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Platelet-derived growth factor receptors (α and β) are included in the family of receptor tyrosine kinases (RTKs). The dimeric PDGF ligands bind to the PDGF receptors and this results in receptor dimerization, autophosphorylation, and thus activation. The autophosphorylated receptor dimer activates numerous signaling pathways. The responses of the cell to the activation of these pathways include proliferation, migration and survival. After ligand stimulated activation, PDGF receptors are internalized mainly via clathrin-coated pits and subsequently degraded in the lysosomes. However, the proteasomes have also been reported to play a role in PDGFRβ degradation. The main aim of this project was to study the mechanism of ligand-stimulated cleavage of PDGFRβ and to explore its possible consequences. By using antibodies that recognize the extracellular and intracellular parts of the receptor, respectively, we showed that ligand stimulation of PDGFRβ leads to the formation of an extracellular (~130 kDa) and an intracellular (~70 kDa) fragment. These fragments were shown to be specific fragments of the receptor located at the plasma membrane and not newly synthesized receptor. The presence of calcium ions intracellularly was found to be essential for cleavage. Interestingly, we observed that with the use of the proteasomal inhibitor called bortezomib, receptor cleavage was inhibited. On the other hand, lysosomal inhibition did not inhibit PDGFRβ fragmentation. Subsequently, we showed that cleavage occurs after the receptor has been endocytosed. Then, we examined whether blocking PDGFRβ cleavage had any effect on the downstream signaling of the receptor. We revealed an increase in receptor, PLCγ and STAT3 phosphorylation and a decrease in Erk phosphorylation upon bortezomib treatment. Moreover, we observed that PDGFRβ undergoes another type of cleavage upon ligand stimulation in fibroblasts, known as ectodomain shedding. Finally, we showed that receptor internalization is decreased upon bortezomib treatment, suggesting that bortezomib blocks receptor cleavage by inhibiting its internalization. The cleavage site, responsible enzyme, as well as potential functional consequences of ligand-stimulated PDGFRβ cleavage, remain to be determined.

Ribosome biogenesis is the maturation and accurate assembly of ribosomal RNA and proteins to form functional ribosomes competent to enter the translation cycle, facilitated by the action of ribosome biogenesis factors. RbfA and KsgA are two factors involved in late stages of the 30S subunit biogenesis, yet before translation initiation. Despite the extensive research on both factors, their precise mechanisms remain unclear in translation initiation. Initiation factors constitute a key component of the translation initiation system, especially IF3 that proofreads the codon-anticodon interaction of the mRNA and initiator tRNA to maintain the accuracy of the initiation step. However, defective ribosomes with knockout RbfA (ΔrbfA) or KsgA (ΔksgA) appear to weaken IF3 binding affinities and subsequent proofreading. Previous results suggest that when defective ribosomes enter in vivo translation with cognate (AUG) or even near-cognate (AGG) start codons could bypass the fidelity checkpoints created by initiation factors, leading to the inaccurate initiation of translation. This study aimed to characterize the assembly defective ΔrbfA and ΔksgA ribosomes in in vitro initiation assays on cognate and near-cognate codons and with the interplay of initiation factors for the rate and accuracy in translation initiation. The two assays, BOP-Met-tRNAfMet binding and subunit association, reveal that ΔrbfA ribosomes are defective in both rate and accuracy in translation initiation, which largely contribute to its growth defect at 37°C. In contrast, ΔksgA ribosomes are not defective in the initiation step of translation when comparing to the wildtype ribosome. Therefore, RbfA seems to have the primary role in ribosome biogenesis compared to KsgA. Further, the results show that the initiation factors, in particular IF3, adds to the fidelity of initiation by dissociating the initiator tRNA on near/non-cognate codons, with no major difference between Wt and mutant ribosomes in selection against near/non-cognate codons. Therefore, we do not see any loss of fidelity for the ΔrbfA and ΔksgA ribosomes in in vitro initiation based assays, contrary to previous in vivo based results.

