The de facto independence of regulatory agencies and its consequences for policy making and regulatory outcomes

This dissertation focuses on the practice of regulatory governance, throughout the study of the functioning of formally independent regulatory agencies (IRAs), with special attention to their de facto independence. The research goals are grounded on a "neo-positivist" (or "reconstructed positivist") position (Hawkesworth 1992; Radaelli 2000b; Sabatier 2000). This perspective starts from the ontological assumption that even if subjective perceptions are constitutive elements of political phenomena, a real world exists beyond any social construction and can, however imperfectly, become the object of scientific inquiry. Epistemologically, it follows that hypothetical-deductive theories with explanatory aims can be tested by employing a proper methodology and set of analytical techniques. It is thus possible to make scientific inferences and general conclusions to a certain extent, according to a Bayesian conception of knowledge, in order to update the prior scientific beliefs in the truth of the related hypotheses (Howson 1998), while acknowledging the fact that the conditions of truth are at least partially subjective and historically determined (Foucault 1988; Kuhn 1970). At the same time, a sceptical position is adopted towards the supposed disjunction between facts and values and the possibility of discovering abstract universal laws in social science. It has been observed that the current version of capitalism corresponds to the golden age of regulation, and that since the 1980s no government activity in OECD countries has grown faster than regulatory functions (Jacobs 1999). Following an apparent paradox, the ongoing dynamics of liberalisation, privatisation, decartelisation, internationalisation, and regional integration hardly led to the crumbling of the state, but instead promoted a wave of regulatory growth in the face of new risks and new opportunities (Vogel 1996). Accordingly, a new order of regulatory capitalism is rising, implying a new division of labour between state and society and entailing the expansion and intensification of regulation (Levi-Faur 2005). The previous order, relying on public ownership and public intervention and/or on sectoral self-regulation by private actors, is being replaced by a more formalised, expert-based, open, and independently regulated model of governance. Independent regulation agencies (IRAs), that is, formally independent administrative agencies with regulatory powers that benefit from public authority delegated from political decision makers, represent the main institutional feature of regulatory governance (Gilardi 2008). IRAs constitute a relatively new technology of regulation in western Europe, at least for certain domains, but they are increasingly widespread across countries and sectors. For instance, independent regulators have been set up for regulating very diverse issues, such as general competition, banking and finance, telecommunications, civil aviation, railway services, food safety, the pharmaceutical industry, electricity, environmental protection, and personal data privacy. Two attributes of IRAs deserve a special mention. On the one hand, they are formally separated from democratic institutions and elected politicians, thus raising normative and empirical concerns about their accountability and legitimacy. On the other hand, some hard questions about their role as political actors are still unaddressed, though, together with regulatory competencies, IRAs often accumulate executive, (quasi-)legislative, and adjudicatory functions, as well as about their performance.

The role of career adaptability and work conditions on general and professional well-being.

This study, conducted with a representative sample of employed and unemployed adults living in Switzerland (N = 2002), focuses on work conditions (in terms of professional insecurity and job demands), career adaptability, and professional and general well-being. Analyses of covariance highlighted that both unemployed and employed participants with low job insecurity reported higher scores on career adaptability and several dimensions (notably on control) than employed participants with high job insecurity. Moreover, structural equation modeling for employed participants showed that, independent of work conditions, adaptability resources were positively associated both with general and professional well-being. As expected professional outcomes were strongly related to job strain and professional insecurity, emphasizing the central role of the work environment. Finally, career adaptability partially mediated the relationship between job strain and professional insecurity, and the outcome well-being.

Plasmacytoid dendritic cells and regulatory T cells in the tumor microenvironment: A dangerous liaison.

Tumor-infiltrating plasmacytoid dendritic cells (pDCs) have been associated with poor patient prognosis. We have recently uncovered the ability of pDCs to activate and expand a subset of tumor-infiltrating FOXP3(+) regulatory T cells that express inducible costimulator (ICOS), providing new insights into the mechanisms that govern the escape of cancer from immunosurveillance.

Prognostic value of arginase-II expression and regulatory T-cell infiltration in head and neck squamous cell carcinoma.

