L'infection chronique par le virus de l'hépatite C (VHC) conduit au développement de pathologies hépatiques, telles que la fibrose dont le terme évolutif est la cirrhose sur laquelle peut se développer un carcinome hépatocellulaire. Les observations cliniques indiquent que le VHC interfère avec la voie de signalisation du Transforming Growth Factor β (TGFβ). Entre autres fonctions, cette cytokine induit la transition épithélio-mésenchymateuse (EMT), ce qui favorise la migration cellulaire et l'invasion tumorale. Le but de cette thèse est d'analyser l'impact des protéines non structurales du VHC sur la voie de signalisation du TGFβ.Nous avons montré que le réplicon subgénomique du VHC induit une augmentation de la signalisation du TGFβ résultant en une plus forte expression de gènes associés à l'EMT et induisant un phénotype d'EMT. L'expression de la protéase virale NS3-4A seule, augmente et prolonge la phosphorylation de Smad2/3 en aval du récepteur du TGFβ et renforce l'expression de certains gènes cibles du TGFβ. L'analyse des interactions entre les protéines du VHC et les protéines de la voie du TGFβ a permis d'identifier l'interaction entre NS3-4A et la protéine Smurf2. Le réplicon subgénomique ou la protéase NS3-4A ont des rôles antagonistes à la protéine Smurf2 sur la voie de signalisation du TGFβ. L'analyse globale des gènes régulés par le TGFβ dans les cellules exprimant le réplicon subgénomique a permis d'identifier, que dans ces cellules, le TGFβ induit une réponse pro-tumorale.Ces résultats montrent que NS3-4A induit une plus forte réponse des cellules au TGFβ, en inhibant la fonction de Smurf2 dans le rétrocontrôle négatif de la voie du TGFβ. Ce nouveau mécanisme d'interférence du VHC avec la voie du TGFβ pourrait contribuer à l'EMT des cellules hépatocytaires infectées favorisant ainsi la cancérisation. Ce travail apporte de nouvelles pistes dans la compréhension des mécanismes associés à la cancérisation chez les patients chroniquement infectés par le VHC.

yes / Type 2 diabetes (T2DM) promotes premature atherosclerosis and inferior prognosis after arterial reconstruction. Vascular smooth muscle cells (SMC) respond to patho/physiological stimuli, switching between quiescent contractile and activated synthetic phenotypes under the control of microRNAs (miRs) that regulate multiple genes critical to SMC plasticity. The importance of miRs to SMC function specifically in T2DM is unknown. This study was performed to evaluate phenotype and function in SMC cultured from non-diabetic and T2DM patients, to explore any aberrancies and investigate underlying mechanisms. Saphenous vein SMC cultured from T2DM patients (T2DM-SMC) exhibited increased spread cell area, disorganised cytoskeleton and impaired proliferation relative to cells from non-diabetic patients (ND-SMC), accompanied by a persistent, selective up-regulation of miR-143 and miR-145. Transfection of premiR-143/145 into ND-SMC induced morphological and functional characteristics similar to native T2DM-SMC; modulating miR-143/145 targets Kruppel-like factor 4, alpha smooth muscle actin and myosin VI. Conversely, transfection of antimiR-143/145 into T2DM-SMC conferred characteristics of the ND phenotype. Exposure of ND-SMC to transforming growth factor beta (TGFβ) induced a diabetes-like phenotype; elevated miR-143/145, increased cell area and reduced proliferation. Furthermore, these effects were dependent on miR-143/145. In conclusion, aberrant expression of miR-143/145 induces a distinct saphenous vein SMC phenotype that may contribute to vascular complications in patients with T2DM, and is potentially amenable to therapeutic manipulation.