Loss of appetite is together with fever and malaise hallmarks of infection. Loosing appetite during an acute infection such as influenza does not result in any longlasting effects, but loosing appetite during chronic diseases such as cancer or AIDS constitutes a risk factor for mortality. Food intake regulation during inflammation is orchestrated by the brain in response to peripheral inflammatory signals. It is known that expression of the prostaglandin synthesizing enzyme cyclooxygenase 2 (COX-2) is crucial for the mechanisms underlying inflammation-induced anorexia, and that prostaglandin E2 (PGE2) is involved in anorexia induced by interleukin-1 beta (IL-1β). In this thesis I examined the prostaglandin-pathways proposed to be involved in anorexia. We show that acute anorexia is dependent on COX-2 expression, while cancer-induced anorexia is mediated by cyclooxygenase 1 (COX-1), at least in the initial stages, suggesting that the signaling pathways for chronic- and acute anorexia are distinct. We were able to demonstrate that the pathway underlying acute anorexia is distinct from that of fever, and that taste aversion is prostaglandin independent. We could also show that both acute and chronic anorexia-cachexia is dependent on expression of myeloid differentiation primary response gene (MyD88) in hematopoietic/myeloid cells. In summary, the findings presented in this thesis suggest that anorexia is a result of many different signaling pathways, as opposed to what is the case for several other inflammatory symptoms such as fever and malaise, where the pathways have been shown to be very exclusive. This provides new insight into the diversity of the pathways underlying inflammatory symptoms, which is fundamental for the ability to present potential, symptom-specific drug targets.

L’anévrisme de l’aorte abdominale (AAA) correspond à une dilatation progressive de l’aorte dont la complication principale, et potentiellement mortelle, est la rupture. La physiopathologie de l’AAA est complexe mais elle comporte une destruction des fibres élastiques liée à l’activité des métalloprotéinases (MMPs) matricielles, une apoptose des cellules musculaires lisses et une infiltration inflammatoire chronique. Actuellement, il n’existe aucun traitement pharmacologique permettant de limiter la progression et la rupture de l’AAA. Les fibroblastes gingivaux (FGs) sont des cellules multipotentes anti-inflammatoires qui permettent une réparation parfaite de la gencive sans cicatrice ni fibrose. Ces propriétés font des FGs un candidat potentiel pour une approche de thérapie cellulaire de l’AAA. Les FGs murins cultivés in vitro produisent une grande quantité de TIMP 1, un inhibiteur des MMPs. Lorsque les FGs sont implantés autour de l’aorte, ils survivent, prolifèrent et s’organisent en une épaisse couche cellulaire au niveau de l’adventice. In vivo, les FGs produisent au niveau de la paroi aortique de l’IL-10, du TGF-β, du collagène et du TIMP-1. Parallèlement les FGs inhibent l’activité protéolytique de la MMP-9. Dans un modèle d’AAA induit par l’élastase, les FGs inhibent la dégradation de l’élastine, l’infiltration macrophagique et lymphocytaire ainsi que la croissance des AAA. L’invalidation génétique de TIMP-1 dans les FG abolit leur effet protecteur sur le remodelage aortique et la formation des AAA. Dans un second modèle d’AAA induit par l’infusion d’angiotensine II et l’injection d’anticorps neutralisant anti-TGF-β, les FGs limitent localement le développement de la maladie vasculaire et préviennent la rupture de l’aorte abdominale. L’ensemble de ces données expérimentales suggère qu’une approche de thérapie cellulaire utilisant les FG pourrait permettre de ralentir la croissance de l’AAA et in fine sa rupture. / Abdominal aortic aneurysm (AAA), frequently diagnosed in old patients, is characterized by chronic inflammation, vascular cell apoptosis and metalloproteinases-mediated extracellular matrix destruction. Depiste improvement in the understanding of the pathophysiology of the aortic aneurysm disease, no pharmacological treatment is available to limit dilatation and/or rupture. In the study reported here, we tested whether periadventitial allograft of GF prevented abdominal aortic aneurysmal growth and rupture in mice and investigated the mechanisms of vascular protection. In vitro, mouse GF proliferated and produced large amounts of anti-inflammatory cytokines and Timp-1, an inhibitor of metalloproteinases. When layed down in the periadventitial abdominal aorta, we documented that GF survived in vivo, proliferated and organized as a thick layer. Furthermore, GF locally produced Il-10, TGF-β and Timp-1. In an elastase-induced AAA, GF prevented both macrophage and lymphocyte infiltration, elastin degradation and aneurysm growth. Specific invalidation of Timp-1 in GF abolished the beneficial effect of cell therapy. In an Angiotensin II/anti-TGF-β model of AAA, GF cell therapy limited AAA development and prevented abdominal rupture. Gingival fibroblast is a promising cell therapy approach to inhibit aneurysmal progression and rupture through the local production of Timp-1.

