Recherche des partenaires moléculaires d’un long ARN non codant, régulé par l’hypoxie, dans les adénocarcinomes pulmonaires

Lung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. It is therefore essential to identify new prognostic markers and new therapeutic targets. My team is interested in gene regulation related to hypoxia, a factor associated with relapse of lung adenocarcinomas (LUAD), the most represented form of lung cancer. While long non-coding RNAs (lncRNA) are increasingly recognized as major gene expression regulators through various mechanisms involving nucleic acid and protein partners, their roles in cancer development and hypoxic response are still largely unexplored. We previously identified a "hypoxaLinc", LincH3 that is significantly up-regulated both in LUAD tumor tissues and cell lines in response to hypoxia. This 10 kb nuclear transcript is regulated by NFB and may participate in the regulation of hypoxic or inflammatory responses. My project focused on the characterization of LincH3 mode of action through the identification of its DNA-binding sites. My goal was to set up a novel technique, the ChIRP (Chromatin Isolation by RNA Purification), based on affinity capture of LncRNA: chromatin complexes by tiling antisense-oligos and mapping of its genomic binding sites at high resolution. Using WT and CRISPR/Cas9 LincH3-deleted LUAD cells, I performed ChIRP-Seq to identify 254 LincH3 binding sites on chromatin. Through transcriptome analysis I showed dysregulation of TGF, NRF2 and NFB signaling pathways in LincH3-deficient LUAD cells. Finally, the comparative analysis of these data identified 11 potential target genes of LincH3 including CTGF, a central regulator of TGFβ and angiogenic pathways

Production de furfural par déshydratation de pentoses hémicellulosiques à l'aide d'un système biphasique réactif

Les biocarburants sont une alternative durable et écologique aux énergies fossiles. Les biocarburants de deuxième génération sont produits à partir de biomasse lignocellulosique, biomasse largement répandue sur terre. En comparaison, la production des biocarburants de première génération est basée sur une biomasse riche en sucres ou en huiles aisément disponibles tels que la canne à sucre ou le colza. Cependant, cette production de biocarburants de première génération est en compétition directe avec le secteur agro-alimentaire. Les procédés de production de biocarburants s’orientent donc actuellement vers la deuxième génération plus disponible et versatile que la première. Ce projet s’inscrit dans cet élan en valorisant les pentoses de la biomasse lignocellulosique en furfural, une molécule plateforme et intermédiaire pour la synthèse de différents produits biosourcés et carburants. Par exemple, l’hydrogénation du furfural produit du 2-méthyltétrahydrofurane, un additif oxygéné pouvant être ajouté à hauteur de 14,5% massique dans l’essence, suivant la réglementation de l’United States Department of Energy. Ce projet vise à optimiser la réaction de déshydratation du xylose en furfural en milieu biphasique à l’aide d’émulsions créées par des ultrasons. En fonction de la nature de la biomasse considérée, le xylane, un polymère d’unité de xylose, peut composer jusqu’à un tier de la plante. Sa valorisation en furfural contribue à la valorisation globale de la biomasse lignocellulosique. En parallèle, l’objectif de ce projet est de fournir les informations nécessaires à la mise au point d’un procédé industrialisable à des rendements en furfural supérieurs à 60% molaire. En effet, le furfural est une molécule très réactive qui polymérise avec ses intermédiaires de réaction. Bien que les rendements obtenus en laboratoire avoisinent les 100%, ceux de production industrielle côtoient les 50%. Cette différence s’explique par une mise en oeuvre difficile des technologies développées au laboratoire comme l’utilisation de catalyseurs ou de solvants coûteux ou encore de temps de résidence démesurément longs. L’innovation de ce projet se base sur l’utilisation d’un mélange biphasique où la phase aqueuse est le siège de la réaction de déshydratation du xylose en furfural et la phase organique agit comme stabilisateur du furfural produit. De plus, la création d’une émulsion augmente la surface d’échange entre les deux phases, permettant ainsi d’extraire le furfural hors de la phase aqueuse avant qu’il ne polymérise. Un traitement préalable aux ultrasons permettra d’émulsionner le mélange réactionnel. Le second point intéressant de ce projet est l’utilisation d’un mélange de solvants organiques verts que sont le carbonate de diméthyle et le méthanol. Par ailleurs, un catalyseur homogène peu coûteux, l’acide sulfurique, est utilisé et recyclé avec la phase aqueuse. Ainsi, ce projet s’inscrit dans l’étude de la mise en place d’un procédé de production industriel de carburant de seconde génération valorisant la totalité de la biomasse lignocellulosique. Plus particulièrement, il se focalise sur la valorisation d’un constituant de la biomasse, le xylose, en furfural en optimisant la réaction de déshydratation dans un système biphasique, émulsionné par ultrasons

