Osteoarthritis is an inflammation-related disease that progressively destroys joint cartilage. This disease causes pain and stiffness of the joints, and at advanced stages, limitations to the movement or bending of injured joints. Therefore, it often restricts daily activities and the ability to work. Currently, there is no cure to prevent its progression, although certain damaged joints, such as fingers, knees and hips, can be treated with joint replacement surgeries. However, joint replacement surgeries of larger joints are very invasive operations and the joint replacements have a limited lifetime. Cell-based therapies could offer a way to treat cartilage injuries before the ultimate damage of osteoarthritis on articular cartilage. The development of novel treatments needs both a good knowledge of articular cartilage biology and tissue engineering methods. This thesis primarily investigates the effects of mechanical cyclic stretching, a 5% low oxygen atmosphere and the Rho-kinase inhibitor, Y-27632, on protein responses in chondrocytic human chondrosarcoma (HCS-2/8) cells. Special focus is placed on Rho-kinase inhibition, relating to its potential to promote and support extracellular matrix production in cultured chondrocytes and its role in fibroblast cells as a part of direct chemical cellular differentiation. The means to enhance the production of cartilage-specific extracellular matrix is needed for cell-based tissue engineering applications, since cultured chondrocytes quickly lose their cartilage-specific phenotype. A mechanical 8% cyclic cell stretching at a 1 Hz frequency was used to model a stretching rhythm similar to walking. The cellular stretching relates to stresses, which are directed to chondrocytes during the mechanical load. The stretch induced changes in proteins related, e.g., to certain cytoskeletal proteins, but also in enzymes associated with protein synthesis, such as eukaryotic elongation factors 1-beta and 1-delta. Hypoxic conditions were used to model the oxygen tension present in healthy cartilage tissue. Long-term hypoxia changed relative amounts in a total of 44 proteins and induced gene expressions of aggrecan and type II collagen, in addition to chondrocyte differentiation markers S100A1 and S100B. A short-term inhibition of Rho-kinase failed to induce extracellular matrix production in fibroblasts or in HCS-2/8 cells, while its long-term exposure increased the expressions of chondrocyte-specific genes and differentiation markers, and also promoted the synthesis of sulfated glycosaminoglycans by chondrocytic cells. Interestingly, Rho kinase inhibition under hypoxic conditions produced a more effective increase in chondrocyte-specific gene expression and synthesis of extracellular matrix components by HCS-2/8 cells. The treatment induced changes in the synthesis of 101 proteins and ELISA analysis revealed a sixfold higher secretion of type II collagen compared to control cells. The secretion of sulfated glycosaminoglycans was simultaneously increased by 65.8%. Thus, Rho-kinase inhibition at low oxygen tension can be regarded as a potential way to enhance extracellular matrix production and maintain a chondrocyte phenotype in cell-based tissue engineering applications.

Most genome-wide association studies search for genetic variants associated to a single trait of interest, despite the main interest usually being the understanding of a complex genotype-phenotype network. Furthermore, many studies collect data on multiple phenotypes, each measuring a different aspect of the biological system under consideration, therefore it can often make sense to jointly analyze the phenotypes. However this is rarely the case and there is a lack of well developed methods for multiple phenotype analysis. Here we propose novel approaches for genome-wide association analysis, which scan the genome one SNP at a time for association with multivariate traits. The first half of this thesis focuses on an analytic model averaging approach which bi-partitions traits into associated and unassociated, fits all such models and measures evidence of association using a Bayes factor. The discrete nature of the model allows very fine control of prior beliefs about which sets of traits are more likely to be jointly associated. Using simulated data we show that this method can have much greater power than simpler approaches that do not explicitly model residual correlation between traits. On real data of six hematological parameters in 3 population cohorts (KORA, UKNBS and TwinsUK) from the HaemGen consortium, this model allows us to uncover an association at the RCL locus that was not identified in the original analysis but has been validated in a much larger study. In the second half of the thesis we propose and explore the properties of models that use priors encouraging sparse solutions, in the sense that genetic effects of phenotypes are shrunk towards zero when there is little evidence of association. To do this we explore and use spike and slab (SAS) priors. All methods combine both hypothesis testing, via calculation of a Bayes factor, and model selection, which occurs implicitly via the sparsity priors. We have successfully implemented a Variational Bayesian approach to fit this model, which provides a tractable approximation to the posterior distribution, and allows us to approximate the very high-dimensional integral required for the Bayes factor calculation. This approach has a number of desirable properties. It can handle missing phenotype data, which is a real feature of most studies. It allows for both correlation due to relatedness between subjects or population structure and residual phenotype correlation. It can be viewed as a sparse Bayesian multivariate generalization of the mixed model approaches that have become popular recently in the GWAS literature. In addition, the method is computationally fast and can be applied to millions of SNPs for a large number of phenotypes. Furthermore we apply our method to 15 glycans from 3 isolated population cohorts (ORCADES, KORCULA and VIS), where we uncover association at a known locus, not identified in the original study but discovered later in a larger one. We conclude by discussing future directions.

