available online via OhioLINK's ETD Center

Theoretical part of this diploma thesis gives a brief characteristic of human mobile elements (transposons), which represents nearly 50% of human genome. It provides basic transposon clasification and describes types of transposons present in hunam genome, as well as mobilization, activation and regulation mechanisms. The work also deals with the domestication of transposons, describes the ways in which TE contribute to DNA damage and summarizes the diseases caused by mutagenic activity of transposons in the human genome. Conclusion of theoretical part describes next-generation sequencing technologies (NGS). As practical part, data from RNA-seq experimet were analyzed in order to compare differen transposon activity in normal and cancer cells from prostate and colorectal tissues. As like as publicly available sophisticated tools (TopHat), new scripts were created to analyze these data. The results show that cancer cells exhibit overexpression of transposons. This corresponds with the published results and suggests a connection of transposon activation with cancer development.

Las enfermedades complejas se encuentran entre las más comunes y son causadas por una combinación de factores genéticos y ambientales (contaminación ambiental, estilo de vida, etc). Entre las enfermedades complejas que se pueden destacar se encuentran la obesidad, el asma, la hipertensión o la diabetes. Diversos estudios científicos sugieren que el hecho de padecer enfermedades complejas está condicionado a la aparición o acumulación de determinados factores ambientales. Asimismo, se ha descrito que los factores ambientales son unos de los principales contribuyentes a la carga mundial de morbilidad. Todo esto nos lleva a definir el término exposoma como el conjunto de factores ambientales a los que un individuo se ve expuesto desde la concepción hasta la muerte. El estudio de la mecánica subyacente que vincula el exposoma con la salud es un campo de investigación emergente con un fuerte potencial para proporcionar nuevos conocimientos sobre la etiología de las enfermedades. La primera parte de esta tesis se centra en la exposición a la radiación ultravioleta. La exposición a la radiación ultravioleta proviene de fuentes tanto naturales como artificiales. La radiación ultravioleta incluye tres subtipos de radiación según su longitud de onda (UVA 315-400 nm, UVB 315-295 nm y UVC 295-200 nm). Si bien la principal fuente natural de radiación ultravioleta es el Sol, la UVC no llega a la superficie de la Tierra debido a su absorción por la capa estratosférica de ozono. En consecuencia, la exposición a radiación ultravioleta a la que estamos usualmente sometidos consisten en una mezcla de UVA (95 %) y UVB (5 %). Los efectos de la radiación ultravioleta en humanos pueden ser beneficiosos o perjudiciales dependiendo de su cantidad y forma. Los efectos perjudiciales y agudos de la radiación ultravioleta incluyen eritema, oscurecimiento del pigmento, retraso en el bronceado y engrosamiento de la epidermis. Repetidas lesiones en la piel producidas por radiación ultravioleta pueden predisponer, en última instancia, a efectos crónicos de fotoenvejecimiento, inmunosupresión y fotocarcinogénesis. El mayor efecto beneficioso de la radiación ultravioleta es la síntesis cutánea de la vitamina D. La vitamina D es necesaria para mantener el calcio fisiológico y del fósforo para la mineralización ósea y para prevenir el raquitismo, la osteomalacia y la osteoporosis. El paradigma del exposoma es trabajar con múltiples exposiciones a la vez en vez centrarse en una sola exposición. Este enfoque permite tener una visión más parecida a la realidad que vivimos. Luego, la segunda parte se centra en las herramientas para explorar cómo caracterizar y analizar el exposoma y cómo probar sus efectos en múltiples capas biológicas intermedias para proporcionar información sobre los mecanismos moleculares subyacentes que vinculan las exposiciones ambientales a los resultados de salud.
Most common diseases are caused by a combination of genetic, environmental and lifestyle factors. These diseases are referred to as complex diseases. Examples of this type of diseases are obesity, asthma, hypertension or diabetes. Several empirical evidence suggest that exposures are necessary determinants of complex disease operating in a causal background of genetic diversity. Moreover, environmental factors have long been implicated as major contributors to the global disease burden. This leads to the formulation of the exposome, that contains any exposure to which an individual is subjected from conception to death. The study of the underlying mechanics that links the exposome with human health is an emerging research field with a strong potential to provide new insights into disease etiology. The first part of this thesis is focused on ultraviolet radiation (UVR) exposure. UVR exposure occurs from both natural and artificial sources. UVR includes three subtypes of radiation according to its wavelength (UVA 315-400 nm, UVB 315-295 nm, and UVC 295-200 nm). While the main natural source of UVR is the Sun, UVC radiation does not reach Earth's surface because of its absorption by the stratospheric ozone layer. Then, exposures to UVR typically consist of a mixture of UVA (95%) and UVB (5%). Effects of UVR on human can be both beneficial and detrimental, depending on the amount and form of UVR. Detrimental and acute effects of UVR include erythema, pigment darkening, delayed tanning and thickening of the epidermis. Repeated UVR-induced injury to the skin, may ultimately predispose one to the chronic effects photoaging, immunosuppression, and photocarcinogenesis. The beneficial effect of UVR is the cutaneous synthesis of vitamin D. Vitamin D is necessary to maintain physiologic calcium and phosphorous for normal bone mineralization and to prevent rickets, osteomalacia, and osteoporosis. But the exposome paradigm is to work with multiple exposures at a time and with one or more health outcomes rather focus in a single exposures analysis. This approach tends to be a more accurate snapshot of the reality that we live in complex environments. Then, the second part is focused on the tools to explore how to characterize and analyze the exposome and how to test its effects in multiple intermediate biological layers to provide insights into the underlying molecular mechanisms linking environmental exposures to health outcomes.