Rôle de Pinhead lors de la mise en place de l’axe dorso-ventral chez l’oursin Paracentrotus Lividus

Dorsal-ventral patterning is an important process during development in bilaterians and relies on an interplay between secreted BMP factors and their antagonists produced in two opposite signaling centers. Studies in Xenopus have shown that the dorsal and ventral signaling centers communicate each other to finely regulate the level of BMP signaling by using an autoregulatory expansion-repression circuit based on the activity of an atypical BMP called ADMP, whose transcription is repressed by BMP signaling. When BMP2/4 signaling decreases, admp expression increases and reactivates the BMP signaling pathway. The molecular mechanisms by which BMP signaling represses ADMP expression remain however, largely enigmatic. Recent studies in the tunicate Ciona intestinalis have uncovered a key role for the secreted factor Pinhead in this process. However, whether the function of Pinhead as a key regulator of admp expression is conserved in other species has never been investigated. To test if the role of Pinhead in mediating the repression of admp is conserved in echinoderms, we cloned the sea urchin pinhead and analyzed its spatiotemporal expression. Our results suggest that the positive regulation of pinhead by BMP signaling is not conserved in the sea urchin. We also perturbed the function of pinhead by injecting mRNA encoding Pinhead and antisense morpholino oligonucleotides targeting the pinhead transcript. Our results do not support the idea that Pinhead is a central regulator of admp expression in the sea urchin and suggest that additional mechanism likely account for the repression of pinhead by BMP signaling in this embryo

Options for Producing a Warm-Water Fish in the UK: limits to "Green-Growth"?

This paper explores the development of a sustainable production system for tilapia and the research implications involved with ensuring commercial viability of such a system for UK farmers. The tilapia is a warm water fish with firm texture, white flesh and mild taste quite similar to a cod or haddock. Whilst tropical in origin it is thought to be highly suitable for low cost aquaculture in temperate zones with the potential to be a more sustainable source of food with fewer environmental impacts than other substitutes. Drawing on a literature review and findings from technical trials the paper will review and compare two production systems - novel Activated Suspension Technology (AST) and conventional Recirculating Aquaculture Systems (RAS) - considering their feasibility in terms of potential and financial viability for scaling up to commercial production of tilapia and their environmental and sustainability benefits. The review concludes that AST based only on microbial floc is currently uncompetitive with RAS in a UK context although the approach has benefits that might be incorporated in a new generation of mixed systems. Refinement of such systems needs to occur with potential adopters and could be part of diversification of mixed farms. Such development might further enhance the ethical values of fish produced in small-scale, modular RAS

L’épave de Port Berteau II (Charente-Maritime)