Tumor-infiltrating lymphocytes are present in a variety of tumors and play a central role in antitumor immune responses. Nevertheless, most cancers progress probably because tumors are only weakly immunogenic and develop multiple immunosuppressive mechanisms. In the present study, on head and neck squamous cell carcinoma, we found high intraepithelial infiltration of regulatory FOXP3(+) T cells, and relatively high levels of BDCA2(+) and FOXP3(+) cells in stromal (peripheral) regions of the tumors. Tumor-infiltrating (intraepithelial) FOXP3(+) T cells were significantly more frequent in patients with oropharynx and oral cavity squamous cell carcinoma and in patients without lymph node metastasis. Furthermore, arginase-II (ARG2) was expressed by 60%, inducible nitric oxide synthetase by 9%, cyclooxygenase-2 by 43%, and B-cell lymphoma 2 (BCL2) by 26% of tumors. Interestingly, the absence of ARG2 expression, enhanced stromal infiltration of CD11c(+) myeloid dendritic cells, and high numbers of FOXP3(+) T cells were each significantly associated with prolonged overall survival, and the latter two parameters were also confirmed by multivariate analysis. For disease-free survival, multivariate analysis revealed significant negative correlations with BCL2 and ARG2 expression by tumor cells. These findings shed new light on mechanisms of cancer progression, and provide rationales for therapeutic inhibition of immunosuppressive mechanisms in head and neck squamous cell carcinoma.

Internet use by patients with psychiatric disorders in search for general and medical informations

BACKGROUND: Internet is commonly used by the general population, notably for health information-seeking. There has been little research into its use by patients treated for a psychiatric disorder. AIM: To evaluate the use of internet by patients with psychiatric disorders in searching for general and medical information. METHODS: In 2007, 319 patients followed in a university hospital psychiatric out-patient clinic, completed a 28-items self-administered questionnaire. RESULTS: Two hundred patients surveyed were internet users. Most of them (68.5%) used internet in order to find health-related information. Only a small part of the patients knew and used criteria reflecting the quality of contents of the websites consulted. Knowledge of English and private Internet access were the factors significantly associated with the search of information on health on Internet. CONCLUSIONS: Internet is currently used by patients treated for psychiatric disorders, especially for medical seeking information.

CTLA-4 controls the thymic development of both conventional and regulatory T cells through modulation of the TCR repertoire.

Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4; CD152) is of pivotal importance for self-tolerance, with deficiency or unfavorable polymorphisms leading to autoimmune disease. Tolerance to self-antigens is achieved through thymic deletion of highly autoreactive conventional T (Tconv) cells and generation of FoxP3(+) regulatory T (Treg) cells. The main costimulatory molecule, CD28, augments the negative selection of Tconv cells and promotes the generation of FoxP3(+) Treg cells. The role of its antagonistic homolog CTLA-4, however, remains a topic of debate. To address this topic, we investigated the thymic development of T cells in the presence and absence of CTLA-4 in a T-cell receptor (TCR) transgenic mouse model specific for the myelin basic protein peptide Ac1-9. We reveal that CTLA-4 is expressed in the corticomedullary region of the thymus. Its absence alters the response of CD4(+)CD8(-) thymocytes to self-antigen recognition, which affects the quantity of the Treg cells generated and broadens the repertoire of peripheral Tconv cells. T-cell repertoire alteration after deletion of CTLA-4 results from changes in TCR Vα and Jα segment selection as well as CDR3α composition in Tconv and Treg cells. CTLA-4, therefore, regulates the early development of self-reactive T cells in the thymus and plays a key role in central tolerance.

Differential effects of immunosuppressive drugs on effector and regulatory T cells.

Purpose: Current experimental data suggest that CD4+CD25+Foxp3+regulatory T cells (Tregs) based immunotherapy would be of greatinterest to promote donor-specific immune tolerance in transplantation(Tx). Whether and how adoptive transfer of Tregs could be bestcombined with current immunosuppressive regimens in clinicalsettings remains to be defined. Using an experimental Tx model,we had previously shown that the transfer of antigen-specific Tregspromoted long-term skin allograft acceptance in lymphopenic mice,in the absence of any immunosuppressive drug. However, allograftsurvival was only slightly prolonged when Tregs were transferredalone into non-lymphopenic mice, suggesting that in more stringentconditions such as in clinical settings adjuvant therapies may beneeded to effectively control alloreactive T cells (Teff).Methods and Materials: Here we have investigated the effects ofvarious immunosuppressive drugs on the survival, proliferation andeffector function of Teff and Tregs in response to alloantigens in in vitroassays and in our in vivo Tx model.Results: Teff proliferation was inhibited in a dose-dependant mannerby rapamycin and cyclosporine A, while anti-CD154 only marginallyaffected Teff proliferation and survival in vitro. Rapamycin promotedapoptosis of Teff as compared to Tregs that were more resistant underthe same culture conditions. In vivo, the transfer of donor-specificTregs could be advantageously combined with rapamycin andanti-CD154 to significantly prolong MHC-mismatched skin allograftsurvival in non-lymphopenic recipients.Conclusion: Taken together, our data indicate thatimmunosuppressive drugs differentially target T-cell subsets and couldpromote Tregs expansion and/or function while controlling the Teff pool.