La tumorigenèse est influencée par des facteurs génétiques et environmentaux. La surnutrition est une cause d'obésité et d'accumulation pathologique de lipides dans le foie, la stéatose, qui peut évoluer en stéato-hépatite. Obésité et stéato-hépatite contribuent à l'augmentation de l'incidence du diabète dans le monde entier. Le diabète et l'obésité sont des facteurs de risque pour le cancer du foie (El-Serag et al., Clin Gastroenterol Hepatol, 2006). Cet projet de thèse élucide les mécanismes moléculaires liant l'activation de la voie de signalisation de l'insuline, les maladies du foie gras et le développement du cancer du foie, et il propose de nouvelles stratégies thérapeutiques. La délétion hépato-spécifique du gène codant le suppresseur de tumeurs Phosphatase and tensin homolog (PTEN) est un modèle de cancer du foie associé à la stéatose (Horie et al., J Clin Invest, 2004). Nous avons utilisé ce modèle, où la cascade de signalisation PI3K/mTOR est activée, pour démontrer que l'induction de l'expression du facteur de transcription et récepteur nucléaire Peroxysome Proliferator-Activated Receptor gamma (PPARγ) est responsable de la stéatose et de la glycolyse aérobie (effet Warburg). Son activité est spécifiquement dépendente de l'activité d'un effectuer en aval de la voie PI3K/mTOR, la protéine sérine/thréonine kinase, AKT2 (Panasyuk et al., Nat Comm, 2012). Sur la base de ces observations, nous avons construit l'hypothèse que PPARγ est un important régulateur de la croissance pathologique et du développement des adénocarcinomes hépatiques associés aux stéato-hépatites. Avec mon travail de thèse j'ai pu démontrer que l'expression et l'activité de PPARγ sont essentielles pour le développement du cancer du foie dans le modèle de souris invalidées pour PTEN dans les hépatocytes. Par ailleurs, la délétion de PPARγ dans le foie dépourvu de PTEN joue un rôle protecteur de la tumorigenèse, confirmant que PPARγ est effectivement placé en aval de AKT2. De plus, nous avons découvert que PPARγ est induit dans des échantillons de carcinome hépatocellulaire humains. Ces échantillons sont caractérisés par leur agressivité (haut taux prolifératif et bas niveau de différentiation) et aussi par l'activation de la voie PI3K/AKT. L'analyse des échantillons humains au stade pré-carcinome (adénomes) nous a permis de démontrer que l'ARN de PPARγ est le plus exprimé dans les adénomes caractérisés par un haut degré de stéatose. Ils sont caractérisés par la perte de fonction du facteur de transcription Hepatocyte Nuclear Factor 1α (HNF1α). Nous avons identifié HNF1α comment un nouveau régulateur négatif de la transcription de PPARγ. Nous avons aussi découvert que l'expression et l'activité de HNF1α sont inhibées par AKT2, induisant l'expression de PPARγ et son activité pro-tumorigénique. Finalement, la sensitibilité de PPARγ aux ligands, naturels et exogènes, nous a encouragé à tester des traitements pharmacologiques pour moduler son activation. La stimulation de l'activité de PPARγ avec l'agoniste synthétique Pioglitazone a conduit à une aggravation des symptomes dans le foie. Par contre, son inhibition par un antagoniste sélectif, SR2595, était thérapeutique, resultant en une réduction de signes pré-tumoraux et tumoraux dans le foie des souris invalidé par PTEN. En résumé, nos études chez l'humain et la souris, révèlent une nouvelle signature d'interaction entre les facteurs de transcription HNF1α et PPARγ et la voie de signalisation de l'insuline, suggérant de nouvelles stratégies thérapeutiques possibles pour le traitement d'une sous-classe spécifique de cancer du foie lié aux stéato-hépatites. / Tumorigenesis is influenced by genetic and environmental factors. Overnutrition leads to obesity and fatty liver disease, contributing to increase diabetes incidence worldwide. Diabetes and obesity are independent risk factors for liver cancer development (El-Serag et al., Clin Gastroenterol Hepatol, 2006). This PhD project elucidates the molecular mechanisms linking activated insulin signalling pathway, fatty liver disease and liver cancer development and proposes novel therapeutic strategies. The hepatocytes-specific deletion of tumour suppressor Phosphatase and tensin homolog (PTEN) is a model of steatosis-associated liver cancer (Horie et al., J Clin Invest, 2004). Using this model of activated PI3K/mTOR signalling, our laboratory discovered that the nuclear receptor transcription factor Proliferator-Activated Receptor gamma (PPARγ) is induced in PTEN-null liver. My group demonstrated that in the liver PPARγ contributes to steatosis and aerobic glycolysis. Its activity specifically requires a downstream effector in the PI3K/mTOR pathway, the serine/threonine-specific protein kinase AKT2 (Panasyuk et al., Nat Comm, 2012). Based on these observations, we hypothesized that PPARγ might be an important regulator of pathological growth and development of steatohepatitis-associated liver adenocarcinomas. In my PhD work, I demonstrated that PPARγ expression and activity is essential for liver cancer in PTEN mutants. Moreover, PPARγ is induced in human samples of Hepatocellular Carcinoma (HCC) characterized by poor differentiation accompanied by the activation of PI3K/AKT pathway. We could attribute to PPARγ a specific role in tumour formation as it is required for abnormal liver growth and steatosis in mice at pre-tumoral age. In addition, deletion of PPARγ in PTEN mutants protected animals form liver tumorigenesis placing PPARγ downstream of