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<p>A microarray analysis of RNA from contact inhibited CEF indicated a hypoxic signature in the contact inhibition program of gene expression (Ghosh <em>et al</em>., 2009). The purpose of this thesis was to investigate whether GAS genes known to be induced during contact inhibition are inducible by hypoxia. The gene p20K was selected as the model for this investigation because it is a growth arrest specific (GAS) gene with a well-characterized promoter (Mao <em>et al</em>., 1993). p20K expression was shown to be positively regulated in hypoxia. It was then determined by transient expression assay that this induction occurred at the promoter level. Interestingly by dissecting the promoter it was found that the quiescent responsive unit (QRU) was required for promoter induction during hypoxia. It has previously been shown that the QRU was required for contact inhibition induction of p20K in a C/EBPβ dependent manner (Mao <em>et al</em>., 1993; Kim <em>et al</em>., 1999).</p> <p>The mechanism behind hypoxic induction of the QRU was then investigated. The kinetics of HIF1α and p20K induction during hypoxia demonstrated that HIF1α was transiently expressed between 2-8 hrs of hypoxia while p20K was induced after 8 hrs of hypoxia. Co-Immuniprecipitation assay was also used to determine if a HIF1α-C/EBPβ interaction occurred, however, this molecular interaction could not be shown. These experiments suggests that HIF1α is not involved with the induction of the QRU. Over-expression of the dominant negative C/EBPβΔ184 repressed p20K induction, thus implicating C/EBPβ in activation in both contact inhibition and hypoxia. We also observed by western blot analysis that the C/EBP family member CHOP was repressed during hypoxia, causing a decrease in the amount of CHOP-C/EBPβ complexes in the cell. It was also found that over-expression of CHOP antagonized the induction of p20K by hypoxia. In conclusion hypoxia represses CHOP levels resulting in an increase of potent C/EBPβ homodimers at the expense of the inactive CHOP-C/EBPβ heterodimers.</p> / Master of Science (MSc)

<p>The study of growth arrest specific (GAS) genes is critical for our understanding of quiescence cell states. C/EBP-β is a transcriptional activator which is central to the expression of GAS genes in growth arrested cells. C/EBP-β is involved in the activation of numerous pathways, including mitogenesis, cytokine signaling, stress response, etc. Thus, it requires signaling cues which confer specificity in terms of gene expression.</p> <p>Here we used the p20K gene in chicken embryonic fibroblasts as a model system to study the control mechanisms of GAS genes. p20K is expressed in conditions such as contact inhibition mediated growth arrest and mild hypoxia. Here we explored the control mechanism mediated by ERK2 at the p20K promoter (QRU), as a mode of regulation which confers C/EBP-β binding specificity.</p> <p>In this study we demonstrate that ERK2 is recruited to the QRU in proliferative cells, i.e. where p20K is repressed. Using ChIP analysis we show that ERK2 binds directly to the QRU in proliferative cell states, but not in growth arrested cell conditions. Using a similar approach we demonstrate that ERK2 binding to the QRU is lost in states of hypoxia, where p20K is strongly induced. Furthermore, we show that this interaction is specific to ERK2 and is not observed with the related ERK1 kinase. Lastly, we employed transient expression assays to illustrate that ERK2 acts as a transcriptional repressor of the QRU. Through these experiments we have illustrated that ERK2 mediated transcriptional repression is a novel control mechanism at the QRU which skews C/EBP-β mediated signaling networks in proliferating cells.</p> / Master of Science (MSc)

We reported previously that prostaglandin E2 (PGE2) upregulates IL-23 in vitro in bone marrow-derived dendritic cells (DC), and in vivo in models of collagen-induced arthritis and inflammatory bowel disease, leading to preferential Th17 development and activity. There is very little information on the molecular mechanisms involved in the PGE2-induced upregulation of Il23a gene expression. In the present study we investigated the signaling pathways and transcription factors involved in the stimulatory effect of PGE2. Although PGE2 does not induce IL-23p19 expression by itself, it synergizes with both extra- and intracellular TLR ligands and with inflammatory cytokines such as TNFα. We established that the effect of PGE2 in conjunction with either LPS or TNFα is mediated through the EP4 receptor and the cAMP-dependent activation of both PKA and EPAC. Using the EP4 agonist PGE1OH in conjunction with TNFα, we found that PKA-induced PCREB and EPAC-induced PC/EBPβ mediate the stimulatory effect of PGE2 on IL-23p19 expression. This is the first report of CREB and C/EBPβ involvement in Il23a promoter activation. Mutation within the putative CREB and C/EBP sites combined with in vivo DNA binding (ChIP) assays identified the distal CREB site (-1125) and the two proximal C/EBP sites (-274 and -232) as essential for PKA-activated CREB and EPAC-activated C/EBPβ induced IL-23p19 expression. / Microbiology and Immunology

