Helicobacter pylori colonizes the human stomach, where it causes gastritis that may develop into peptic ulcer disease or cancer when left untreated. Neisseria gonorrhoeae colonizes the urogenital tract and causes the sexually transmitted disease gonorrhea. In contrast, Lactobacillus species are part of the human microbiota, which is the resident microbial community, and are considered to be beneficial for health. The first host cell types that bacteria encounter when they enter the body are epithelial cells, which form the border between the inside and the outside, and macrophages, which are immune cells that engulf unwanted material.       The focus of this thesis has been the interaction between the host and bacteria, aiming to increase our knowledge of the molecular mechanisms that underlie the host responses and their effects on bacterial pathogenicity. Understanding the interactions between bacteria and the host will hopefully enable the development of new strategies for the treatment of infectious disease. In paper I, we investigated the effect of N. gonorrhoeae on the growth factor amphiregulin in cervical epithelial cells and found that the processing and release of amphiregulin changes upon infection. In paper II, we examined the expression of the transcription factor early growth response-1 (EGR1) in epithelial cells during bacterial colonization. We demonstrated that EGR1 is rapidly upregulated by many different bacteria. This upregulation is independent of the pathogenicity, Gram-staining type and level of adherence of the bacteria, but generally requires viable bacteria and contact with the host cell. The induction of EGR1 is mediated primarily by signaling through EGFR, ERK1/2 and β1-integrins. In paper III, we described the interactions of the uncharacterized protein JHP0290, which is secreted by H. pylori, with host cells. JHP0290 is able to bind to several cell types and induces apoptosis and TNF release in macrophages. For both of these responses, signaling through Src family kinases and ERK is essential. Apoptosis is partially mediated by TNF release. Finally, in paper IV, we showed that certain Lactobacillus strains can reduce the colonization of H. pylori on gastric epithelial cells. Lactobacilli decrease the gene expression of SabA and thereby inhibit the binding mediated by this adhesin. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 4: Manuscript.</p>

Le mésothéliome pleural malin (MPM) est une tumeur primitive de la plèvre, rare, très agressive, avec un pronostic sombre. Nous avons souhaité au cours de ce travail de thèse, identifier de nouveaux biomarqueurs du MPM en testant l’influence de l’inactivation des membres de la famille RASSF/Hippo sur la survie des 448 patients inclus dans l’essai clinique MAPS (IFCT-GFPC-0701). Nous souhaitions également comprendre quelles fonctions et signalisations essentielles à l’homéostasie cellulaire, auxquelles participe la voie de signalisation RASSF/Hippo, sont perturbées lors du processus de transformation des cellules mésothéliales. L’inactivation des membres de la voie a été étudiée par PCR spécifique de méthylation (MS-PCR) et leur influence sur la survie des 448 patients inclus dans l’essai clinique MAPS testée en analyse uni- et multivariée avant d’être validée par boostrap. D’autre part, nous avons mimé in cell, l’inactivation par ARN interférence de plusieurs membres de la voie Hippo dans des cellules de lignées mésothéliales humaines (MSTO-211H, H2452, H28 et H2052). Nous avons pu identifier plusieurs biomarqueurs du MPM : i) la kinase MST1 dont l’inactivation est un facteur de mauvais pronostic, ii) l’amphiréguline dont l’expression cytoplasmique est au contraire un facteur de bon pronostic et enfin iii) le CD44 dont l’expression élevée constitue un outil diagnostique du MPM. Les approches in cell, nous ont permis de démontrer que les altérations de la voie RASSF/Hippo induisent une activité inappropriée de l’effecteur terminal YAP : le moins bon pronostic des patients présentant une inactivation de MST1 s’explique ainsi par le fait qu’en régulant l’activité de YAP, MST1 contrôle la balance apoptose/prolifération et prévient l’invasion et la croissance sans adhésion. En son absence, ces processus cellulaires sont dérégulés. Ce travail démontre l’importance de l’axe CD44/RASSF1A/MST1 dans le contrôle d’une activité appropriée de YAP et de l’homéostasie des cellules mésothéliales. La compréhension des désordres cellulaires induits par la dérégulation de le voie RASSF/Hippo, désigne YAP comme cible thérapeutique