<p>Epithelial-mesenchymal transition (EMT) is a pathological process leading to the formation of anterior subcapsular cataract (ASC). Mediated by transforming growth factor beta (TGFβ), EMT involves the transformation of the monolayer of lens epithelial cells (LECs) into spindle-shaped myofibroblasts, which manifest as plaques directly beneath the lens capsule. TGFβ-induced EMT leading to ASC has been associated with the upregulation of two specific matrix metalloproteinases (MMPs), MMP-2 and MMP-9. Having identified MMP-2 and MMP-9 as participants in the formation of cataracts, the specific roles of either of these MMPs have yet to be determined.</p> <p>The current study utilized MMP-2 and -9 knockout (KO) mice to determine their unique roles in TGFβ-induced EMT. First, adenoviral injection of active TGFβ1 into the anterior chamber of MMP-2 KO mice led to the formation of distinct αSMA-positive anterior subcapsular plaques, in contrast to treated MMP-9 KO eyes, which were resistant. Additionally, an <em>ex vivo </em>mouse LEC explant system was established in these KO lines. In the isolated lens epithelial explants, TGFβ triggered a transformation of LECs from a tightly packed cuboidal monolayer to an elongated mesenchymal phenotype. This process involved a disruption in epithelial cell contacts indicated by a loss of E-cadherin, and an acquisition of myofibroblast marker, αSMA. In the absence of MMP-2, TGFβ was still able to induce EMT with E-cadherin loss and concurrent αSMA expression. In contrast, LEC explants from MMP-9 KO mice treated with TGFβ did not acquire a characteristic spindle-like phenotype and showed substantially less αSMA expression. Results from both of these approaches were consistent; MMP-2, but not MMP-9, KO mice stimulated with TGFβ exhibited phenotypic changes typical of those described in ASC formation, namely a loss in cell attachments, multilayering of previously epithelial-like cells, and αSMA reactivity. Therefore, while MMP-2 is not necessary, MMP-9 is critical to TGFβ-induced EMT in LECs.</p> / Master of Science (MSc)

Being the longest-lived rodent, naked mole-rats (NMR; Heterocephalus glaber) are an exceptional model for biogerontological research. However, unlike other rodents, not much is known about their wound healing process. To investigate that, full-thickness wounds were created in the back skin of naked mole rats. Our initial data confirmed that wound closure in NMR skin was achieved primarily by reepithelialization and granulation tissue formation, with only ~26% wound contraction, making them an excellent model to study human cutaneous wound healing. Similar to mice and human skin, changes in wound epithelial tongue included progressive enlargement of wound epithelium, increased proliferation and changes in the expression pattern of epidermal markers including K14, K17, integrin α6 and E-cadherin. Further analysis revealed characteristics of reduced scarring in NMR wounds including low collagen I to III ratio, increased HA expression (HMW) and increased fibronectin expression. Transcriptional profiling of TGFβ isoforms and different pro/anti-inflammatory cytokines revealed a balance in the expression and repression of different cytokines, potentially contributing into reduced scarring. Comparison of RNA-seq data from NMR and human fullthickness wounds revealed a delay in the activation of important biological processes and pathways in NMR skin in response to injury. Further analysis based on cultured human and NMR cells revealed differential regulation of TGFβ signalling pathway between both species. 3-D collagen gel contraction assay revealed that NMR fibroblast showed noticeable contraction but independently of TGFβ treatment, while human fibroblast showed marked increased in gel contraction in the presence of TGFβ. In conclusion, NMR can serve as a very useful model to study human cutaneous wound healing. The reduced scarring in NMR could be a result of multiple factors including HMW-HA, balanced cytokine expression and differential regulation of different TGFβ cytokines as observed in the in vitro studies. / The full text will be available at the end of the embargo: 3rd May 2024