Les cancers du sein triple négatifs (CSTN) sont un sous-type hétérogène représentant 12% à 24% de tous les cancers du sein. Ils ont les plus mauvais pronostics et touchent souvent les femmes jeunes. Le traitement au stade localisé repose essentiellement sur la chimiothérapie, sans thérapie ciblée (hors cas de mutations germinales de BRCA). Quasiment toutes les patientes reçoivent la même chimiothérapie néoadjuvante (CNA) avec anthracyclines et taxanes, ce qui grève leur survie en l’absence de réponse complète pathologique (RCp). L’intensification thérapeutique est la tendance actuelle, notamment avec l’addition d’immunothérapie, afin d’améliorer taux de RCp et survie. Les signatures d’expression génique standard ne pas utilisables en pratique clinique pour prédire la chimiorésistance des CSTNs à la CNA, alors qu’elles permettraient de sélectionner les patientes pour une intensification thérapeutique. D’où l’intérêt d’explorer les transcrits issus des 90% du génome restant, constitué de régions non codantes et non référencées. Parmi eux, les ARNs longs non-codant (lnc) qui se caractérisent par une longueur d’au moins 200 nucléotides présentent l’intérêt pour certains d’entre eux d’avoir une expression tissu- voire cancer- spécifique. De plus, certains ARNs lnc ont un rôle démontré dans différents mécanismes de chimiorésistance. Les ARNs lnc ne sont cependant pas bien annotés dans le génome humain et de nouveaux transcrits non référencés, codant ou non, et de nouvelles isoformes de gènes connus, sont découverts quotidiennement.Mon 1er objectif de thèse était d’évaluer le transcriptome non référencé en tant que réservoir potentiel de biomarqueurs prédictifs de chimiorésistance des CSTN à la CNA. Nous avons analysé une cohorte de 78 CSTNs avant CNA, et comparé les cas chimiosensibles (chS) et chimiorésistants (chR) selon le score RCB (Residual Cancer Burden). Nous avons comparé l’analyse d’expression génique différentielle standard (DE-seq) sur les gènes annotés, et sur de nouveaux ARNs lnc détectés grâce à un profiler ARN, et une analyse non biaisée par les annotations génomiques, de fragments de transcrits différentiels. L’analyse non biaisée a permis d’obtenir la meilleure séparation des cas chS et chR dans notre cohorte exploratoire. Nous avons ensuite évalué cette approche sur une cohorte indépendante, et nous l’avons optimisée, en considérant les régions génomiques d’expression différentielle. Ceci nous a permis d’obtenir une signature reproductible entre les deux cohortes. Au total, ces résultats montrent le potentiel d’une approche non référencée pour générer une signature de chimiorésistance précoce des CSTN. Des validations supplémentaires sont nécessaires sur des cohortes plus larges.Mon 2nd objectif de thèse était d’évaluer les ARNs lnc en tant que potentiels acteurs/cibles thérapeutiques dans les CSTNs chR. Nous avons sélectionné les ARNs lnc surexprimés dans les CSTNs chR pre-CNA (versus les CSTNs chS pre-CNA) et dans les CSTNs chR post-CNA (versus les CSTNs chR pre-CNA). En intégrant leurs niveaux et spécificités d’expression, leurs localisations génomiques et les données préexistantes suggérant une fonction potentielle, nous avons retenu trois ARNs lnc (AL450326.1, LINC02609 et MIR503HG). Nous avons évalué l’impact de l’inactivation de leur expression sur la cytotoxicité du Docetaxel dans un modèle de lignée cellulaire de CSTN. L’inactivation de l’expression de chacun des trois ARNs lnc a induit une cytotoxicité accrue du Docetaxel. Une clonogénicité spontanée diminuée et une mort cellulaire accrue sous Docetaxel ont de plus été observées après la déplétion d’AL450326.1 et de LINC02609. Au total, nous avons identifié trois ARNs lnc jouant un rôle dans la chimiorésistance des CSTN. Des tests fonctionnels supplémentaires sont nécessaires pour en décrypter les mécanismes, avec pour objectif à long terme d’identifier de nouvelles approches thérapeutiques contrant la chimiorésistance des CSTNs à la CNA
Triple-negative breast cancers (TNBC) represent a heterogeneous subtype of breast cancers including 12% to 24% of all cases, having the poorest prognoses and often affecting young women. Treatment at localized stage is mainly based on chemotherapy, with no targeted therapy (except germline BRCA mutated patients). Nearly all patients receive the same Neo-Adjuvant Chemotherapy (NAC) with anthracyclines and taxanes, that badly impacts survival in the absence of pathological complete response (pCR). Therapeutic intensification, notably with addition of immunotherapy, is the current trend to increase pCR rate and improve survival. Standard gene expression signatures have failed to provide effective tools to predict TNBC chemoresistance, probably due to their incomplete nature, as they are mostly based on expression of protein coding genes and/or referenced transcripts and up to date there is no clinically useful transcriptomic signature predicting TNBC chemoresistance to NAC. Such a predictive signature would allow patient selection for therapeutic intensification. Therefore, it is important to explore the remaining 90% of the genome consisting of non-coding and non-referenced regions. One class of non-coding RNAs that is of great interest are long non-coding (lnc) RNAs, that are at least 200 nucleotides long, some of them being specifically expressed in cancer. Moreover, some lncRNAs have been shown to be implicated in different mechanisms of chemoresistance. LncRNAs are not fully well annotated in the human genome and new unreferenced transcripts, coding or not, and new isoforms of known genes are discovered daily.Therefore, the first goal of my PhD was to assess reference-free transcriptome as a potential reservoir of predictive biomarkers of TNBC chemoresistance. A cohort of 78 TNBCs before NAC was analyzed, comparing chemosensitive (chS) and chemoresistant (chR) cases based on international Residual Cancer Burden (RCB) score. A standard differential gene expression analysis (DE-seq) on annotated genes, and on new lncRNAs detected with a de novo RNA-profiler, and a reference-free analysis of differential fragments of transcripts without annotation bias were compared. Reference-free approach showed best separation of chS and chR patients in the training cohort. Further, based on comparison with an independent validation cohort, an optimized approach was proposed, where specific genomic regions with differential expression were selected. This technique gave a