Découverte en 1973 dans la Charente, en aval de Saintes et par 7 m de fond, l’épave de Port Berteau II a été fouillée de 1992 à 1997 avec une double problématique reposant sur la mise en relation des vestiges de la coque avec son environnement.Conduite selon les techniques classiques de l’archéologie subaquatique, l’étude de l’épave, de l’étrave à l’étambot, a permis de dresser le -portrait architectural » d’un caboteur du début du Vile s. Long de 1430 m et bâti à franc-bord embrure première », ce bateau a été construit pour la navigation fluvio-maritime et pouvait transporter une cargaison d’une dizaine de tonnes. L’analyse du milieu fluvial, relevant de ce que l’on pourrait qualifier d’archéologie nautique, a abouti quant à elle à une restitution du paysage naturel contemporain de l’épave. Le caboteur de Port Berteau II est désormais une référence pour l’histoire de la construction à carvel dans le Ponant uniquement attestée jusqu’alors par des textes du XVe s.Discovered in 1973, the Port Berteau wreck lies at a depth of 7 metres in the Charente river below Samtes. Excavations (1992-1997) were carried out with the dual objective of studying both the hull’s remains and its natural environment.Using the traditional techniques of ship archaeology to examine the wreck from the stem to the stern-post has enabled archaeologists to draw an ‘architectural portrait" of an early 7th century coaster. The 14.30 metre carvel built "frame-first" boat was intended for river navigation and built to carry a cargo of up to ten tonnes. Using what could be called a naval archaeology approach, researchers have analysed the nver environment to reconstruct the natural landscape of the period. The Port Berteau II coaster thus provides a reference for carvel construction history in the Ponant, reviously attested only in 15th century texts

Comprehensive analysis of long non-coding RNAs expression associated with hypoxia and prognosis in non-small cell lung cancer

Lung cancer is the leading cause of cancer death worldwide, with poor prognosis and a high rate of recurrence despite early surgical removal. It is therefore essential to identify new prognostic markers and new therapeutic targets. We are interested in gene regulation related to hypoxia, a factor associated with relapse of lung adenocarcinomas (LUAD). While long non-coding RNAs (lncRNA) are increasingly recognized as major gene expression regulators through various molecular mechanisms, their roles in cancer development and hypoxic response are still largely unexplored. Combining profiling studies on A549 LUAD cell line cultured in normoxic or hypoxic conditions and on early stages LUAD biopsies, we identified a signature of lncRNAs that are regulated by hypoxia in vitro and correlated to i) the hypoxic status of tumors and/or ii) the overall survival of patients. We focused on a candidate, LINC01116, and confirmed this dysregulation on public datasets from larger LUAD and LUSC (Lung Squamous Cell Carcinomas) cohorts of "The Cancer Genome Atlas". We showed that LINC01116 is a short cytosolic transcript composed of 3 exons. Using RNA FISH imaging, RNA interference knock-down and transcriptome analyses, we suggest its potential implication in cellular morphology and cell-to-cell contact. In addition, we found a correlation between LINC01116 expression and an alpha catenin gene network involved in cell-to-cell signaling

Functional characterization of long non coding RNAs induced by hypoxia and implicated in lung cancer agressiveness