L'adénocarcinome canalaire pancréatique (PDAC) est une maladie agressive dans le monde. En raison du manque d'outils de détection hautement sensibles et spécifiques à un stade précoce de la maladie et de traitements peu curatifs, la plupart des patients sont diagnostiqués à un stade incurable avec une mortalité élevée. Comprendre la pathologie moléculaire, en particulier le stade précoce de cette maladie mortelle, est d’une importance capitale pour améliorer les bons pronostics de ces patients. La première et principale partie de mes travaux reposait sur une analyse de profilage génétique dans laquelle ils mettaient en évidence le rôle de la signalisation TGFβ / Activine et du récepteur ALK4 au cours du développement de la PDAC, en plus des quatre gènes oncogéniques courants (KRAS, CDKN2A, TP53 et SMAD4). ). D'autre part, INHBA (codant pour ActivinA) en tant que membre clé des ligands de la superfamille TGFβ est une cible de l'activation oncogénique de Kras dans les cellules du canal pancréatique et a été désigné comme composant potentiel de la SASP par une analyse au sécrétome de cellules subissant une sénescence induite par Kras. . Le but de mes travaux est donc d’étudier l’impact de la signalisation ActivinA lors de l’initiation de la PDAC. Dans la première partie de mon projet, j'ai démontré qu'ActivinA était un facteur SASP protecteur de la tumeur, produit par les cellules sénescentes d'ADM / PanIN, qui limitent leur prolifération et leur expansion dans les lésions hautement prolifératives via son récepteur ALK4 lors de l'initiation du PDAC. De plus, nos travaux ont révélé que les cellules sénescentes Dclk1 + limitent l'expansion des lésions prénéoplasiques et ont un impact partiel sur le composant stromal soutenu par la signalisation ActivinA. En outre, j'ai également exploré les fonctions autocrines et paracrines de la signalisation ActivinA au dernier stade de la PDAC. Nous avons constaté que le manque de signalisation ActivinA-ALK4 chez la souris entraînait une augmentation de la capacité de prolifération des cellules tumorales et une accumulation réduite de cellules stromales, ce qui pourrait aider à expliquer leurs métastases variées et un temps de survie relativement plus long. En conclusion, mon travail donne un autre aperçu de la signalisation ActivinA et Dclk1 dans l'initiation de la PDAC et leur existence dans des cellules sénescentes au sein de lésions précurseurs pancréatiques de bas grade devrait être considérée comme une étape cruciale pour développer de nouvelles stratégies diagnostiques et thérapeutiques pour les patients atteints de PDAC / Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease worldwide and remains the ranking of 7th leading cause of cancer related death in the past 5 years. Because of the lack of highly sensitive and specific detective tools in an early stage of this disease and low curative treatments, most of the patients are diagnosed in an incurable stage and end up with a high mortality. Understanding the molecular pathology especially the early stage of this lethal disease is of great importance to improve the outcome of these patients. The first and major part of my work was based on gene profiling analysis in which they emphasized the role of TGFβ/Activin signalling and ALK4-receptor during the development of PDAC in addition to the four common oncogenic genes (KRAS, CDKN2A, TP53 and SMAD4). On the other hand, INHBA(encoding for ActivinA) as a key member of TGFβ-superfamily ligands is a target of Kras oncogenic activation in pancreatic duct cell and has been pointed a potential SASP candidate component through a secretome analysis of cells undergoing Kras induced senescence. Therefore, the aim of my work is to investigate the impact of ActivinA-signalling during PDAC initiation. In the first part of my project, I demonstrated that ActivinA as a tumour-protecting SASP factor, produced by ADM/PanINs senescent cells, that limit their proliferation and expansion into highly proliferative lesions through its ALK4 receptor in the initiation of PDAC. Furthermore, our work revealed that senescent Dclk1+ cells limit the expansion of preneoplastic lesions and partly impact the stromal component which is sustained by ActivinA-signalling. Besides, I also explored the autocrine and paracrine functions of ActivinA-signalling in the late stage of PDAC. We found that lack of ActivinA-ALK4 signalling in mice leads to an increased proliferative capacity in tumor cells and a reduced accumulation of stromal cells, which might help to explain their diverse metastasis and a relative longer survival time. To conclude, my work gives another insight of ActivinA-signalling and Dclk1 in the onset of pancreatic cancer and their existence in senescent cells within low grade pancreatic precursor lesions should be considered a crucial step for developing novel diagnostic and therapeutic strategies for PDAC patients