reproducible signature of chemoresistance between the two cohorts. In all, these results show the potential of a reference-free approach to generate a transcriptomic signature as predictive biomarker of early TNBC chemoresistance. Further investigation is needed to validate the signature using larger validation cohorts.The second objective of my PhD was to assess lncRNAs as potential actors/therapeutic targets in chR TNBCs. For that we selected lncRNAs upregulated in chR pre-NAC TNBCs (compared with chS pre-NAC TNBCs) and in chR post-NAC TNBCs (compared with chR pre-NAC TNBCs). Considering lncRNAs level and specificity of expression, genomic position, and pre-existing data of their potential function, three lncRNAs (AL450326.1, LINC02609 and MIR503HG) were retained for functional analysis. By knocking down levels of these lncRNAs in TNBC cell line model, an impact on Docetaxel cytotoxicity was assessed. All three lncRNAs knock downs showed an improved Docetaxel induced cytotoxicity. Knock down of AL450326.1 and LINC02609 resulted in a decreased spontaneous clonogenicity and increased Docetaxel induced cell death, giving a first indication of their mode of action. In all, we identified three lncRNAs playing a role in NAC chemoresistance. Further functional studies will allow to decipher the mechanisms by which the identified lncRNAs affect chemoresistance with the ultimate goal to identify new therapeutic approaches to circumvent NAC chemoresistance of TNBCs

Les progrès technologiques en séquençage haut débit et en manipulation cellulaire permettent d'analyser simultanément et indépendamment le contenu de nombreuses cellules (ARN, ADN,...). Cette révolution "omique" offre un nouveau cadre pour revisiter la "Théorie Cellulaire", essentiellement basée sur des caractéristiques morphologiques et fonctionnelles. Les nombreuses modalités cellulaires désormais accessibles au niveau de la cellule unique, telles que leur transcriptome, leur localisation spatiale, leurs trajectoires développementales, enrichissent considérablement cette définition, et établissent un contexte totalement renouvelé pour réévaluer la définition de "types" ou d’"états" cellulaires ainsi que leurs interactions. \Mon travail de thèse a été de mettre en place des approches statistiques appropriées pour analyser ces données transcriptomiques sur cellule unique caractérisées par une forte variance, la présence d'un pourcentage élevé de valeurs nulles et un grand volume de données. Mon travail s’est focalisé sur le modèle expérimental central de mon laboratoire d’accueil, l'épithélium des voies respiratoires humaines. Les voies respiratoires humaines sont bordées d'un épithélium pseudo-stratifié composé principalement de cellules basales, sécrétrices, à gobelet et multiciliées. Les voies respiratoires constituent en outre un véritable écosystème cellulaire, dans lequel la couche épithéliale interagit étroitement avec les cellules immunitaires et mésenchymateuses. Cette coordination entre les cellules assure une bonne défense du système respiratoire et sa correcte régénération en cas d'agressions extérieures. Une meilleure compréhension des situations cellulaires normales et pathologiques peut améliorer les approches pour lutter contre des pathologies telles que la maladie pulmonaire obstructive chronique, l'asthme ou la mucoviscidose.J'ai d'abord pu caractériser au niveau de la cellule unique la séquence précise et spécifique des événements conduisant à la régénération fonctionnelle de l'épithélium, en utilisant un modèle 3D de cellules humaines. J'ai identifié des hiérarchies de lignées cellulaires et j'ai pu reconstruire les différentes trajectoires possibles de différentiation cellulaire. J'ai confirmé des trajectoires cellulaires décrites précédemment, mais j'ai aussi découvert une nouvelle trajectoire reliant les cellules à gobelet aux cellules multiciliées, identifiant de nouvelles populations cellulaires et de nouvelles interactions moléculaires impliquées dans le processus de régénération de l'épithélium sain des voies aériennes humaines. J'ai ensuite construit un atlas des différents types cellulaires qui tapissent les voies respiratoires humaines saines, du nez jusqu’à la 12ième génération de bronches. Le profilage de 10 volontaires sains a généré un ensemble de données de 77 969 cellules, provenant de 35 emplacements distincts, qui comprend plus de 26 types cellulaires épithéliaux, immunitaires et mésenchymateuses. Cet atlas illustre l'hétérogénéité cellulaire présente dans les voies respiratoires. Son analyse révèle une différence d'expression des gènes entre le nez et les voies respiratoires pulmonaires que j’ai caractérisé dans les cellules suprabasales, sécrétrices et multiciliées. Mes travaux ont également permis d'améliorer la caractérisation de certaines populations de cellules rares, comme les cellules "hillock", déjà décrites chez la souris. En conclusion, mon travail contribue à une meilleure compréhension des dynamiques de différenciation et d'hétérogénéité cellulaire dans les voies respiratoires humaines saines. La ressource ainsi constituée sera extrêmement utile dans tout projet futur visant à analyser avec précision les conditions spécifiques des maladies respiratoires
Improvements made in nucleic acid sequencing and cell handling technologies now offer the opportunity to analyze simultaneously the content of numerous single cells (RNA, DNA, ...) by global and unbiased approaches. This single-cell ‘omics’ revolution provides a new framework to revisit the “Cell Theory”, elaborated over several centuries, and essentially based on morphological and functional features. The many cell modalities now accessible at single- cell level, such as their transcriptome, spatial localization, developmental trajectories, enrich considerably this definition, and set a renewed context to precisely reassess the definition of ‘cell types’, ‘cell states’ as well as their different interactions and fates.My thesis work initially set up ad hoc approaches and statistical framework to analyze appropriately these single-cell data, which deeply differ from standard bulk RNA-seq. High variance, presence of a huge percentage of null values, large volume of data are among the specific characteristics of these datasets. My work was centered on the main experimental model of my host laboratory, e.g. the human airway epithelium. Human airways are lined by a pseudostratified epithelium mainly composed of