activated AKT2. Analysing human samples of pre-carcinoma lesions characterized by high steatotic rate, we demonstrated that PPARγ transcript levels are increased in a specific subgroup of adenomas characterized by loss-of-function mutations in the Hepatocyte Nuclear Factor 1α (HNF1α). We identified HNF1α as a novel direct negative regulator of PPARγ transcription. We also revealed HNF1α expression and activity inhibited by AKT2 and thereby inducing PPARγ pro-tumorigenic action. Finally, the sensitivity of PPARγ to natural and exogenous ligands encouraged us to perform treatments to pharmacologically modulate PPARγ activity. Further activation of PPARγ with its synthetic ligand pioglitazione dramatically aggravates liver disease. While PPARγ inhibition by selective antagonist SR2595 allowed to reduce the pre-tumoral and tumoral signs of PTEN-null mice. In sum, our studies in men and mice reveal a novel pro-tumorigenic network of transcription factors HNF1α and PPARγ downstream of activated insulin signalling pathway, suggesting possible strategies for treatment of a subgroup of steatohepatitis-associated liver cancer.

<p>Transmission of bacterial strains between patients is a serious problem in hospitals and with the increasing rate of antibiotic resistance the problem has farther escalated. Enterobacteriaceae produced extended-spectrum beta-lactamses (ESBLs), especially Escherichia coli (E-coli), are increasingly important nosocomial pathogens (7, 8). These bacteria are often multiple resistant and are responsible for many intestinal infections and urinary tract infections (2, 5). With the more frequent use of invasive devices in hospital care, these types of nosocomial infections have increased, particularly in seriously ill patients.</p><p>In order to diminish transmission of bacterial strains between patients and to study the epidemiology of these bacteria, it is of great importance to develop rapid and specific methods to be able to subtype on strain-level, i.e. to create a fingerprint of the isolates. The method may be based on phenotypic or genotypic characteristics of the microorganism. Any typing method must have high reproducibility and discrimination power to differentiate unrelated strains and also to demonstrate relationship of organisms deriving from the same source. In the present project, a Pulsed-Field Gel Electrophoresis (PFGE) assay for genotyping clinical E. coli isolates was used. PFGE can be used as a genotyping tool and is widely used to type bacteria and trace nosocomial infection. However, the method is time-consuming and relatively expensive in compare with other methods like PCR. In this study, a total of 93 strains were collected. The study was aimed to investigate the genotypes of the collected isolates and to identify and potential the outbreak strains.</p><p>The isolates investigated were genotypically diverse shown by a variety of PFGE banding patterns. However, clusters of closely related isolates involved in outbreaks were also identified.</p><p>In conclusion, when analyzing a large number of strains, a combination of a rapid phenotyping or genotyping method and a powerful genotyping method like PFGE would be an appropriate strategy for studying clonal relationship among isolates e.g. for detecting cross-transmission of nosocomial pathogens.</p>

Transmission of bacterial strains between patients is a serious problem in hospitals and with the increasing rate of antibiotic resistance the problem has farther escalated. Enterobacteriaceae produced extended-spectrum beta-lactamses (ESBLs), especially Escherichia coli (E-coli), are increasingly important nosocomial pathogens (7, 8). These bacteria are often multiple resistant and are responsible for many intestinal infections and urinary tract infections (2, 5). With the more frequent use of invasive devices in hospital care, these types of nosocomial infections have increased, particularly in seriously ill patients. In order to diminish transmission of bacterial strains between patients and to study the epidemiology of these bacteria, it is of great importance to develop rapid and specific methods to be able to subtype on strain-level, i.e. to create a fingerprint of the isolates. The method may be based on phenotypic or genotypic characteristics of the microorganism. Any typing method must have high reproducibility and discrimination power to differentiate unrelated strains and also to demonstrate relationship of organisms deriving from the same source. In the present project, a Pulsed-Field Gel Electrophoresis (PFGE) assay for genotyping clinical E. coli isolates was used. PFGE can be used as a genotyping tool and is widely used to type bacteria and trace nosocomial infection. However, the method is time-consuming and relatively expensive in compare with other methods like PCR. In this study, a total of 93 strains were collected. The study was aimed to investigate the genotypes of the collected isolates and to identify and potential the outbreak strains. The isolates investigated were genotypically diverse shown by a variety of PFGE banding patterns. However, clusters of closely related isolates involved in outbreaks were also identified. In conclusion, when analyzing a large number of strains, a combination of a rapid phenotyping or genotyping method and a powerful genotyping method like PFGE would be an appropriate strategy for studying clonal relationship among isolates e.g. for detecting cross-transmission of nosocomial pathogens.