The early IL-4 response to Leishmania major responsible for progressive disease in BALB/c mice is subject to the control of autocrine IL-2 production and regulatory CD4+C25+ T cells

Résumé : La majorité des souches de souris de laboratoire sont résistantes à l'infection par le parasite Leishmania major (L. major). A l'opposé, les souris de la souche BALB développent une maladie évolutive. La résistance et la sensibilité sont corrélées avec l'apparition de lymphocytes T CD4+ spécifiques du parasite, Th1 (de l'anglais T helper) ou Th2 respectivement. La réponse aberrante Th2 chez les souris de la souche BALB/c dépend, au moins en partie, de façon critique de la production rapide d'IL-4 suite à l'infection. Ce pic précoce d'IL-4 est produit par une population de lymphocytes T CD4+ restreinte aux molécules du MHC de classe II, exprimant les chaînes du récepteur des cellules T Vß4-Va8. Ces lymphocytes sont spécifiques d'un épitope de l'homologue Leishmania de la molécule RACK1 des mammifères, appelée LACK. Il a été clairement démontré que l'IL-4 rapidement produite par ces cellules T CD4+ Vß4-Va8 induit la maturation Th2 responsable de la sensibilité vis-à-vis de L. major. Des expériences ont été entreprises pour étudier la régulation de cette réponse précoce d'IL-4. Dans ce travail, nous avons documenté, dans les cellules provenant des ganglions de souris sensibles infectées par L. major, une augmentation de la transcription de l'ARNm de l'IL-2 qui précède la réponse précoce d'IL-4. La neutralisation de l'IL-2 durant les premiers jours d'infection induit la maturation des cellules Thl et la résistance vis-à-vis de L. major. Ces effets de l'anticorps anti-IL-2 neutralisant sont liés à sa capacité d'interférer avec la transcription rapide d'IL-4 des cellules CD4+ réactives à l'antigène LACK. Une augmentation similaire d'IL-2 survient chez les souris résistantes C57BL/6 qui sont incapables de générer la réponse précoce d'IL-4. Cependant, la protéiné LACK induit une transcription précoce d'IL-2 uniquement chez les souris sensibles. Des expériences de reconstitution utilisant des souris C.B.-17 SCID et des cellules T CD4+ réactives à LACK provenant de souris BALB/c IL-2-~démontrent un mode d'action autocrine de l'IL-2 sur la régulation de la réponse précoce d'IL4. Par conséquent, chez les souris C57BL/6, l'absence du pic précoce d'ARNm de l'IL-4 important pour la progression de la maladie paraît liée à l'incapacité des cellules T CD4+ réactives à LACK de produire de l'IL-2. Un rôle dans le contrôle de la production précoce d'IL-4 par les cellules T régulatrices CD4+CD25+ a été investigué en déplétant in vivo cette population de cellules. La déplétion induit une élévation du pic précoce de l'ARNm de l'IL-4 dans les ganglions drainant de souris BALB/c, ainsi qu'une exacerbation du cours de la maladie avec des taux augmentés d'IL-4 dans les ganglions. La réponse rapide d'IL-2 vis-à-vis de L. major est aussi significativement augmentée chez les souris BALB/c déplétées en cellules CD4+CD25+. De plus, nous avons démontré que le transfert de 10puissance(7) cellules provenant de la rate de souris BALB/c déplétées en cellules T régulatrices CD4+CD25+ rend les souris SCID sensibles à l'infection et permet la différentiation Th2. Au contraire, les souris SCID reconstituées avec 10' cellules de la rate de souris BALB/c contrôle sont résistantes à infection par L. major et développent une réponse Thl. Chez les souris SCID reconstituées avec des cellules de rate déplétées en cellules exprimant le marqueur CD25, le traitement avec un anticorps neutralisant l'IL-4 au moment de l'infection par L. major prévient le développement de la réponse Th2 et rend ces souris résistantes à l'infection. Ces résultats démontrent que les cellules T régulatrices CD4+CD25+ jouent un rôle dans la régulation du pic précoce d'IL-4 responsable du développement cellulaire Th2 dans ce modèle d'infection. Summary Mice from most strains are resistant to infection with Leishmania major (L. major). In contrast, BALB mice develop progressive disease. Resistance and susceptibility result from parasite-specific CD4+ Thl or Th2 cells, respectively. The aberrant Th2 response in BALB/c mice depends, at least in part, upon the production of IL-4 early after infection. The CD4+ T cells responsible for this early IL-4 response to L. major express a restricted TCR repertoire (Vß4-Va8) and respond to an I-Ad-restricted epitope of the Leishmania homologue of mammalian RACK1, designated LACK. The role of these cells and the IL-4 they produce for subsequent Th2 cell development and disease progression in BALB/c mice was demonstrated. Experiments have been undertaken to study the regulation of the rapid IL-4 production to L. major. In this report, we document an IL-2 mRNA burst, preceding the reported early IL-4 response, in draining lymph nodes of susceptible mice infected with L. major. Neutralization of IL-2 during the first days of infection redirected Thl cell maturation and resistance to L. major, through interference with the rapid IL-4 transcription in LACKreactive CD4+ cells. A burst of IL-2 transcripts also occurred in infected C57BL/6 mice that do not mount an early IL-4 response. However, although the LACK protein induced IL-2 transcripts in susceptible mice, it failed to trigger this response in resistant C57BL/6 mice. Reconstitution experiments using C.B.-17 SCID mice and LACK-reactive CD4+ T cells from IL-2-/- BALB/c mice showed that triggering of the early IL-4 response required autocrine IL2. Thus, in C57BL/6 mice, the inability of LACK-reactive CD4+ T cells to express early IL-4 mRNA transcription, important for disease progression, appears due to an incapacity of these cells to produce IL-2. A role for CD4+CD25+ regulatory T cells in the control of this early IL-4 production was investigated by depleting in vivo this regulatory T cell population. Depletion induced an increase in the early burst of IL-4 mRNA in the draining lymph nodes of BALB/c mice, and exacerbated the course of disease with higher levels of IL-4 mRNA and protein in their lymph nodes. The rapid IL-2 response to L. major is also significantly enhanced in BALB/c mice depleted of CD4+CD25+ cells. We further showed that transfer of 10~ BALB/c spleen cells that were depleted of CD4+CD25+ regulatory T cells rendered SCID mice susceptible to infection and allowed Th2 differentiation while SCID mice reconstituted with 10 control BALB/c spleen cells were resistant to infection with L. major and developed a Thl response. Treatment with a mAb against IL-4 upon infection with L. major in SCID mice reconstituted with CD25-depleted spleen cells prevented the development of Th2 polarization and rendered them resistant to infection. These results demonstrate that CD4+CD25+ regulatory T cells play a role in regulating the early IL-4 mRNA and the subsequent development of a Th2 response in this model of infection.