Chondroplasia, the development of cartilage-like characteristics in tendinous tissue, is a form of degeneration found in tendons including those of the rotator cuff. The molecular mechanism of its development is currently unknown. An examination of the features of the torn rotator cuff and the cartilage literature led to the identification of several potential drivers of chondroplasia including cell shape change/actin cytoskeleton and hypoxia. Lovastatin caused actin cytoskeleton disruption and promoted cartilage matrix deposition in the ATDC5 model. It was the most effective member of a panel of cytoskeletal inhibitors, increasing expression of the chondrocytic markers Sox5 and Sox9 and decreasing expression of COL1A1 and COL3A1 in primary human tenocytes. Its effects were dose dependent, reversible by mevalonate addition and long term treatment induced de novo expression of collagen II. Short term hypoxia upregulated VEGF-A and chondrocytic marker gene DEC1 expression but not other chondrocyte markers. Combination treatment with hypoxia did not enhance the effects of lovastatin. These data suggest that modulation of pathways that regulate the actin cytoskeleton and cell shape may alter tenocyte phenotype.

L’hyperplasie et l’hypertrophie contribuent à l'augmentation de la masse de muscle lisse bronchique observée dans le souffle. Les cellules musculaires lisses (CML) présentent deux phénotypes; prolifératif ou contractile. Le serum response factor (SRF), un facteur de transcription impliqué dans l’activation de nombreux gènes, contribuerait à cette modulation phénotypique. Notamment, lorsqu'associé au cofacteur Elk-1, un phénotype prolifératif serait observé, alors qu'en présence de la myocardine (MYOCD) il y aurait induction d'un profil contractile. Récemment, il a été démontré que SRF est surexprimé dans les voies périphériques chez les chevaux atteints du souffle suite à une exposition antigénique. Cette étude vise à caractériser l'expression protéique et génique de SRF, Elk-1 et MYOCD dans les CML des voies respiratoires centrales et périphériques chez des chevaux atteints du souffle et des chevaux contrôles. L'évaluation de l’expression protéique de SRF, Elk-1 et MYOCD s’est effectuée par immunodétection sur des tissus provenant de biopsies thoracoscopiques ou endobronchiques, et ce, avant, à 1 et 30 jours du défi antigénique. L'expression génique a été étudiée par qPCR sur du muscle lisse disséqué de la trachée, et des bronches, ainsi que sur des voies respiratoires intermédiaires et périphériques. Les expressions génique et protéique de MYOCD sont augmentées uniquement dans les voies périphériques. L’expression génique de SRF et Elk-1 varient dans les voies centrales alors que le taux de protéines demeure stable. En conclusion, SRF et MYOCD pourraient être impliquées dans l’hypertrophie des voies respiratoires périphériques dans le souffle alors que l’hyperplasie ne semble pas être activée par Elk-1. / Airway smooth muscle (ASM) cells hyperplasia and hypertrophy contribute to the increased airway smooth muscle mass present in heaves. ASM cells express either a synthetic proliferative or a contractile phenotype. Serum response factor (SRF) is a transcription factor that has been shown to regulate myocyte differentiation in vitro in vascular and intestinal smooth muscles. When SRF is associated with Elk-1, it promotes ASM proliferation while myocardin (MYOCD) promotes the expression of contractile elements. Recently, SRF was shown to be overexpressed in the peripheral airways of heaves affected horses following an antigenic challenge. The objective of this study was to characterize the protein and gene expression of SRF, Elk-1 and MYOCD in ASM cells from central and peripheral airways of heaves affected horses and controls. Protein expression of Elk-1 and MYOCD was evaluated using immunohistochemistry while immunofluorescence was used for SRF detection in pulmonary peripheral and endobronchial biopsies before and at 1 and 30 days of antigenic exposure. Gene expression was investigated in ASM cells dissected from trachea and bronchi as well as from intermediate and peripheral airways using qPCR. MYOCD gene and protein expressions are increased only in peripheral airways. SRF and Elk-1 gene expression varied in the central airways while the positive cell percentage remains stable. In conclusion, the pulmonary peripheral airways hypertrophy observed in heaves seems to implicate SRF and MYOCD while the hyperplasia doesn’t seem to be activated by Elk-1.