potentielle chez les patients atteints de MPM et présentant des altérations de cette voie de signalisation. / Malignant pleural mesothelioma (MPM) is a rare, very aggressive, primary tumor with a poor prognosis. During this thesis, we wanted to identify new biomarkers of MPM by testing the influence of the RASSF/Hippo pathway inactivation on the survival of the 448 patients included in the clinical trial MAPS (IFCT- GFPC-0701). We also wanted to understand which functions and signals essential to cellular homeostasis, linked to RASSF/Hippo signaling pathway, are disturbed during the mesothelial cell transformation process. Inactivation of RASSF/Hippo members was studied by methylation-specific PCR (MS-PCR) and their influence on the survival of the 448 patients included in the MAPS clinical trial tested in uni- and multivariate analysis before being validated by bootstrap. We also mimed in cell, by RNA interference, several members of the Hippo pathway inactivation in human mesothelial cells lines (MSTO-211H, H2452, H28 and H2052). We have identified several biomarkers of MPM: i) MST1 kinase whose inactivation is a factor of poor prognosis, ii) amphiregulin whose cytoplasmic expression is on the contrary a factor of good prognosis and finally iii) CD44 whose high expression is a diagnostic tool for MPM. In cell we demonstrate that RASSF/Hippo pathway alterations induce an inappropriate activity of YAP, one Hippo end effector: the poorer prognosis of patients with inactivation of MST1 is thus explained by the fact that, by regulating YAP activity, MST1 controls the apoptosis/proliferation balance and prevents invasion and growth without adhesion from mesothelial cells. In its absence, these cellular processes are deregulated. This work finally demonstrates the importance of the CD44/RASSF1A/MST1 axis in controlling appropriate YAP activity and mesothelial cell homeostasis. The understanding of the cellular disorders induced by the of the RASSF/Hippo pathway deregulation designates YAP as a potential therapeutic target in patients with MPM and presenting alterations of this signaling pathway.

AREG und EREG sind Liganden des EGFR, deren Expression mit einem positiven EGFR-zielgerichtetem Therapieansprechen in Darmkrebs korreliert. Ziel dieser Arbeit war es, einen epigenetischen Einfluss auf die AREG und EREG Expression zu klären. Es wurde gezeigt, dass AREG und EREG in verschiedenen kolorektalen Krebszelllinien differenziell exprimiert sind, und dass die Expression beider Gene durch epigenetische Inhibitoren erhöht werden kann. Eine Analyse in fünf Zelllinien zeigte jedoch, dass die Promotoren beider Gene hauptsächlich unmethyliert vorlagen. Hingegen wurden kurze Regionen im Gen als differentiell methyliert identifiziert. Im AREG Gen liegt diese Region im Exon 2, was auf einen ungewöhnlichen Regulationsmechanismus hindeutet. Promotorfunktionsanalysen zeigten dann, dass diese Region eine methylierungs- und orientierungsabhängige Promotorfunktion hat, in die das MDB-Protein CTCF involviert sein könnte. Expressionsanalysen wiesen darauf hin, dass auch ZBTB33, ein anderes MDB-Protein, in die AREG Regulation involviert sein könnte. Die ZBTB33 Expression korrelierte negativ mit der AREG Expression in den Zelllinien. Eine ZBTB33-Bindungsstelle konnte ausserdem bioinformatorisch im AREG Exon 2 identifiziert werden. Des weiteren wurde gezeigt, dass die Behandlung der Zelllinie LIM1215 mit HDAC Inhibitoren in vitro zu einer Erhöhung der Sensitivität gegenüber EGFR-zielgerichteten Medikamenten führt, begleitet von einer Erhöhung der AREG und EREG Expression. Im in vivo Versuch konnte die Sensitivität von LIM1215 Zellen durch die Behandlung mit DNMT Inhibitoren erhöht werden. Begleitet wurde dies hier mit einer Verringerung der Methylierung der AREG und EREG intragenischen CpGs. Diese Ergebnisse zeigen auf, dass Patienten, die resistent gegenüber EGFR-zielgerichteten Therapien sind, möglicherweise sensitiv gemacht werden können. In dem Fall könnten AREG und EREG als prädiktive Marker eingesetzt werden, um den Effekt der epigenetischen Inhibitoren zu evaluieren. / AREG and EREG are ligands of the EGFR whose expression correlates with a positive EGFR-targeted therapy response in colorectal cancer. Aim of this work was to define the influence of epigenetic mechanisms on AREG and EREG gene expression. It could be shown that AREG and EREG are differentially expressed in a set of colorectal cancer cell lines and that the expression of both genes increases after treatment with epigenetically interfering compounds such