Lung cancers, and notably Lung Adenocarcinomas (LUAD) are the leading cause of cancer death worldwide. Their high rate of recurrence despite early, requires new prognostic markers and new therapeutic targets. The combined study of local cohort (CHU of Nice) and large scale (TCGA) transcriptomes of LUAD allowed the identification of a shortlist of 28 long non-coding RNAs (lncRNA) correlated with hypoxia, a factor of tumor aggressiveness, and a poor prognosis. LncRNAs are transcripts that modulate gene expression through the recruitment of proteins and/or nucleic acids and represent an interesting source of new therapeutic targets. Two lncRNAs candidate were selected and molecular characterization was undertaken by sequencing, RT-PCR and smRNA FISH and concern the nuclear lncRNA NLUCAT1 of 9,8kb and the cytosolic lncRNA LINC01116 of 1,2kb. Experiments with loss of function via CRISPR/Cas9 systems, interference RNA and gain of function allowed to characterize the function of these transcripts. Invalidation of NLUCAT1 by CRISPR/Cas9 reduces proliferation, migration, invasion and increases cisplatin sensitivity and ROS production. Bioinformatic analysis of transcriptomes from cells invalidated or not for NLUCAT1 has demonstrated its involvement in the mechanisms of regulation of oxidative stress via a positive feedback from the NRF2 antioxidant pathway. On the other hand, lncRNA LINC01116 is mainly expressed in endothelial cells of the tumor microenvironment and its inhibition by interfering RNA reduces the adhesion capacities and increases the permeability of the endothelium. Mechanistic characterization was perform for LINC01116 via RNA pulldown-MS co-precipitation experiments and idenfied a list of potential partner proteins. The proteins of RNA metabolism and stability, ILF3 and PABPC1 were identified and their interactions with LINC01116 were validated by RNA immunoprecipitation (RIP). Overall, during my thesis, I determine the pro-tumoral action of the NLUCAT1 in LUAD and the involvement of LINC01116 in the modification of the tumor microenvironment. These two transcripts could represent potential therapeutic targets in the management of lung cancer

Functional characterization of hypoxia-regulated lncRNAs in Non-Small Cell Lung Carcinomas

Hypoxia triggers the activation of several signaling pathways inducing a complex transcriptomic response with multiple consequences on cell survival, migration, invasion and metabolic reprogramming. This regulatory network integrates specific feedbacks and checkpoint signaling loops including epigenetic factors and non-coding RNAs. However, while a large set of miRNAs have been reported as crucial molecular regulators of the hypoxic response, the precise functional characterization of hypoxia-regulated lncRNAs is still in its infancy. Our study aimed at comprehensively characterize the whole set of hypoxia-regulated lncRNAs in lung adenocarcinoma. Using a combination of experimental profiling approaches in both tumor cell lines and patient samples as well as exploring in silico TCGA datasets, we identified a validated signature of lncRNAs correlated to i) the hypoxic status of tumors and ii) the overall survival of patients. Molecular characterization of a subset of these “hypoxa-lcnRNAs” using RNA-Seq, RT-PCR, northern blot and single molecule RNA FISH pointed to 2 interesting candidates with different subcellular localization pattern: i) NLUCAT1, a large nuclear transcript composed of 6 exons and ii) LINC01116, a short cytosolic transcript composed of 3 exons. We next used CRISPR/Cas9-mediated invalidation and transcriptomic analyses as well as RNA-pulldown approaches to functionally characterize their function and mode of action. While NLUCAT1 was mainly identified as a regulator of the NRF2-mediated anti-oxidant response with an impact on cisplatin resistance, LINC01116 was mostly associated to the regulation of cellular morphology, cell-to-cell contact and cell invasion through an alpha catenin gene network. Overall, our study strongly supports the central role of non-coding RNAs in the cellular response to hypoxia and provides the first molecular characterization of 2 hypoxa-lncRNAs contributing to an aggressive phenotype in hypoxic tumors

Caractérisation fonctionnelle de longs ARNs non codants induits par l’hypoxie et impliqués dans l’agressivité des cancers pulmonaires non à petites cellules