L’inflammation fait partie des processus réactionnels de défense dont dispose l’organisme en réponse aux agressions, assurant l’intégrité de l’hôte. En réponse au dommage tissulaire, plusieurs médiateurs inflammatoires interviennent dans le processus de l’inflammation. Lors de ces dommages, des signaux de dangers provenant de cellules endommagées sont relâchés dans l’environnement tissulaire, pouvant causer des dommages cellulaires et tissulaires. Les macrophages, tout comme d’autres cellules, peuvent être activés par ces signaux de danger, menant à la sécrétion de molécules telles que des cytokines et des chimiokines pouvant modifier le microenvironnement tissulaire. Les insultes au tissu sain peuvent entrainer la mort cellulaire telle que l’apoptose. Les molécules pouvant être relâchées lors de celle-ci contribuent au microenvironnement, notamment de par l’influence de celles-ci sur le macrophage. Parmi ces médiateurs, nous avons identifié le Milk Fat Globule-Epidermal growth factor 8 (MFG-E8), un acteur important dans la résolution de l’inflammation, comme étant relâché spécifiquement par les cellules apoptotiques. Nous avons émis l’hypothèse que le microenvironnement apoptotique tissulaire, via la relâche de MFG-E8, module le phénotype du macrophage, modifiant le microenvironnement, la réponse inflammatoire ainsi que le devenir de l’insulte tissulaire. Nos objectifs sont 1) de caractériser ce microenvironnement apoptotique tissulaire et la cinétique de relâche du MFG-E8 par les cellules apoptotiques, 2) d’en évaluer son rôle dans la modulation du phénotype du macrophage ainsi que 3) d’en étudier, in vivo, son influence sur l’environnement inflammatoire et le devenir tissulaire. Dans le premier article présenté, nous avons démontré que les cellules endothéliales apoptotiques relâchent le MFG-E8 de façon Caspase-3 dépendante. La stimulation des macrophages par l’environnement conditionné par les cellules endothéliales apoptotiques mène à l’adoption d’un profil macrophagien davantage anti-inflammatoire et moindrement pro-inflammatoire. Ce phénotype est réduit par l’inhibition de la Caspase-3 et il dépend de la présence de MFG-E8. De plus, le potentiel du MFG-E8 à la reprogrammation du macrophage pro-inflammatoire a été démontré via un modèle expérimental de péritonite. Ce changement phénotypique médié par MFG-E8 implique une signalisation STAT3. Ayant démontré que les cellules épithéliales apoptotiques, à l’instar des cellules endothéliales apoptotiques, relâchent elles aussi de façon apoptose-dépendante le MFG-E8, nous avons étudié plus exhaustivement un modèle in vivo riche en apoptose épithéliale, l’obstruction urétérale unilatérale. Dans ce deuxième article présenté, nous rapportons l’implication bénéfique de MFG-E8 dans ce modèle de pathologie rénale obstructive. Nous avons constaté que la présence ou l’administration de MFG-E8 réduit le dommage tissulaire et la fibrose. La protection conférée par MFG-E8 est médiée via la modulation de l’activation de l’inflammasome. De plus, nos résultats illustrent l’importance du phénotype anti-inflammatoire du macrophage médié par le MFG-E8 dans la régulation négative de l’activation de l’inflammasome rénal et du dommage tissulaire. Cette thèse présente la première description de la relâche Caspase-3-dépendante de MFG-E8 par les cellules apoptotiques. Elle démontre également l’importance du MFG-E8 dans le microenvironnement apoptotique inflammatoire dans l’atténuation du phénotype pro-inflammatoire du macrophage. De plus, nous avons démontré son rôle protecteur dans des modèles in vivo de transplantation aortique et de réparation tissulaire, de même que dans un modèle de maladie rénale chronique où nous avons montré que cette