basal, secretory, goblet and multiciliated cells. Airways also constitute a true cellular ecosystem, in which the epithelial layer interacts closely with immune and mesenchymal cells. This coordination between cells ensures proper defense of the respiratory system and its correct regeneration in case of external aggression and injuries. A better understanding of the operating sequences in normal and physiopathological situations is relevant in pathologies such as chronic obstructive pulmonary disease, asthma or cystic fibrosis.First, I characterized at a single cell level the precise and cell-specific sequence of events leading to functional regeneration of the epithelium, using a 3D model of human cells. I then built a single-cell atlas of the different cell types that are lining healthy human airways from the nose to the 12th generation of bronchi.By applying computational and statistical approaches, I have identified cell lineage hierarchies and was able to reconstruct a comprehensive cell trajectory roadmap in human airways. I not only confirmed previously described cell lineages, but I have also discovered a novel trajectory that links goblet cells to multiciliated cells, identifying novel cell populations and molecular interactors involved in the process of healthy human airway epithelium regeneration. The profiling of 12 healthy volunteers then generated a dataset of 77,969 cells, derived from 35 distinct locations. The resulting atlas is composed of more than 26 epithelial, immune and stromal cell types demonstrating the cellular heterogeneity present in the airways. Its analysis has revealed a strong proximo-distal gradient of expression in suprabasal, secretory, or multiciliated cells between the nose and lung airways. My work has also improved the characterization of rare cells, including “hillock” cells that have been previously described in mice.In conclusion, this work probably represents one of the first single-cell investigations in human airways. It brings original contributions to our understanding of differentiation’s dynamics and cellular heterogeneity in healthy human airways. The resulting resource will be extremely useful for any future single-cell investigators and also for establishing a very useful joint between clinical and biological works. As such, it will constitute a reference in any future project aiming to precisely analyze specific disease conditions

CCCTC-binding factor (CTCF) binds DNA, thereby helping to partition the mammalian genome into discrete structural and regulatory domains. In doing so, it insulates chromatin and fine-tunes gene activation, repression, and silencing. Complete removal of CTCF from mammalian cells causes catastrophic genomic dysregulation, most likely due to widespread collapse of 3D chromatin looping within the nucleus. In contrast, Ctcf hemizygous mice with lifelong reduction in CTCF expression are viable but have an increased incidence of spontaneous multi-lineage malignancies. In addition, CTCF is mutated in many human cancers and is thus implicated as a tumour suppressor gene. This study aimed to interrogate the genome-wide consequences of a reduced genomic concentration of Ctcf and its implications for carcinogenesis. In a genetically engineered mouse model, Ctcf hemizygous cells showed modest but robust changes in almost a thousand sites of genomic CTCF occupancy; these were enriched for lower affinity binding events with weaker evolutionary conservation across the mouse lineage. Furthermore, several hundred genes concentrated in cancer-related pathways were dysregulated due to changes in transcriptional regulation. Global chromatin structure was preserved but some loop interactions were destabilised, often around differentially expressed genes and their enhancers. Importantly, these transcriptional alterations were also seen in human cancers. These findings were then examined in a hepatocyte-specific mouse model of Ctcf hemizygosity with diethylnitrosamine-induced liver tumours. Ctcf hemizygous mice had a subtle liver-specific phenotype, although the overall tumour burden in Ctcf hemizygous and wild-type mice was the same. Using whole genome sequencing, the highly reproducible mutational signature caused by DEN exposure was characterised, revealing that Braf(V637E), orthologous to BRAF(V600E) in humans, was the predominant oncogenic driver in these liver tumours. Taken together, while Ctcf loss is partially physiologically compensated, chronic CTCF depletion dysregulates gene expression by subtly altering transcriptional regulation. This study also represents the first comprehensive genome-wide and histopathological characterisation of this commonly used liver cancer model.

Par sa capacité à capturer la diversité complète des transcrits produits par chaque cellule, la technologie de séquençage d’ARN à haut-débit (RNA-seq) a révolutionné notre vision de l’expression des gènes. Les données RNA-seq sont de plus en plus utilisées en médecine de précision afin d'établir les profils moléculaires des tumeurs, ou pour étudier des réseaux de gènes régissant l'adaptation d'une cellule à son environnement. Cependant, l’analyse RNA-seq qui classiquement se base sur la comparaison avec des séquences géniques de référence, est incapable d'identifier une grande part des ARN aberrants produits dans les maladies par altération du génome ou des processus de maturation. Notre projet vise à exploiter un nouveau concept pour l'analyse du transcriptome fondé sur des "étiquettes", ou k-mers, représentant l'intégralité des variations de séquences observées dans un transcriptome. Nous avons appliqué ce concept à la découverte de signatures diagnostiques ou pronostiques à partir de données RNA-seq du cancer de la prostate. A cette fin, nous avons appliqué différentes méthodes de réduction de dimension et de sélection de variable utilisées dans l'analyse transcriptomique classique. En raison de la très grande dimension des matrices de k-mers, ces méthodes ont nécessité des adaptations afin de réduire de manière drastique le nombre de variables à analyser. Nous sommes parvenu à établir un protocole informatique capable de réduire efficacement une matrice de k-mers issue du séquençage de plusieurs centaines de transcriptomes. A l'aide de ce protocole, nous avons pû produire de nouvelles signatures diagnostiques et pronostiques pour le cancer de la prostate. Ces signatures "sans référence" ne nécessitent pas de connaissance a priori sur le génome ou le transcriptome humain et sont au moins aussi performantes que les signatures géniques conventionnelles. De plus ces signatures contiennent des séquences d'ARN jamais identifiées, correspondant notamment à des variants d'ARNm ou à de nouveaux longs ARN non-codants qui pourront orienter les biologistes vers de nouveaux mécanismes d'oncogénèse