The evolution of alternative splicing patterns and regulatory mechanisms

Alternative splicing produces multiple isoforms from the same gene, thus increasing the number of transcripts of the species. Alternative splicing is a virtually ubiquitous mechanism in eukaryotes, for example more than 90% of protein-coding genes in human are alternatively spliced. Recent evolutionary studies showed that alternative splicing is a fast evolving and highly species- specific mechanism. The rapid evolution of alternative splicing was considered as a contribution to the phenotypic diversity between species. However, the function of many isoforms produced by alternative splicing remains unclear and they might be the result of noisy splicing. Thus, the functional relevance of alternative splicing and the evolutionary mechanisms of its rapid divergence among species are still poorly understood. During my thesis, I performed a large-scale analysis of the regulatory mechanisms that drive the rapid evolution of alternative splicing. To study the evolution of alternative splicing regulatory mechanisms, I used an extensive RNA-sequencing dataset comprising 12 tetrapod species (human, chimpanzee and bonobo, gorilla, orangutan, macaque, marmoset, mouse, opossum, platypus, chicken and frog) and 8 tissues (cerebellum, brain, heart, kidney, liver, testis, placenta and ovary). To identify the catalogue of alternative splicing eis-acting regulatory elements in the different tetrapod species, I used a previously defined computational approach. This approach is a statistical analysis of exons/introns and splice sites composition and relies on a principle of compensation between splice sites strength and the presence of additional regulators. With an evolutionary comparative analysis of the exonic eis-acting regulators, I showed that these regulatory elements are generally shared among primates and more conserved than non-regulatory elements. In addition, I showed that the usage of these regulatory elements is also more conserved than expected by chance. In addition to the identification of species- specific eis-acting regulators, these results may explain the rapid evolution of alternative splicing. I also developed a new approach based on evolutionary sequence changes and corresponding alternative splicing changes to identify potential splicing eis-acting regulators in primates. The identification of lineage-specific substitutions and corresponding lineage-specific alternative splicing changes, allowed me to annotate the genomic sequences that might have played a role in the alternative splicing pattern differences among primates. Finally, I showed that the identified splicing eis-acting regulator datasets are enriched in human disease-causing mutations, thus confirming their biological relevance.