Der TGFβ-Signalweg ist ein multifunktionales System, das zelluläre Prozesse reguliert, die von Proliferation und Migration bis zu Differenzierung und Zelltod reichen. Nach Ligandenbindung und Rezeptoraktivierung translozieren SMAD-Proteine zum Zellkern und induzieren die Expression zahlreicher Zielgene. Während viele Komponenten des TGFβ-Signalweges identifiziert wurden, verstehen wir noch nicht genau, wie die Aktivierung des Signalwegs in verschiedene zelluläre Antworten übersetzt wird. Da die zelluläre Antwort auf einen gegebenen Stimulus oft sogar in genetisch identischen Zellen variiert, konzentrierte ich mich auf die Messung der Signalwegaktivität auf der Einzelzellebene. Durch die Kombination fluoreszierender Reporterzelllinien mit Zeitraffer-Lebendzellmikroskopie und automatisierter Bildanalyse beobachtete ich die zytoplasmatische und nukleäre Translokation von SMADs mit hoher zeitlicher und räumlicher Auflösung in Hunderten einzelner Zellen. Unsere Experimente zeigten, dass die Signalwegaktivität in eine erste synchrone Phase der SMAD-Translokation, gefolgt von einer Adaption und einer zweiten Signalphase mit hoher Variabilität in Stärke und Dauer der nuklearen Akkumulation unterteilt werden kann. Darüber hinaus beobachtete ich, dass Zellen, die aufgrund ihrer dynamischen Eigenschaften in Subpopulationen gruppiert sind, unterschiedliche phänotypische Reaktionen zeigen. Ich war nun daran interessiert, die Netzwerkinteraktionen zu identifizieren, die diese Dynamiken formen und fokussierte mich auf den Crosstalk mit nicht-kanonischen Komponenten des TGFβ-Signalweges. Ich konnte zeigen, dass die Hemmung der MAP Kinasen p38 und ERK die zweite Signalphase spezifisch aufhebt. Diese dynamische Remodellierung führt zu Veränderungen in der Zielgenexpression und den Zellschicksalen. Dies wird zu einem tieferen Verständnis der molekularen Netzwerke führen, die die TGFβ-Signaltransduktion regulieren und Möglichkeiten eröffnen, es in erkrankten Zellen zu modulieren. / The TGFβ pathway is a multi-functional signaling system regulating cellular processes ranging from proliferation and migration to differentiation and cell death. Upon ligand binding and receptor activation, SMAD proteins translocate to the nucleus and induce expression of numerous target genes. While many components of the TGFβ pathway have been identified, we are still challenged to understand how pathway activation is translated into distinct cellular responses. As the cellular response to a given stimulus often varies even in genetically identical cells, I focused on measuring pathway activity on the single cell level. By combining fluorescent reporter cell lines with time-lapse live-cell microscopy and automated image analysis, I monitored the cytoplasmic to nuclear translocation of SMADs with high temporal and spatial resolution in hundreds of individual cells. Our experiments demonstrated that pathway activity can be divided into a first synchronous phase of SMAD translocation, followed by adaptation and a second signaling phase with high variability in the extent and duration of nuclear accumulation. Furthermore, I observed that cells clustered into subpopulations according to their dynamic features show different phenotypic responses. I was interested in identifying the network interactions that shape these dynamics and focus on crosstalk with non-canonical components of the TGFβ pathway. I could show that inhibition of the MAP kinases p38 and ERK specifically abrogates the second signaling phase. This dynamic remodeling led to changes in target gene expression and cell fate decisions. I explored the molecular mechanisms underlying this interaction of the canonical and non-canonical pathways. This will provide a deeper understanding of the molecular networks regulating TGFβ signaling and open opportunities to modulate it in diseased cells.

The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this mainly through a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. Another major way in which 1,25(OH)2D3 inhibits cell growth in prostate cancer is via membrane-initiated steroid signalling, which triggers activation of signal cascades upon steroid binding to a receptor complex, leading to induction of genes regulating cell growth, proliferation and apoptosis. The main prostate cancer inhibiting membrane-initiated route is the TGFβ signalling pathway, elicited by the protein TGFβ. In this experiment the activating effects of 1,25(OH)2D3 on TGFβ in prostate cancer cells, as well as two other important proteins downstream in this cascade, Smad2 and 3, were investigated. PC-3 cells were incubated for 3, 5, 10, 30 and 60 minutes as well as 38 hours both together with 1,25(OH)2D3 of the concentrations 10-10 and 10-7 M and without. As the downstream cascade protein JNK is a known activator of Smad2/3, this procedure was also repeated with a JNK inhibitor. An ELISA assay scanning for activated TGFβ was then performed on supernatants from the cells treated without JNK inhibitor. In addition, a Western Blot scanning for activated Smad2 and 3 was performed on supernatants from all groups of treatment. The analysis of the result values showed that 10-10 M 1,25(OH)2D3 significantly lowered the content of active TGFβ in PC-3 cells within 3 and 5 minutes. Unfortunately the Western Blot was unsuccessful and needs therefore be repeated.