With its ability to capture the full diversity of transcripts produced by each cell, high-throughput RNA sequencing (RNA-seq) has revolutionized our view of gene expression. RNA-seq data are increasingly used in precision medicine to establish the molecular profiles of tumors, or to study gene networks governing the adaptation of a cell to its environment. However, RNA-seq analysis, which is conventionally based on comparison with reference gene sequences, is unable to identify a large fraction of abnormal RNA transcripts produced in disease tissues, through defects in the genome or in RNA processing. Our project aims to exploit a new concept for the analysis of transcriptomes based on short sequence labels, or k-mers, representing all of the sequence variations observed in a given transcriptome dataset. We applied this concept to the discovery of diagnostic or prognostic signatures from RNA-seq data of prostate cancer. To this end, we applied different dimension reduction and variable selection methods used in classical transcriptomic analysis. Due to the very large dimension of the k-mer matrices, these methods required specific adaptations in order to drastically reduce the number of variables to be analyzed. We established a computer pipeline capable of effectively reducing a k-mer matrix obtained from the sequencing of several hundred transcriptomes. Using this pipeline, we were able to produce new diagnostic and prognostic signatures for prostate cancer. These "reference-free" signatures do not require a priori knowledge of the human genome or transcriptome and are at least as effective as conventional gene signatures. In addition, these signatures contain novel RNA sequences corresponding to mRNA variants or new long non-coding RNAs. These novel RNAs involved in cancer risk may orient biologists towards new oncogenesis mechanisms

Ces dernières années, l’émergence des approches en cellules uniques (scRNA-seq) a favorisé la caractérisation de l’hétérogénéité cellulaire avec une précision inégalée. Malgré leur démocratisation, l’analyse de ces données reste complexe, en particulier pour les organismes dont les annotations sont incomplètes. Au cours ma thèse, j’ai observé que les annotations génomiques du poulet sont lacunaires, ce qui engendre la perte d’un grand nombre de lectures de séquençage. J’ai évalué à quel point une annotation améliorée affecte les résultats biologiques et les conclusions issues de ces analyses. Nous proposons une nouvelle approche basée sur la ré-annotation du génome à partir de données scRNA-seq et de RNA-seq bulk en lectures longues. Ce projet de biologie computationnelle s’appuie sur une étroite collaboration avec l’équipe expérimentale de Xavier Morin (IBENS). Le principal objectif biologique est la recherche de signatures de mode de division symétrique et asymétrique au sein de progéniteurs neuronaux. Afin d’identifier les principaux changements transcriptionnels, j’ai mis en place des approches dédiées à la recherche de signatures géniques à partir de données scRNA-seq
In recent years, single-cell RNA-seq (scRNA-seq) has fostered the characterization of cell heterogeneity at a remarkable high resolution. Despite their democratization, the analysis of scRNA-seq remains a challenge, particularly for organisms whose genomic annotations are partial. During my PhD, I observed that the chick genomic annotations are often incomplete, thus resulting in a loss of a large number of sequencing reads. I investigated how an enriched annotation affects the biological results and conclusions from these analyses. We developed a novel approach based on the re-annotation of the genome with scRNA-seq data and long reads bulk RNA-seq. This computational biology project capitalises on a tight collaboration with the experimental team of Xavier Morin (IBENS). The main biological focus is the search for signatures of symmetric versus asymmetric division mode in neural progenitors. In order to identify the key transcriptional switches that occur during the neurogenic transition, I have implemented bioanalysis approaches dedicated to the search for gene signatures from scRNA-seq data

Helicobacter pylori (H. pylori) est une bactérie qui infecte l’estomac et induit une gastrite inflammatoire, qui peut être chronique et évoluer vers un cancer gastrique. La sévérité de l’infection et son évolution clinique sont associées aux différents facteurs notamment le statut immunitaire de l’hôte. La réponse inflammatoire initiale à l'infection à H. pylori entraîne la sécrétion d'un large panel de cytokines, notamment l'interleukine-1β (IL-1β), l'IL-8 et l'IL-17A. qui semblent jouer un rôle clé dans l'initiation et la progression du cancer gastrique. Parmi ces cytokines, l'IL-1β est une cytokine clé au cours de l’infection à H. pylori dont l’expression est étroitement associée à l'inflammation gastrique et à la carcinogenèse. La production de cette cytokine dépend de l'activation de l'inflammasome, en particulier l'inflammasome NLRP3. Ce dernier, responsable de l’activation des processus inflammatoires, est essentiel pour le maintien de l'homéostasie contre diverses infections pathogènes telles les infections bactérienne et virale.L’objectif général de ce travail est i) d’étudier l’expression et le polymorphisme des gènes de cytokines comme IL-1β, IL-17 et IL-8 chez des patients marocains infectés par H. pylori. ii) explorer l’activation de l'inflammasome NLRP3 par H. pylori et déterminer les mécanismes impliqués dans l'activation de ce complexe par des virus à ARN ; connus comme des activateurs définis de NLRP3.Nos résultats ont souligné une prévalence élevée de H. pylori et ont mis en évidence une signature cytokinique : elle peut prédire la métaplasie au cours de la progression de l'infection à H. pylori impliquant une diminution de l’expression de l'IL17A dans l’antre et une augmentation de l’expression de l'IL-1β dans le fundus. Plus particulièrement, les polymorphismes génétiques de l’IL-1β (IL-1β -31 et -511) ne semblent pas influencer l’expression de l’IL-1β de manière significative.Au regard