Political market and regulatory uncertainty: Insights and implications for integrated strategy

Managers can craft effective integrated strategy by properly assessing regulatory uncertainty. Leveraging the existing political markets literature, we predict regulatory uncertainty from the novel interaction of demand and supply side rivalries across a range of political markets. We argue for two primary drivers of regulatory uncertainty: ideology-motivated interests opposed to the firm and a lack of competition for power among political actors supplying public policy. We align three, previously disparate dimensions of nonmarket strategy - profile level, coalition breadth, and pivotal target - to levels of regulatory uncertainty. Through this framework, we demonstrate how and when firms employ different nonmarket strategies. To illustrate variation in nonmarket strategy across levels of regulatory uncertainty, we analyze several market entry decisions of foreign firms operating in the global telecommunications sector.

The contribution of individual and professional characteristics to general and work-related well-being

Cette thèse porte sur la contribution des caractéristiques individuelles et des situations professionnelles au bien-être. En combinant différentes perspectives théoriques, notamment la théorie de la construction de la carrière, la théorie de la justice organisationnelle, les modèles du bien-être au travail, et les conceptualisations de l'incivilité au travail, un certain nombre d'hypothèses sont proposées concernant le lien entre certaines caractéristiques individuelles et situationnelles et le bien-être général et professionnel. Les deux premières études se focalisent sur la validation d'une échelle d'adaptabilités de carrière et sur le rôle médiateur de cette adaptabilité dans la relation entre des dispositions et le bien-être. La troisième étude évolue l'hypothèse d'un possible effet de médiation de l'adaptabilité mais cette fois de la relation entre insécurité professionnelle et charge de travail d'une part et le bien-être d'autre part. La quatrième étude adopte une perspective longitudinale et analyse les associations entre les dimensions de la personnalité, l'adaptabilité de carrière et le bien-être dans quatre parcours professionnels différents. La cinquième étude porte sur une autre caractéristique individuelle, à savoir la croyance en un monde juste. Cette étude illustre comment la croyance en un monde juste influence les perceptions de justice organisationnelle une année après, qui ont une incidence importante sur le bien-être. Enfin, la dernière étude se concentre sur une population spécifique, les immigrants en Suisse, et souligne qu'être la cible d'incivilité sur le lieu de travail est généralement liée au pays d'origine. Globalement, cette thèse met en évidence que les caractéristiques individuelles ont des effets tant directs qu'indirects sur le bien-être et que ces mêmes caractéristiques explique en partie, les relations entre la situation professionnelle et le bien-être. Plus spécifiquement, des situations professionnelles peuvent influencer l'expression de certaines caractéristiques individuelles, soit en contribuant à leurs activations ou à leurs inhibitions. De plus, l'impact des caractéristiques individuelles sur le bien-être semble dépendre de la situation professionnelle. Il est donc important de considérer les influences simultanées et réciproques des caractéristiques individuelles et de la situation contextuelle et professionnelle pour rendre compte du bien-être général et professionnel. -- This thesis explores how individual characteristics and professional situations correspond to well-being. Drawing from various theoretical backgrounds, such as career construction theory, justice theory, models of job strain, and theories on subtle discrimination, a number of specific hypotheses are put forward pertaining to a selection of individual and professional aspects as well as general and work-related well-being. The six studies presented in this thesis focus on specific aspects and adopt different methodological and theoretical approaches. The first two studies concern the validation of the career adapt-abilities scale and test the potential of career adapt-abilities to mediate the relationship between dispositions and outcomes. The third study extends the hypothesis of career adapt-abilities as a mediator and finds that it mediates the effects of job insecurity and job strain on general and professional well-being. The fourth study adopts a longitudinal approach and tests the associations between personality traits and career adaptability and well-being in four different professional situations. Study five concerns another individual characteristic, belief in a just world, and illustrates how justice beliefs drive perceptions of organizational justice, which in turn impact, on well-being outcomes one year later. The final study focuses on the professional experiences of a specific population, immigrants in Switzerland, and confirms that being a target of incivilities is related to national origin. Globally, this thesis finds that individual characteristics have direct and indirect influences on well-being and that these characteristics may also mediate the associations between professional situations and outcomes. In particular, the professional situation may alter the display of individual characteristics, either by contributing to their activation or their depletion, and the ways in which individual factors influence well-being does seem to depend on the professional situation. It is thus necessary to adopt a "both...and" perspective when studying the impact of individual and professional characteristics as these factors mutually influence each other.