protection conférée par MFG-E8 est médiée par la régulation négative de l’inflammasome tissulaire. Nos résultats suggèrent ainsi que le MFG-E8 pourrait être considéré comme un interrupteur inflammatoire et ainsi comme une cible potentielle dans la modulation de maladies inflammatoires. / Inflammation is an important component of the « response to injury » process, allowing host integrity. In response to injury, released inflammatory mediators from damaged cells play a crucial role in the modification of the inflammatory microenvironment, which can lead to more cellular and tissue damages. Macrophages can be activated by those danger signals, leading to a spectrum of cytokines and chemokines secretion and modulating the tissular microenvironment. Tissue injuries can lead to cell death such as apoptosis. Mediators released during apoptosis contribute to the nature of the microenvironment, by their influence on macrophage amongst others. We have identified that Milk Fat Globule-Epidermal growth factor 8 (MFG-E8), an important actor in inflammation resolution, is specifically released by apoptotic cells. We hypothesized that tissular apoptotic microenvironment, through MFG-E8 release, modulates macrophage phenotype, resulting in the modification of microenvironment, inflammatory response and tissu injury outcome. Thus, our objectives were to 1) characterize this tissular apoptotic microenvironment by studying MFG-E8 release kinetic by apoptotic cells, to 2) evaluate its role in macrophage phenotype modulation and to 3) study, in vivo, its influence on inflammatory environment and tissu damage outcome. In the first study, we demonstrated that MFG-E8 is released by apoptotic endothelial cells in a caspase-3-dependent manner. When macrophages were exposed to conditioned media from apoptotic endothelial cells, they adopt a high anti-inflammatory, low pro-inflammatory cytokine/chemokine secreting phenotype that is lost if apoptosis is inhibited or if MFG-E8 is absent from the media. Furthermore, MFG-E8 potential to anti-inflammatory macrophage reprogramming has been demonstrated in the experimental peritonitis model. This MFG-E8-mediated reprogramming of macrophages occurs through increased phosphorylation of STAT-3. As apoptotic endothelial cells, apoptotic epithelial cells also release MFG-E8 in an apoptotic-dependent manner. Thus, we investiguated more exhaustively an in vivo epithelial apoptosis rich model, the unilateral ureteral obstruction. In this second study, we report the positive impact of MFG-E8 in this renal obstructive model. MFG-E8 administration reduced kidney damage and fibrosis compared to the control, whereas its absence in MFG-E8 KO mice was associated with more severe disease. Moreover, we demonstrated that the protective role of MFG-E8 is mediated through inflammasome activation modulation in the kidney. Furthermore, our results showed the importance of the anti-inflammatory macrophage phenotype that results in decreased inflammasome activation, preventing severe tissue damage. This thesis presents the first description of apoptosis-dependent release of MFG-E8 by apoptotic cells. It also demonstrate the importance of MFG-E8 in inflammatory apoptotic microenvironment, leading to pro-inflammatory macrophage phenotype attenuation. Moreover, we demonstrated MFG-E8 protective role in an aortic transplantation and a tissue repair models, as well as in a chronic kidney disease model where we showed that this MFG-E8 confered protection is mediated by negative regulation of tissular inflammasome activation. These data provide valuable insight for identifying MFG-E8 as a novel target in the modulation of inflammatory diseases and could be considerate as an inflammatory switch.