des difficultés rencontrés pour l’isolement et la culture de H. pylori, nous avons utilisé le LPS de H. pylori pour stimuler l’inflammasome. Nos résultats montrent que la transfection des cellules in vitro par le LPS bactérien induit la production de l’IL-1β qui semble être modulée par la caspase 4, NOD1 et NOD2. Par ailleurs, bien qu’il soit clairement établi que les virus à ARN induisent l’activation de l’inflammasome NLRP3, les mécanismes par lesquels ces virus induisent la production d'IL-1β ne sont pas bien compris et restent à confirmer. Les résultats de cette partie du travail ont montré que la réplication des virus à ARN cytopathogènes tels que le virus de la stomatite vésiculaire (VSV) ou le virus de l'encéphalomyocardite (EMCV) induit une mort cellulaire lytique conduisant à un efflux de potassium qui déclenche l'activation de l'inflammasome NLRP3. Ainsi, les virus à forte capacité de réplication et qui ont un effet cytopathique sont capables d'induire l'activation de la caspase-1 conduisant à la production d'IL-1β. A l'inverse, les virus qui induisent une très bonne réponse IFN de type I sont de très mauvais inducteurs de l'inflammasome NLRP3.Une meilleure compréhension de l’activation de l’inflammasome pourrait aider dans la mise au point de stratégies thérapeutiques ciblées utilisables dans la lutte contre les infections bactérienne et virale.Mots clés : Helicobacter pylori, inflammation, inflammasome NLRP3, IL-1β, virus à ARN
Helicobacter pylori (H. pylori) is a bacteria that infects the stomach and induces inflammatory gastritis, which can be chronic and progress to gastric cancer. The severity of the infection and its clinical course are associated with various factors including the immune status of the host. The initial inflammatory response to H. pylori infection results in the secretion of a wide range of cytokines, including interleukin-1β (IL-1β), IL-8 and IL-17A. which appear to play a key role in the initiation and progression of gastric cancer. Among these cytokines, IL-1β is a key cytokine during H. pylori infection whose expression is associated with gastric inflammation and carcinogenesis. The production of this cytokine depends on the activation of the inflammasome, in particular the NLRP3 inflammasome. The latter, responsible of the activation of inflammatory processes, is essential for the maintenance of homeostasis against various pathogenic infections such as bacterial and viral infections.The general objective of this work is i) to study the expression and polymorphism of genes for cytokines such as IL-1β, IL-17 and IL-8 in Moroccan patients infected with H. pylori. ii) explore the activation of the NLRP3 inflammasome by H. pylori and determine the mechanisms involved in the activation of this complex by RNA viruses; known as defined activators of NLRP3.Our results underlined a high prevalence of H. pylori and demonstrated a cytokine signature: it can predict metaplasia during the progression of H. pylori infection involving a decrease in IL17A expression in the antrum and increased expression of IL-1β in the fundus. In particular, the genetic polymorphisms of IL-1β (IL-1β -31 and -511) do not appear to influence IL-1β expression significantly.In view of the difficulties encountered in isolating and culturing H. pylori, we used LPS from H. pylori to stimulate the inflammasome. Our results show that the transfection of cells in vitro with bacterial LPS induces the production of IL-1β which appears to be modulated by caspase 4, NOD1 and NOD2. Furthermore, while it is clearly established that RNA viruses induce activation of the NLRP3 inflammasome, the mechanisms by which these viruses induce IL-1β production are not well understood and remain to be confirmed. The results of this part of the work showed that the replication of cytopathogenic RNA viruses such as vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) induces lytic cell death leading to an efflux of potassium which triggers activation of the NLRP3 inflammasome. Thus, viruses with a high replication capacity and which have a cytopathic effect are capable of inducing the activation of caspase-1 leading to the production of IL-1β. Conversely, viruses which induce type I IFN response are very poor inducers of the NLRP3 inflammasome.A better understanding of the activation of the inflammasome could help in the development of targeted therapeutic strategies for use in the fight against bacterial and viral infections.Key words: Helicobacter pylori, inflammation, NLRP3 inflammasome, IL-1β, RNA virus

Dissertação de Mestrado em Biologia Celular e Molecular apresentada à Faculdade de Ciências e Tecnologia
Os linfócitos T reguladores (Tregs) desempenham um papel crucial na manutenção da homeostasia imunológica, impedindo ou limitando respostas imunes. As Tregs são particularmente eficazes a suprimir as células T convencionais (Tconvs), e desta forma limitam a imunopatologia associada à imunidade contra patogéneos e células cancerígenas, bem como os processos alérgicos, autoimunes e inflamatórios. Uma população significativa de Tregs é gerada durante o desenvolvimento das células T no timo conhecidas como Tregs naturais (ou derivadas do timo), sendo definidas pela presença da proteína FOXP3. Este fator de transcrição desempenha um papel crucial na diferenciação destas células, não só através da repressão de genes normalmente expressos em Tconvs, mas também promovendo a ativação de genes específicos de Tregs, incluindo IL2RA (CD25) e CTLA4. No entanto, vários estudos demostraram que a diferenciação e comprometimento destas células pode ser independente de FOXP3, indicando a existência de outros fatores, atualmente desconhecidos, os quais são suficientes para promover o desenvolvimento das Tregs. Neste estudo, investiguei a diferenciação e o comprometimento das células Treg no timo humano, a fim de identificar novos fatores potencialmente envolvidos nesta decisão. Para isso, isolámos Tregs e Tconvs tímicas maduras CD4 positivas com base na expressão dos marcadores CD27, CD25 e CD127, a partir de tecido tímico humano removido durante cirurgias cardíacas pediátricas corretivas de três indivíduos, e geramos os seus respetivos perfis de expressão génica através da sequenciação do RNA (RNA-seq).A nossa análise da comparação da transcrição identificou 1047 genes significativamente e diferencialmente expressos entre tTregs e tTconvs, dos quais 648 com sobre expressão em tTregs. Destes 