Le gène AFP est exprimé sélectivement par le foie fœtal et dans l’hépatome, formant un excellent modèle pour étudier les mécanismes de différenciation qui sont déréglés dans le cancer. L’hépatome, par exemple, est réfractaire à l’action anti-proliférative et AFP-suppressive des hormones glucocorticoïdes. Cette thèse porte sur la régulation transcriptionnelle du locus AFP et plus particulièrement sur l’identification des facteurs de différenciation hépatique l’induisant, avec l’objectif ultime de forcer la différenciation tumorale. À l’aide d’empreintes génomiques in vivo, je démontre pour la première fois l’occupation d’un site C/EBP au promoteur AFP, contrairement au dogme voulant que HNF1 occupe cette position critique. Des études d’immunoprécipitation de chromatine ont établi la présence de C/EBP au promoteur et aux amplificateurs du gène AFP. De plus, j’ai montré par transfection et transgenèse que C/EBP active le gène AFP en tandem avec le récepteur nucléaire FTF et que cette coopérativité dépend du positionnement précis des deux facteurs l’un par rapport à l’autre au promoteur AFP. Une substitution ou une modification de la distance entre les deux sites de fixation des facteurs change radicalement le comportement du gène pendant le développement et dans l’hépatome : ceci semble suggérer une contrainte nucléosomale déterminante dans l’activité onco-fœtale du promoteur AFP. On observe aussi une diminution du recrutement de C/EBP ou des corégulateurs CBP/p300 aux régions régulatrices AFP au cours du développement hépatique et de la croissance d’une lignée d’hépatome. La suite de ces travaux a permis d’identifier HNF4 sur les régions amplificatrices du gène AFP dans un modèle d’hépatome et chez des rats adultes. Ces résultats suggèrent un rôle négatif de HNF4 sur la transcription du gène AFP et une dérégulation possible de ce facteur dans les hépatomes. Nos travaux indiquent aussi que les complexes C/EBP présents aux régions amplificatrices AFP sont directement ciblés par le récepteur des glucocorticoïdes (via son domaine de liaison à l’ADN) dont la présence empêche le recrutement des cofacteurs sur ces régions. L’ensemble des résultats présentés dans cette thèse suggère donc un rôle central des protéines C/EBP dans la régulation développementale et hormonale du locus AFP. Celles-ci, en combinaison avec FTF, constituent ainsi de bons candidats pour forcer la différenciation tumorale. / The AFP gene is expressed specifically in the fetal liver as well as in some hepatoma cells, which makes this gene an excellent model to study differentiation processes that are deregulated in cancer. Hepatoma cells, for example, are often refractory to the AFP-suppressive as well as anti-proliferative effects of glucocorticoid hormones. This thesis explores transcriptional regulation of the AFP locus with a particular attention given to the identification of hepatic differentiation factors involved in the activation of this locus, the ultimate goal being to enforce tumor differentiation. Using in vivo footprinting experiments, I demonstrate for the first time the occupation of a C/EBP site in the AFP promoter, a site which was generally believed to be occupied by HNF1 proteins. Chromatin immunoprecipitation experiments confirmed the binding of C/EBP proteins to the AFP gene promoter as well as its enhancers. Furthermore, we showed by using transfection and transgenesis experiments that C/EBP proteins activate the AFP gene in tandem with the nuclear receptor FTF and that this cooperativity depends on the precise positionning of the two sites relative to one another. A deletion or modification of the distance between these two factors drastically modifies the expression timing of the AFP gene during hepatic development, suggesting a nucleosome position effect crucial for the onco-fetal activity of the gene. We also observe a differential recruitement of C/EBP or the coregulators CBP/p300 on AFP regulatory regions during hepatic development and growth of a hepatoma cell line. We then identified HNF4 on the enhancer regions of the AFP locus in a rat hepatoma model and in adult rat liver. This suggests that HNF4 negatively influences AFP gene transcription and underscores a possible deregulation of this factor in hepatoma cells. Our work also indicates that the C/EBP complexes present at two enhancer regions of the AFP gene are directly targeted by the glucocorticoid receptor (via its DNA-binding domain), leading to a loss of cofactor recruitement and AFP gene repression. On the whole, this thesis underscores a central role for C/EBP proteins in AFP locus developmental and hormonal regulation. Therefore, C/EBP combined with FTF represent good candidates to enforce tumor cell differentiation.