Interleukin-6 and chondrocyte mineralisation act in tandem to promote experimental osteoarthritis.

OBJECTIVES: Basic calcium phosphate (BCP) crystal and interleukin 6 (IL-6) have been implicated in osteoarthritis (OA). We hypothesise that these two factors may be linked in a reciprocal amplification loop which leads to OA. METHODS: Primary murine chondrocytes and human cartilage explants were incubated with hydroxyapatite (HA) crystals, a form of BCP, and the modulation of cytokines and matrix-degrading enzymes assayed. The ability of IL-6 to stimulate chondrocyte calcification was assessed in vitro. The mechanisms underlying the effects of HA on chondrocytes were investigated using chemical inhibitors, and the pathways mediating IL-6-induced calcification characterised by quantifying the expression of genes involved in chondrocyte mineralisation. The role of calcification in vivo was studied in the meniscectomy model of murine OA (MNX), and the link between IL-6 and cartilage degradation investigated by histology. RESULTS: In chondrocytes, BCP crystals stimulated IL-6 secretion, further amplified in an autocrine loop, through signalling pathways involving Syk and PI3 kinases, Jak2 and Stat3 molecules. Exogenous IL-6 promoted calcium-containing crystal formation and upregulation of genes involved in calcification: the pyrophosphate channel Ank, the calcium channel Annexin5 and the sodium/phosphate cotransporter Pit-1. Treatment of chondrocytes with IL-6 inhibitors significantly inhibited IL-6-induced crystal formation. In meniscectomised mice, increasing deposits of BCP crystals were observed around the joint and correlated with cartilage degradation and IL-6 expression. Finally, BCP crystals induced proteoglycan loss and IL-6 expression in human cartilage explants, which were reduced by an IL-6 inhibitor. CONCLUSIONS: BCP crystals and IL-6 form a positive feedback loop leading to OA. Targeting calcium-containing crystal formation and/or IL-6 are promising therapeutic strategies in OA.

Large scale assessment of regulatory evolution and transcriptome complexity in mammals