648 genes, observei a expressão proeminente de alguns genes associados a este tipo celular, incluindo FOXP3, IL2Rα (CD25), CTLA4, TNFRSF4 (OX40), TNFRSF18 (GITR), IKZF2 (HELIOS) e IKZF4 (EOS). Para alem disso, identifiquei um conjunto de 196 genes unicamente expressos em tTreg em comparação com tTconv, alguns dos quais codificam proteínas com conhecida relevância na biologia destas células, incluindo TNFRSF8 (CD30), LRRC32 (GARP) e CCR8, bem como genes cuja função em tTregs é desconhecida, nomeadamente DNAH8 e TNFRSF11A. Enquanto a expressão de DNAH8 pode ser indicativa da formação de sinapses imunológicas, a expressão de TNFRSF11A pode sugerir um mecanismo adicional de supressão através dos quais as tTregs previnem a ativação das tTconvs.Dos genes encontrados sobre expressos nas tTregs em comparação com as tTconvs, 46 codificam fatores de transcrição. Estes incluem alguns já conhecidos como estando diretamente envolvidos na diferenciação destas células, como FOXP3, IKZF2, IKZF4, FOXO1 e NR4A3, bem como fatores de transcrição envolvidos na ativação e diferenciação de Tconvs, como o TBX21, IRF4, STAT4, BATF e RORA. Além disso, identifiquei também membros da via de sinalização NF-kB (REL, RELB e NFKB2), indicando o seu estado ativo durante a diferenciação destas células. Por fim, encontrei um grupo de fatores de transcrição sobre expressos em Tregs e sem funções previamente descritas nestas células, nomeadamente IRF5, ZBTB38, KLF6 e CREB3L2, o que sugere a presença de novas vias de regulação da transcrição envolvidas nos processos de diferenciação e função das células T reguladorasEm conclusão, esta tese apresenta o primeiro perfil de transcrição de Tregs e Tconvs de timo humano, cujas análises serão fundamentais para a compreensão do seu desenvolvimento. Até à data, estes dados permitiram a identificação de novos genes com funções desconhecidas nestes grupos celulares, o que poderá representar fatores adicionais envolvidos na definição das células T no timo. No futuro, estes dados permitirão explorar novas linhas de investigação com o objetivo de esclarecer o desenvolvimento da linhagem Treg no timo humano, bem como ajudar na definição da assinatura de expressão destas células.
Regulatory T cells (Tregs) are key players in maintaining immune homeostasis, by preventing or limiting immune responses. They are particularly efficient in suppressing conventional T cells (Tconvs), and in this way control the immunopathology associated with immunity against pathogens and cancer as well as preventing allergy, autoimmune diseases, and chronic inflammation. An important Treg subset is generated during T cell development in the thymus, known as thymic-derived Treg. It is best defined by the expression of the forkhead box protein FOXP3, a transcription factor that plays a crucial role in Treg cell differentiation by repressing the expression of genes otherwise upregulated in Tconv cells, as well as by promoting to the activation of Treg specific genes, including IL2Rα (CD25) and CTLA4. However, recent studies have shown that Treg commitment may occur independently of FOXP3, indicating that other factors, presently unknown, are sufficient for the generation of Tregs. Here I have investigated the differentiation and commitment of Treg cells in the human thymus, in order to identify novel factors potentially involved in this decision. To do this, we FACS sorted mature CD4 single-positive thymic Tregs (tTregs) and their conventional counterparts (tTconvs) based on the expression of CD27, CD25, and CD127 markers, from three human thymuses collected during pediatric corrective cardiac surgery, and generated their respective genome-wide expression profiles by RNA-seq. We ensure that these thymuses have an immunophenotype representative of all stages of T cell development and consistent with the one described in the literature.Our comparative transcriptomic analysis identified 1047 genes significantly differentially expressed between tTreg and tTconv subsets, with 648 of these up-regulated in tTregs. Amongst these, I observed the prominent expression of Treg-associated genes, including FOXP3, the IL2Rα (CD25), CTLA4, TNFRSF4 (OX40), TNFRSF18 (GITR), IKZF2 (HELIOS) and IKZF4 (EOS). From these, I identified a set of 196 genes that are uniquely expressed in tTreg compared to tTconv, encoding proteins with relevance to Treg biology, such as TNFRSF8, LRRC32, and CCR8, as well as others with no previously reported activity in tTreg cells, as DNAH8 and TNFRSF11A. Whilst DNAH8 expression may be indicative of the formation of immunological synapses, the expression of TNFRSF11A may indicate an additional suppression mechanism by which Tregs prevents Tconv cell activation. From the genes found to be up-regulated in tTreg cells compared to tTconvs, 46 were transcription factors. These include some known to be directly involved in Treg development, such as FOXP3, IKZF2, IKZF4, FOXO1, and NR4A3, as well as transcription factors involved in Tconv cell activation and differentiation, such as TBX21, IRF4, STAT4, BATF and RORA. In addition, several members of the NF-kB pathway (REL, RELB, NFKB2) are also up-regulated in tTregs, indicating the activated state of this pathway during tTreg differentiation. Importantly, a set of transcription factors with no previous reported role in human regulatory T cells, IRF5, ZBTB38, KLF6, and CREB3L2, are overexpressed in tTregs, suggesting additional layers of transcriptional regulation of Treg cell differentiation and function. Altogether, this thesis presents the first transcriptomic profile of the human thymic Treg and Tconv subsets, which analyses are absolutely necessary to the understanding of their development. So far, they allowed the identification of novel genes with unreported functions in these subsets, which might represent additional factors involved in the definition of thymic T cells; in the near future, these data will open several new lines of research aiming to clarify the pathways of Treg lineage commitment in the human thymus and will help in the definition of the expression signature of human tTreg subset.
Outro - This work is supported by grants PAC-PRECISE-LISBOA-01-0145-FEDER-016394 and LISBOA-01-0145-FEDER-007391 cofunded by FEDER through POR Lisboa 2020 - Programa Operacional Regional de Lisboa PORTUGAL 2020 and Fundação para a Ciência e a Tecnologia (FCT); and by funds from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No.: 675395.