as DNMT inhibitors and HDAC inhibitors. Methylation analysis showed that the promoters of both genes were mainly unmethylated. Nevertheless, short intragenic regions were identified to be differentially methylated. For AREG, this region is located within exon 2, indicating an uncommon epigenetic regulatory mechanism. Promoter function analyses showed that the AREG exon 2 region harbor methylation- and orientation dependent promoter function and they suggested CTCF, an MDB-protein, to be involved in this mechanism. Expression analysis experiments suggested also ZBTB33, another MDB-protein, to be involved in AREG regulation. ZBTB33 was differentially expressed in the cells and it correlated inversely with the AREG expression. Additionally, bioinformatic analyses identified a ZBTB33 binding site within AREG exon 2. It was also shown in this work that LIM1215 cells treated with HDACis were more sensitive towards EGFR inhibitors in vitro. This effect was accompanied by an increased AREG and EREG expression. In vivo, an increased sensitivity towards EGFR inhibitors was achieved in LIM1215 cells by treatment with a DNMT inhibitor. Here the effect was accompanied by a reduced methylation within the AREG and EREG intragenic CpGs. Together, the results suggested a new possibility to potentially make EGFR-targeted therapy resistant patients suitable for this therapy by epigenetic compound treatment. In that case AREG as well as EREG might be predictive markers to evaluate the effect of the epigenetic compounds during therapy.

Responsable de 1,6 million de décès par an dans le monde, le cancer du poumon constitue aujourd’hui la première cause de mortalité par cancer. Les cancers bronchiques non-à-petites cellules représentent 85% des cancers du poumon et ont un pronostic vital très mauvais. Les EGFR-TKI (inhibiteurs de l’activité tyrosine kinase de l’EGFR, gefitinib) constituent un réel progrès thérapeutique pour le traitement des cancers du poumon. Cependant, ces traitements ne sont efficaces que dans un petit sous-groupe de patients. Un des enjeux actuels est donc d’identifier les mécanismes de résistance primaire mis en jeu par les tumeurs.Les récepteurs à activité tyrosine kinase (RTK) activent des voies de signalisation intracellulaires depuis la membrane plasmique. Ces dernières années, une translocation nucléaire des RTK a également été mise en évidence. Ces travaux récents suggèrent que la signalisation nucléaire des RTK pourrait contribuer à la résistance des tumeurs en réponse aux thérapies anti-cancéreuses.Dans l’équipe, il a été montré que l’activation de l’IGF-1R est associée à la progression tumorale des adénocarcinomes pulmonaires et que le gefitinib induit une accumulation nucléaire de l’IGF-1R dans un modèle d’adénocarcinome mucineux. Sur la base de ces résultats, nous avons émis l’hypothèse que l’IGF-1R nucléaire pourrait jouer un rôle dans la résistance aux EGFR-TKI des adénocarcinomes pulmonaires mucineux.Nos résultats indiquent que plus de 70% des adénocarcinomes pulmonaires présentent un marquage nucléaire de l’IGF-1R. A l’aide de différents modèles cellulaires résistants aux EGFR-TKI, nous montrons que le gefitinib induit l’accumulation nucléaire de l’IGF-1R dans les adénocarcinomes pulmonaires mucineux. Cette translocation nucléaire implique l’endocytose clathrines-dépendante de l’IGF-1R et la formation d’un complexe entre l’IGF-1R, l’importine β1 et la pro-amphiréguline. La neutralisation de l’amphiréguline prévient le transport nucléaire de l’IGF-1R et resensibilise les cellules à l’apoptose induite par le gefitinib in vitro et in vivo. L’ensemble de ces résultats identifient le trafic intracellulaire de l’IGF-1R comme un nouveau composant de la réponse aux EGFR-TKI et suggèrent que la signalisation nucléaire IGF-1R/Areg contribue à la progression des adénocarcinomes mucineux sous EGFR-TKI. / Responsible of 1.6 million deaths each year worldwide, lung cancer is today the leading cause of cancer mortality in the world. Non-small-cell lung cancers account for about 85% of lung cancer and have a very bad prognosis (5-year survival rate inferior to 10%). EGFR-TKI (EGFR tyrosine kinase inhibitors, gefitinib) are a real medical advance for lung cancers treatment. However, these treatments are efficient in a small subgroup of patients. So, one of the current issues is to identify primary resistance mechanisms involved in tumors.Tyrosine kinase receptors (RTK) activate intracellular signaling pathways from the plasma membrane. These last years, a nuclear translocation of the RTK was