AbstractIn addition to genetic changes affecting the function of gene products, changes in gene expression have been suggested to underlie many or even most of the phenotypic differences among mammals. However, detailed gene expression comparisons were, until recently, restricted to closely related species, owing to technological limitations. Thus, we took advantage of the latest technologies (RNA-Seq) to generate extensive qualitative and quantitative transcriptome data for a unique collection of somatic and germline tissues from representatives of all major mammalian lineages (placental mammals, marsupials and monotremes) and birds, the evolutionary outgroup.In the first major project of my thesis, we performed global comparative analyses of gene expression levels based on these data. Our analyses provided fundamental insights into the dynamics of transcriptome change during mammalian evolution (e.g., the rate of expression change across species, tissues and chromosomes) and allowed the exploration of the functional relevance and phenotypic implications of transcription changes at a genome-wide scale (e.g., we identified numerous potentially selectively driven expression switches).In a second project of my thesis, which was also based on the unique transcriptome data generated in the context of the first project we focused on the evolution of alternative splicing in mammals. Alternative splicing contributes to transcriptome complexity by generating several transcript isoforms from a single gene, which can, thus, perform various functions. To complete the global comparative analysis of gene expression changes, we explored patterns of alternative splicing evolution. This work uncovered several general and unexpected patterns of alternative splicing evolution (e.g., we found that alternative splicing evolves extremely rapidly) as well as a large number of conserved alternative isoforms that may be crucial for the functioning of mammalian organs.Finally, the third and final project of my PhD consisted in analyzing in detail the unique functional and evolutionary properties of the testis by exploring the extent of its transcriptome complexity. This organ was previously shown to evolve rapidly both at the phenotypic and molecular level, apparently because of the specific pressures that act on this organ and are associated with its reproductive function. Moreover, my analyses of the amniote tissue transcriptome data described above, revealed strikingly widespread transcriptional activity of both functional and nonfunctional genomic elements in the testis compared to the other organs. To elucidate the cellular source and mechanisms underlying this promiscuous transcription in the testis, we generated deep coverage RNA-Seq data for all major testis cell types as well as epigenetic data (DNA and histone methylation) using the mouse as model system. The integration of these complete dataset revealed that meiotic and especially post-meiotic germ cells are the major contributors to the widespread functional and nonfunctional transcriptome complexity of the testis, and that this "promiscuous" spermatogenic transcription is resulting, at least partially, from an overall transcriptionally permissive chromatin state. We hypothesize that this particular open state of the chromatin results from the extensive chromatin remodeling that occurs during spermatogenesis which ultimately leads to the replacement of histones by protamines in the mature spermatozoa. Our results have important functional and evolutionary implications (e.g., regarding new gene birth and testicular gene expression evolution).Generally, these three large-scale projects of my thesis provide complete and massive datasets that constitute valuables resources for further functional and evolutionary analyses of mammalian genomes.

Cardiac expression of ms1/STARS, a novel gene involved in cardiac development and disease, is regulated by GATA4.

Ms1/STARS is a novel muscle-specific actin-binding protein that specifically modulates the myocardin-related transcription factor (MRTF)-serum response factor (SRF) regulatory axis within striated muscle. This ms1/STARS-dependent regulatory axis is of central importance within the cardiac gene regulatory network and has been implicated in cardiac development and postnatal cardiac function/homeostasis. The dysregulation of ms1/STARS is associated with and causative of pathological cardiac phenotypes, including cardiac hypertrophy and cardiomyopathy. In order to gain an understanding of the mechanisms governing ms1/STARS expression in the heart, we have coupled a comparative genomic in silico analysis with reporter, gain-of-function, and loss-of-function approaches. Through this integrated analysis, we have identified three evolutionarily conserved regions (ECRs), α, SINA, and DINA, that act as cis-regulatory modules and confer differential cardiac cell-specific activity. Two of these ECRs, α and DINA, displayed distinct regulatory sensitivity to the core cardiac transcription factor GATA4. Overall, our results demonstrate that within embryonic, neonatal, and adult hearts, GATA4 represses ms1/STARS expression with the pathologically associated depletion of GATA4 (type 1/type 2 diabetic models), resulting in ms1/STARS upregulation. This GATA4-dependent repression of ms1/STARS expression has major implications for MRTF-SRF signaling in the context of cardiac development and disease.

Dynamical modeling and analysis of large cellular regulatory networks.

The dynamical analysis of large biological regulatory networks requires the development of scalable methods for mathematical modeling. Following the approach initially introduced by Thomas, we formalize the interactions between the components of a network in terms of discrete variables, functions, and parameters. Model simulations result in directed graphs, called state transition graphs. We are particularly interested in reachability properties and asymptotic behaviors, which correspond to terminal strongly connected components (or "attractors") in the state transition graph. A well-known problem is the exponential increase of the size of state transition graphs with the number of network components, in particular when using the biologically realistic asynchronous updating assumption. To address this problem, we have developed several complementary methods enabling the analysis of the behavior of large and complex logical models: (i) the definition of transition priority classes to simplify the dynamics; (ii) a model reduction method preserving essential dynamical properties, (iii) a novel algorithm to compact state transition graphs and directly generate compressed representations, emphasizing relevant transient and asymptotic dynamical properties. The power of an approach combining these different methods is demonstrated by applying them to a recent multilevel logical model for the network controlling CD4+ T helper cell response to antigen presentation and to a dozen cytokines. This model accounts for the differentiation of canonical Th1 and Th2 lymphocytes, as well as of inflammatory Th17 and regulatory T cells, along with many hybrid subtypes. All these methods have been implemented into the software GINsim, which enables the definition, the analysis, and the simulation of logical regulatory graphs.