Le récepteur nucléaire Fetoprotein Transcription Factor (FTF) identifié par notre laboratoire et exprimé principalement dans le système digestif est un régulateur important du métabolisme des lipides et des stéroïdes, de la prolifération cellulaire et du développement embryonnaire. Plusieurs groupes ont constaté que l’influence du récepteur FTF sur la synthèse de stéroïdes et la régulation du cycle cellulaire stimule la prolifération tumorale de cellules d’origine tissulaire diverse. Mes études de doctorat ont porté sur l’expression tissulaire de FTF, sur la caractérisation d’un nouvel élément régulateur de son promoteur et sur l’identification par immunoprécipitation de chromatine (ChIP-chip) des cibles transcriptionnelles de FTF dans le foie de souris fœtale et adulte et dans les cellules d’hépatome humain. Ces études ont permis de mieux définir le rôle métabolique de FTF ainsi que son rôle développemental et son implication potentielle dans la carcinogenèse hépatique. L’expression de FTF par les organes du système digestif et par certaines structures nerveuses, sa régulation par des récepteurs nucléaires métaboliques et sa liaison aux promoteurs de multiples enzymes et transporteurs impliqués dans le métabolisme énergétique placent FTF dans une position clé dans l’homéostasie métabolique et énergétique de l’organisme. Le facteur de transcription C/EBPpartenaire de FTF au promoteur de l’AFP et impliqué lui aussi dans le développement hépatique et le métabolisme énergétique, est lié au promoteur de 20% des cibles transcriptionnelles de FTF. De plus, C/EBP lie le promoteur de FTF formant ainsi une autre boucle activatrice s’ajoutant au réseau transcriptionnel hépatique. Dans les cellules d’hépatome, FTF lie les promoteurs de plusieurs gènes impliqués dans la prolifération et le maintien des cellules tumorales, soit des régulateurs de la réplication, de la croissance et de l’apoptose cellulaire. FTF fait donc partie intégrante du réseau transcriptionnel hépatique régissant le développement et la différenciation hépatique et le maintien du métabolisme énergétique chez l’adulte et est vraisemblablement impliqué dans la promotion de la cancérogenèse hépatique. / FTF is a nuclear receptor principally expressed in adult digestive organs that has been shown to act as a major regulator of lipids and steroids metabolism, cellular proliferation and embryonic development. FTF involvement in steroid synthesis and cell cycle regulation tends toward the stimulation of tumor proliferation in neoplasic tissues in which FTF is expressed. However, more studies of FTF function in normal and disease states and on its regulation are needed to draw a complete picture of FTF activity in cell physiology. Within the context of my studies, I delineated the FTF adult and fetal tissular expression, characterized a novel Ftf promoter element and identified FTF direct hepatic transcriptional targets in fetal, adult and tumor cell lines by using chromatin immunoprecipitation (ChIP-on-chip). These studies defined new FTF functions in metabolism, fetal development and hepatic carcinogenesis. FTF expression in digestive system and in neural structures controlling eating behavior, its transcriptional regulation by metabolic nuclear receptors and its binding to enzyme and transporter gene promoters driving energy metabolism, puts FTF in a key location for governing cellular and organismal energy metabolism. C/EBP, a transcriptional FTF partner on the Afp gene promoter and also involved in energy metabolism, is bound to 20% of the FTF targets including FTF itself thus adding branches to the complex hepatic transcriptional network. In hepatoma cells, FTF binds to proliferation and tumor cell maintenance genes like replication, growth and apoptosis regulators. Therefore, FTF belongs to the hepatic transcription network that governs hepatic development, differentiation and adult energy metabolism and is likely to be involved in promoting hepatic tumorogenesis.

The aim of the study was to illustrate how evidence-based praxis is implemented in the police agencies activities with crime prevention work. We asked two questions: a) What actions does the Swedish Police authority take when implementing evidence-based praxis in its operations? and b) What opportunities contra difficulties do the managers, community police officers and teachers at the police institute see with evidence-based praxis? This was investigated through qualitative interviews in order to capture the participants personal opinions and experiences about the subject. Evidence-based policies are practiced through the crime-prevention work that takes place through local cooperations. The role of community police officers constitutes an important strategic role as being cosmopolitan, which ensures that communication between the police and society works, which in turn forms the basis for the crime-prevention work. The result also shows that the police cannot take care of law enforcement on their own initiative; responsibility also rests on individuals, groups and other organizations. We see patterns of a majorly positive attitude within the police, which contradicts previous research. There are not enough evidence-based methods within all working fields of the police, which has emerged as an important factor in order to implement evidence-based praxis. Whilst the police may be positive towards evidence-based methods, they can not solely rely on them. The resources are too lacking in order for the evidence-based praxis to also permeate the police authority's work. In order for the police authority's work to be carried out by evidence-based methods, community police officers need to have a higher status internally. They also need a mandate in the form of being able to make decisions about operative resources. The main contribution of the study´s is that evidence-based praxis needs to be strategically implemented, and the key people who work primarily with evidence-based praxis needs a higher status within the organization.