L’expression du phénotype des organismes dépend en partie d’organismes symbiotiques avec qui ils sont en interaction étroite. Selon le mode de transmission du symbiote, ce dernier va être en conflit d’intérêt plus ou moins intense avec l’hôte pour l’expression du phénotype, conduisant parfois le symbiote à évoluer vers la manipulation du phénotype de l’hôte. Nous avons tenté d’identifier différents facteurs génétiques et environnementaux influençant l’expression et l’évolution de la manipulation chez l’insecte parasitoïde de larves de drosophiles, Leptopilina boulardi, et son virus manipulateur du comportement, LbFV. Ce virus bénéficie d’une transmission mixte, verticale et horizontale, cette dernière étant favorisée par l’induction de superparasitisme induite par le virus. L’étude de la contribution du génotype du parasitoïde dans l’expression de la manipulation a révélé la présence de gènes de résistance partielle à la manipulation. Le potentiel évolutif de cette résistance a ensuite été évalué par des expériences d’évolution expérimentale. Nous avons également montré que LbFV augmente la virulence du parasitoïde envers les larves de drosophiles, révélant ainsi une évolution vers une forme de mutualisme sur ce trait. Par ailleurs, le travail montre qu’un même parasitoïde peut être non seulement infecté par plusieurs souches du virus LbFV mais également infecté par un virus à ARN, décrit pour la première fois dans cette thèse. La transmission verticale, la prévalence élevée et les forts effets phénotypiques de ce virus soulignent de nouveau l’importance des virus dans l’expression du phénotype en population naturelle. / The expression of the phenotype of an individual depends partly on the presence of symbiotic organisms with which it engages in intimate interactions. According to the symbiont’s mode of transmission, the conflict of interest between the host and its symbiont for the expression of the phenotype can be more or less stronger, sometimes leading to the evolution of manipulation of the host phenotype by the symbiont. We attempted to identify different genetic and environmental factors influencing the expression and the evolution of manipulation in the Drosophila parasitoid wasp, Leptopilina boulardi, and its behavior manipulating virus, LbFV. The virus undergoes both vertical and horizontal transmission, the latter being favoured by the induction of superparasitism behaviour by the virus. The study of the contribution of the parasitoid genotype to the expression of the manipulation revealed a polymorphism in the resistance to the manipulation. The evolutionary potential of this resistance was then investigated using a protocol of experimental evolution. We also demonstrated that LbFV increases the virulence of the parasitoid towards its Drosophila host, revealing a form of mutualistic interaction on this trait. Additionally, we showed that an individual parasitoid can be coinfected by several LbFV strains but also by an RNA virus, described for the first time in this thesis. The vertical transmission, the high prevalence and the strong phenotypic effects of this new virus further highlights the importance of viruses in the expression of the phenotype in natural populations.

No description available.

There remain inconsistencies in the literature concerning the intelligence of subjects infected with latent toxoplasmosis. The main goals of this work are to find out whether, a) latent toxoplasmosis influences the intelligence of infected subjects and b) whether Rh negative and Rh positive subjects respond to the infection in different ways. In this work we used a complex test of intelligence, The Structure Intelligence Test I-S-T 2000 R. We were able to statistically control for the confounding variable the size of the place of residence in childhood. This had benefits when compared to previous studies. The differences in intelligence were measured in students of The Faculty of Science. The sample used included 46 toxoplasma-infected and 188 toxoplasma-free individuals. Using nonparametric tests we found lower numerical, fluid and general intelligence in toxoplasma-infected subjects compared to noninfected subjects. In addition, these tendencies were also observed in the results of parametric tests. Further to these components of intelligence there was also found to be a lower component of numerical knowledge in toxoplasma-infected males. By contrast, no differences between infected and noninfected individuals was found to occur in the female test subjects. No statistically significant...