During osteoclast differentiation and bone resorption the redox status in the cell display a decrease in reduction and a shift to an oxidized state. Structure, metabolism and function are some of the extensive changes that cells undergo during differentiation which alters both the extra- and intracellular redox environment. Osteoclasts express enzymes such as TRAP and NADPH oxidase which generates reactive oxygen species (ROS). ROS are molecules formed by oxygen reduction which gives these radicals at least one unpaired electron and makes them very reactive and chemically unstable. These are factors which stimulates differentiation of osteoclasts and bone resorption. RAW 264.7 cells will differentiate to osteoclasts when stimulated with RANKL and to activated macrophages when stimulated with LPS. The aim of this project was to analyze if the redox environment is affected during differentiation of RAW 264.7 cells to osteoclasts and macrophages. The reason for this was that we aimed to se if RAW 264.7 cells could be used as an in vitro system to study the effects of redox changes in osteoclasts and macrophages and their activation. Results from Western blot showed that protein expression of the Cysteine/Glutamate transporter xCT was up regulated with LPS and downregulated with RANKL. Results from the GSH/Cys assay show that the treatments with redox modulators did not affect the levels of GSH and Cys to a measurable extent. However the levels increased for both intracellular and extracellular GSH and Cys forms at day 4 in the control and stimulated cells. Addition of the disulfide reductant DTT affected differentiation to osteoclasts, leading to smaller osteoclasts probably due to interference with fusion of mononuclear pre-osteoclasts. Thus, down regulation of the xCT transporter could be an important mechanism to maintain a low level of free thiols shown to interfere with the differentiation to osteoclasts.

Previous PhD students in the lab created a method for large-scale, high-content microscopy screening of a cell library consisting of over 3000 single mutant strains of the fission yeast, Schizosaccharomyces pombe. Each strain has one nonessential gene knocked-out, allowing investigation of the resulting phenotypes. I report the implementation and completion of this screen; developing methods to ensure reliable and accurate results through inclusion of many controls across multiple screening repeats. In total, over 4.5 million images from approximately 19 000 biologically independent cell populations were imaged and analysed. All strains screened contained GFP-labelled tubulin (GFP-Atb2) allowing visualisation of the microtubule polymer network and its organisation in cells, a feature that is conserved across eukaryotes and simplified in S. pombe, making it easy to study. Examination of cell outlines and microtubule patterns was used to study three cell processes: the shape of cells, the organisational pattern of interphase microtubules and the cell cycle stage of cells, as judged by microtubule pattern. Comparison with extensive data from wild-type cells led to the identification of 262 factors that influence one or more of these cell processes. I go on to biologically validate some of the outcomes from the screen, leading to a publication in Developmental Cell reporting the screen, its findings and the online genomic resource SYSGRO. I then focus on a group of mutants that suggest a connection between the DNA damage response (DDR) and microtubule organisation. From here I show that the DDR induces elongation of microtubule bundles in response to the DDR kinases, ATM and ATR. I begin to reveal factors that may mediate this response and finally, I provide evidence to suggest that the same mechanism is conserved in cultured human cells (Hc3716-hTERT), which may go some way to explaining clinical results showing a beneficial effect of microtubule destabilisation in conjunction with cancer therapies.