shown. Recent works suggest that RTK nuclear signaling could contribute to tumors resistance in response to anti-cancerous therapies.In our team, it was shown that activation of IGF-1R signaling is associated with lung adenocarcinoma progression and that gefitinib induces IGF-1R nuclear accumulation in a mucinous adenocarcinoma cell line. On the basis of these results, we hypothesize that nuclear IGF-1R could play a role in the resistance of mucinous lung adenocarcinoma to EGFR-TKI.Our results indicate that more than 70% lung adenocarcinoma tumors present a positive IGF-1R nuclear staining. Thanks to EGFR-TKI-resistant cell lines, we show that gefitinib induces the nuclear accumulation of IGF-1R in mucinous adenocarcinoma. This nuclear translocation involves clathrin-mediated endocytosis and a complex between IGF-1R, importin β1 and pro-amphiregulin. Amphiregulin silencing prevents IGF-1R nuclear translocation in response to gefitinib and restores gefitinib-induced apoptosis in vitro and in vivo. Our whole results identify that IGF-1R intracellular trafficking is a new component of response to EGFR-TKI and strongly suggest that a nuclear IGF-1R/amphiregulin signaling contributes to mucinous lung adenocarcinoma progression in response to EGFR-TKI.

In vitro, meotic maturation of porcine oocytes and cumulus expansion are induced by FSH and EGF-like peptides AREG and EREG. FSH and EGF-like peptides induce expression of cumulus expansion-related genes (HAS2, PTGS2 and TNFAIP6). To define signaling pathways that control FSH- and AREG-induced cumulus expansion, porcine cumulus-oocyte complexes were treated with specific protein kinase inhibitors. Inhibitors of MAPK3/1, MAPK14 and ERBB1 significantly reduced both FSH- and AREG-induced expression of HAS2, PTGS2 and TNFAIP6. These inhibitors decreased FSH/LH-induced expression of AREG and EREG in mural granulosa cells. Surprisingly, inhibitor of PKA had no effect on AREG expression in cumulus-oocyte complexes but the inhibitor decreased expression of TNFAIP6 induced by AREG. Inhibitor of PI3K increased expression levels of AREG and PTGS2 but EREG, HAS2 and TNFAIP6 were reduced. Expression levels of the cumulus expansion-related genes were not affected by an analog of cGMP (8-CPT-cGMP). However, 8-CPT-cGMP blocked spontaneous in vitro meiotic maturation of porcine oocytes and its effect was abolished by FSH. Key words: cumulus expansion, cumulus expansion-related genes, meotic maturation, FSH, amphiregulin, cGMP

Health is often defined in terms of absence of disease or pathological processes, but this is a definition of exclusion and incomplete. For example, SARS-CoV-2 viral load does not reliably predict disease severity, and so individuals must vary in their ability to control inflammation and maintain normal tissue homeostasis. This host defense strategy is called disease tolerance, and better understanding of disease tolerance mechanisms could change the way that we treat disease and work to maintain health. The first project presented in this dissertation found that after accounting for effects of age and sex, innate lymphoid cells (ILCs), but not T cells, were lower in adults and children sick with COVID-19 or MIS-C, independent of lymphopenia. Furthermore, abundance of ILCs, but not of T cells, correlated inversely with disease severity. These blood ILCs were shown to produce amphiregulin, a protein implicated in disease tolerance and tissue homeostasis, and the percentage of amphiregulin-producing ILCs was lower in males. These results suggest that, by promoting disease tolerance, homeostatic ILCs decrease morbidity and mortality associated with SARS-CoV-2 infection, and that lower ILC abundance accounts for increased COVID-19 severity with age and in males. The second project describes a novel mouse model of epigenetic inheritance wherein paternal influenza A virus (IAV) infection results in less severe influenza disease in IAV infected offspring. This offspring phenotype was not attributable to differences in viral load, indicating a possible difference in disease tolerance. Paternal caloric deprivation decreased, and influenza B virus infection increased, offspring influenza disease severity, and in vitro fertilization demonstrated sperm are sufficient to transfer IAV-associated epigenetic inheritance phenotypes. These findings represent a foundation for further work that, by continuing to elucidate the mechanisms of disease tolerance and epigenetic inheritance, could provide novel therapeutic interventions to help promote and maintain health.