<p>The vitamin D metabolite 1,25(OH)2D3 has long been known to inhibit growth of prostate cancer cells and this mainly through a VDR-mediated pathway controlling target gene expression, resulting in cell cycle arrest, apoptosis and differentiation. Another major way in which 1,25(OH)2D3 inhibits cell growth in prostate cancer is via membrane-initiated steroid signalling, which triggers activation of signal cascades upon steroid binding to a receptor complex, leading to induction of genes regulating cell growth, proliferation and apoptosis. The main prostate cancer inhibiting membrane-initiated route is the TGFβ signalling pathway, elicited by the protein TGFβ. In this experiment the activating effects of 1,25(OH)2D3 on TGFβ in prostate cancer cells, as well as two other important proteins downstream in this cascade, Smad2 and 3, were investigated. PC-3 cells were incubated for 3, 5, 10, 30 and 60 minutes as well as 38 hours both together with 1,25(OH)2D3 of the concentrations 10-10 and 10-7 M and without. As the downstream cascade protein JNK is a known activator of Smad2/3, this procedure was also repeated with a JNK inhibitor. An ELISA assay scanning for activated TGFβ was then performed on supernatants from the cells treated without JNK inhibitor. In addition, a Western Blot scanning for activated Smad2 and 3 was performed on supernatants from all groups of treatment. The analysis of the result values showed that 10-10 M 1,25(OH)2D3 significantly lowered the content of active TGFβ in PC-3 cells within 3 and 5 minutes. Unfortunately the Western Blot was unsuccessful and needs therefore be repeated.</p>

The present thesis discusses how gene regulation by transforming growth factor β (TGFβ) family cytokines is affected by post-translational modifications of different transcription factors. The thesis also focuses on gene regulation by transcription factors involved in TGFβ signaling. The importance of the poly ADP-ribose polymerase (PARP) family in controlling gene expression in response to TGFβ and bone morphogenetic protein (BMP) is analyzed first. PARP2, along with PARP1, ADP-ribosylates Smad2 and Smad3, the signaling mediators of TGFβ. On the other hand, poly ADP-ribose glycohydrolase (PARG) removes the ADP-ribose from Smad2/3 and antagonizes PARP1 and PARP2. ADP-ribosylation of Smads in turn affects their DNA binding capacity. We then illustrate how PARP1 and PARG can regulate gene expression in response to BMP that signals via Smad1, 5. Over-expression of PARP1 suppressed the transcriptional activity of Smad1/5. Knockdown of PARP1 or over-expression of PARG enhanced the transcriptional activity of BMP-Smads on target genes. Hence our data suggest that ADP-ribosylation of Smad proteins controls both TGFβ and BMP signaling.  I then focus on elucidating novel genes that are regulated by ZEB1 and Snail1, two key transcriptional factors in TGFβ signaling, known for their ability to induce EMT and cancer metastasis. Chromatin immunoprecipitation-sequencing (ChIP-seq) and targeted whole genome transcriptomics in triple negative breast cancer cells were used, to find binding regions and the functional impact of ZEB1 and Snail1 throughout the genome. ZEB1 binds to the regulatory sequences of a wide range of genes, not only related to cell invasion, pointing to new functions of ZEB1. On the other hand, Snail1 regulated only a few genes, especially related to signal transduction and cellular movement. Further functional analysis revealed that ZEB1 could regulate the anchorage-independent growth of the triple negative breast cancer cells, whereas Snail1 could regulate the expression of BMP6 in these cells. We have therefore elucidated novel functional roles of the two transcription factors, Snail1 and ZEB1 in triple negative breast cancer cells.

No description available.