Huntington's disease (HD) and Parkinson's disease (PD) are devastating neurodegenerative disorders that are characterized pathologically by degeneration of neurons in the brain and clinically by loss of motor function and cognitive decline in mid to late life. The cause of neuronal degeneration in these diseases is unclear, but both are histologically marked by aggregation of specific proteins in specific brain regions. In HD, fragments of a mutant Huntingtin protein aggregate and cause medium spiny interneurons of the striatum to degenerate. In contrast, PD brains exhibit aggregation of toxic fragments of the alpha synuclein protein throughout the central nervous system and trigger degeneration of dopaminergic neurons in the substantia nigra. Considering the commonalities and differences between these diseases, identifying common biological patterns across HD and PD as well as signatures unique to each may provide significant insight into the molecular mechanisms underlying neurodegeneration as a general process. State-of-the-art high-throughput sequencing technology allows for unbiased, whole genome quantification of RNA molecules within a biological sample that can be used to assess the level of activity, or expression, of thousands of genes simultaneously. In this thesis, I present three studies characterizing the RNA expression profiles of post-mortem HD and PD subjects using high-throughput mRNA sequencing data sets. The first study describes an analysis of differential expression between HD individuals and neurologically normal controls that indicates a widespread increase in immune, neuroinflammatory, and developmental gene expression. The second study expands upon the first study by making methodological improvements and extends the differential expression analysis to include PD subjects, with the goal of comparing and contrasting HD and PD gene expression profiles. This study was designed to identify common mechanisms underlying the neurodegenerative phenotype, transcending those of each unique disease, and has revealed specific biological processes, in particular those related to NFkB inflammation, common to HD and PD. The last study describes a novel methodology for combining mRNA and miRNA expression that seeks to identify associations between mRNA-miRNA modules and continuous clinical variables of interest, including CAG repeat length and clinical age of onset in HD.

Les microARN (miARN) sont des petits ARN non-codants pour des protéines qui permettent d’inhiber la traduction d’ARN messagers. Pour obtenir un miARN, un gène de miARN passe à travers une voie de maturation dans laquelle il sera coupé à deux reprises par les enzymes Drosha et Dicer. Pour interagir avec les enzymes, les gènes de miARN possèdent une structure générale en tige-boucle. Cependant, les détails de cette structure sont encore peu connus. L’objectif principal de ce projet était d’établir s’il y a une relation entre l’efficacité de maturation et la dynamique de la structure. Pour cela, l’efficacité de maturation de plusieurs variants de miARN a été évaluée par Northern Blot. La dynamique de la structure a été mesurée par un programme informatique à partir de l’information de la séquence. Une corrélation de 0,74 avec une valeur p de 0,02206 a été obtenue entre les dynamiques et les ratios d’efficacités de maturation de miARN. Cette corrélation est supérieure à celle obtenue basée sur l’énergie libre des structures prédites les plus stables qui n’atteignent pas 0,6. Les mutants de miR128-1 et miR188 ont été découverts comme diminuant la maturation. De plus, les mutants de miR125a, miR188 et miR330 affectent le site de clivage de Drosha. Une meilleure connaissance de la dynamique de l’ARN impliquée dans la maturation permettrait de définir l’impact des mutations dans les séquences de miARN ou encore de prédire les séquences pouvant générer des miARN.
MicroRNAs (miRNAs) are small non-coding RNAs, which can inhibit target messenger RNAs translation. In order to obtain a miRNA, two enzymes, Drosha and Dicer cut the gene of miRNA. The RNA interacts with the proteins by its general hairpin structure. However, the details of the structure are still missing. The objective of this project is to establish if there is a relation between the efficiency of maturation and the RNA’s structural dynamics. In order to do this, the maturation efficiency of miRNA variants is measured by Northern Blot. The structural dynamics is measured by a program assessing the information of the sequence. The correlation between the dynamics and the maturation efficiency of the miRNA is 0.74 with a p-value of 0.02206. This correlation is superior to those based on free energy, which does not reach 0.6. The tested mutants of miR128-1 and miR188 have inhibited maturation; also, those of miR125a, miR188 and miR330 have modified the cleavage site of Drosha. A better knowledge of the dynamic structure involved in maturation would help define the impact of miRNA mutation or to predict sequences that are able to generate miRNAs.

Des évidences expérimentales récentes indiquent que les ARN changent de structures au fil du temps, parfois très rapidement, et que ces changements sont nécessaires à leurs activités biochimiques. La structure de ces ARN est donc dynamique. Ces mêmes évidences notent également que les structures clés impliquées sont prédites par le logiciel de prédiction de structure secondaire MC-Fold. En comparant les prédictions de structures du logiciel MC-Fold, nous avons constaté un lien clair entre les structures presque optimales (en termes de stabilité prédites par ce logiciel) et les variations d’activités biochimiques conséquentes à des changements ponctuels dans la séquence. Nous avons comparé les séquences d’ARN du point de vue de leurs structures dynamiques afin d’investiguer la similarité de leurs fonctions biologiques. Ceci a nécessité une accélération notable du logiciel MC-Fold. L’approche algorithmique est décrite au chapitre 1. Au chapitre 2 nous classons les impacts de légères variations de séquences des microARN sur la fonction naturelle de ceux-ci. Au chapitre 3 nous identifions des fenêtres dans de longs ARN dont les structures dynamiques occupent possiblement des rôles dans les désordres du spectre autistique et dans la polarisation des œufs de certains batraciens (Xenopus spp.).
Recent experimental evidence indicates that RNA structure changes, sometimes very rapidly and that these changes are both required for biochemical activity and captured by the secondary structure prediction software MC-Fold. RNA structure is thus dynamic. We compared RNA sequences from the point of view of their structural dynamics so as to investigate how similar their biochemical activities were by computing a signature from the output of the structure prediction software MC-Fold. This required us to accelerate considerably the software MC-Fold. The algorithmic approach to this acceleration is described in chapter 1. In chapter 2, point mutations that disrupt the biochemical activity of microRNA are explained in terms of changes in RNA dynamics. Finally, in chapter 3 we identify dynamic structure windows in long RNA with potentially significant roles in autism spectrum disorders and separately in Xenopus ssp. (species of frogs) egg polarisation.