Lung cancers, and notably Lung Adenocarcinomas (LUAD) are the leading cause of cancer death worldwide. Their high rate of recurrence despite early, requires new prognostic markers and new therapeutic targets. The combined study of local cohort (CHU of Nice) and large scale (TCGA) transcriptomes of LUAD allowed the identification of a shortlist of 28 long non-coding RNAs (lncRNA) correlated with hypoxia, a factor of tumor aggressiveness, and a poor prognosis. LncRNAs are transcripts that modulate gene expression through the recruitment of proteins and/or nucleic acids and represent an interesting source of new therapeutic targets. Two lncRNAs candidate were selected and molecular characterization was undertaken by sequencing, RT-PCR and smRNA FISH and concern the nuclear lncRNA NLUCAT1 of 9,8kb and the cytosolic lncRNA LINC01116 of 1,2kb. Experiments with loss of function via CRISPR/Cas9 systems, interference RNA and gain of function allowed to characterize the function of these transcripts. Invalidation of NLUCAT1 by CRISPR/Cas9 reduces proliferation, migration, invasion and increases cisplatin sensitivity and ROS production. Bioinformatic analysis of transcriptomes from cells invalidated or not for NLUCAT1 has demonstrated its involvement in the mechanisms of regulation of oxidative stress via a positive feedback from the NRF2 antioxidant pathway. On the other hand, lncRNA LINC01116 is mainly expressed in endothelial cells of the tumor microenvironment and its inhibition by interfering RNA reduces the adhesion capacities and increases the permeability of the endothelium. Mechanistic characterization was perform for LINC01116 via RNA pulldown-MS co-precipitation experiments and idenfied a list of potential partner proteins. The proteins of RNA metabolism and stability, ILF3 and PABPC1 were identified and their interactions with LINC01116 were validated by RNA immunoprecipitation (RIP). Overall, during my thesis, I determine the pro-tumoral action of the NLUCAT1 in LUAD and the involvement of LINC01116 in the modification of the tumor microenvironment. These two transcripts could represent potential therapeutic targets in the management of lung cancer.Les cancers broncho-pulmonaires non à petites cellules, et plus particulièrement les adénocarcinomes (ADC), constituent la première cause de mortalité due au cancer dans les pays développés. Malgré des prises en charge précoces, leur fort taux de récidive, nécessite l'identification de nouveaux marqueurs pronostics et de nouvelles cibles thérapeutiques. Dans cette optique, nous nous intéressons à l'hypoxie, un facteur d’agressivité tumoral, et à la famille émergente des longs ARNs non codants qui régulent l’expression génique par le biais de complexes ribonucléoprotéiques. Des analyses transcriptomiques de cohortes de patients d’ADC pulmonaires ont permis l’identification d'une quarantaine de longs ARNs non codants (lncARN) corrélés au statut hypoxique des tumeurs et/ou à un mauvais pronostic vital. Nous nous sommes focalisés sur deux candidats, NLUCAT1 et LINC01116, induits par l'hypoxie et associés à une diminution de la survie des patients. NLUCAT1 est un transcrit nucléaire de 9800 nt dont l’invalidation par le système CRISPR/Cas9 réduit les propriétés prolifératives et invasives des cellules et augmente la production de RLO et l'apoptose induite par le cisplatine ou un stress oxydatif. L’analyse comparative des transcriptomes de cellules invalidées ou non pour NLUCAT1 a révélé son implication dans la régulation du stress oxydatif via un rétrocontrôle positif sur des gènes de la réponse anti-oxydante. Par ailleurs, LINC01116 est cytosolique et exprimé conjointement dans les cellules cancéreuses et dans les cellules endothéliales du microenvironnement tumoral. J'ai montré que des siARNs réduisent l'adhésion et augmentent la perméabilité trans-endothéliale suggérant des défauts au niveau des contacts intercellulaires. J'ai ensuite entrepris l'étude du mode d'action de LINC01116 par l'identification de ses partenaires protéiques. Des expériences de "RNA pulldown" couplées à de la spectrométrie de masse ont permis d’identifier les protéines ILF3 et PABPC1. Ces interactions ont été validées par des expériences inverses d'immunoprécipitation d'ARN (RIP). L’implication de ces protéines dans le mode d’action de LINC01116 nécessite des études complémentaires.L’ensemble des résultats collectés durant ma thèse ont mis en évidence l’action pro-tumorale du transcrit NLUCAT1 dans les ADC et l'implication de LINC01116 dans la modification du microenvironnement vasculaire tumoral. Ces transcrits, associés à un mauvais pronostic des ADC, pourraient représenter des cibles thérapeutiques potentielles pour la prise en charge des patients