Interleukin-1alpha (IL-1alpha) is a well-known proinflammatory mediator acting as a secreted molecule. However, in addition to its ability to activate its membrane-bound receptor, there is growing evidence on its noncanonical nuclear function, which classifies IL-1alpha as a "dual function cytokine". This nuclear action depends on the evolutionary conserved N-terminal domain of IL-1alpha. After proteolytic processing, the N-terminal domain of IL-1alpha translocates into nucleus. Histone acetyltransferase (HAT) complexes were previously identified as nuclear targets of IL-1alpha precursor. However, the specific protein which is responsible for the interaction between IL-1alpha and HAT complexes has not been identified yet. To dissect this interaction, the N-terminal domain of IL-1alpha was produced in yeast. Suitability of this experimental setup for testing the interaction between IL-1alpha and eukaryotic HAT complexes was evaluated in this study. IL-1alpha has been analyzed in this study using bioinformatics approaches as well. Putative amphipatic acidic helixes of IL-1alpha have been characterized. One of the potential binding partners of these domains is protein Ada2. Protein Ada2, mature IL-1alpha and IL-1alpha precursor in fusion with epitopes suitable for affinity purification were produced in...

Mycobacterium tuberculosis (Mtb) is the pathogen causing tuberculosis (TB), a disease most often affecting the lung. 1.5 million people die annually due to TB, mainly in low-income countries. Usually considered a disease of the poor, also developed nations recently put TB back on their agenda, fueled by the HIV epidemic and the global emergence of drug-resistant Mtb strains. HIV-coinfection is a predisposing factor for TB, and infection with multi-drug resistant and extremely drug resistant strains significantly impedes and lengthens antibiotic treatment, and increases fatality. Mtb is transmitted from a sick individual via coughing, and resident macrophages are the first cells to encounter the bacterium upon inhalation. These cells phagocytose intruders and subject them to a range of destructive mechanisms, aiming at killing pathogens and protecting the host. Mtb, however, has evolved to cope with host pressures, and has developed mechanisms to submerge macrophage defenses. Among these, inhibition of phagosomal maturation and adaptation to the intracellular environment are important features. Mtb profoundly alters its phenotype inside host cells, characterized by altered metabolism and slower growth. These adaptations contribute to the ability of Mtb to remain dormant inside a host during latent TB infection, a state that can last for decades. According to recent estimates, one third of the world’s population is latently infected with Mtb, which represents a huge reservoir for active TB disease. Mtb is also intrinsically tolerant to many antibiotics, and adaptation to host pressures enhances tolerance to first-line TB drugs. Therefore, TB antibiotic therapy takes 6 to 9 months, and current treatment regimens involve a combination of several antibiotics. Patient noncompliance due to therapeutic side effects as well as insufficient penetration of drugs into TB lesions are reasons for treatment failure and can lead to the rise of drug-resistant populations. In view of the global spread of drug-resistant strains, new antibiotics and treatment strategies are urgently needed. In this thesis, we studied the interplay of the primary host cell of Mtb, human macrophages, and different Mtb phenotypes. A low-burden infection resulted in restriction of Mtb replication via phagolysosomal effectors and the maintenance of an inactive Mtb phenotype reminiscent of dormant bacteria. Macrophages remained viable for up to 14 days, and profiling of secreted cytokines mirrored a silent infection. On the contrary, higher bacterial numbers inside macrophages could not be controlled by phagolysosomal functions, and intracellular Mtb shifted their phenotype towards active replication. Although slowed mycobacterial replication is believed to render Mtb tolerant to antibiotics, we did not observe such an effect. Mtb-induced macrophage cell death is dependent on ESAT6, a small mycobacterial virulence factor involved in host cell necrosis and the spread of the pathogen. Although well-studied, the fate of ESAT6 inside infected macrophages has been enigmatic. Cultivation of Mtb is commonly carried out in broth containing detergent to avoid aggregation of bacilli due to their waxy cell wall. Altering cultivation conditions revealed the presence of a mycobacterial capsule, and ESAT6 situated on the mycobacterial surface. Infection of macrophages with this encapsulated Mtb phenotype resulted in rapid ESAT6-dependent host cell death, and ESAT6 staining was lost as bacilli were ingested by macrophages. These observations could reflect the earlier reported integration of ESAT6 into membranes followed by membrane rupture and host cell death. In conclusion, the work presented in this thesis shows that the phenotype of Mtb has a significant impact on the struggle between the pathogen and human macrophages. Taking the bacterial phenotype into account can lead to the development of drugs active against altered bacterial populations that are not targeted by conventional antibiotics. Furthermore, deeper knowledge on Mtb virulence factors can inform the development of virulence blockers, a new class of antibiotics with great therapeutic potential.