Das Lipopolysaccharid Bindende Protein (LBP) ist ein überwiegend in der Leber synthetisiertes Akutphaseprotein. Es bindet den Zellwandbestandteil Lipopolysaccharid (LPS) Gram-negativer Bakterien und transportiert es zu zellulären Rezeptoren, wodurch das angeborene Immunsystem aktiviert wird. In dieser Arbeit wird die Regulation der LBP-Expression in Interleukin (IL)-1, IL-6 und Dexamethason (Dex) stimulierten humanen Hepatomzelllinien HuH-7 und HepG2 untersucht. Der wichtigste Stimulator ist dabei IL-6, dessen Wirkung über die Transkriptionsfaktoren (TF) Stat-3, C/EBP-beta und AP-1 vermittelt wird. Für alle 3 TF konnten aktive Bindungsstellen auf dem LBP-Promotor nachgewiesen werden. Für IL-1-Effekte die u. a. über den TF NF-kappaB vermittelt werden, konnten ebenfalls aktive Bindungsstellen nachgewiesen werden. Die Wirkung von Dex wird über Glucocorticoid Responsive Elements (GREs) vermittelt. Auf dem LBP-Promotor befinden, sich wie gezeigt werden konnte, mehrere aktive GREs, wobei einige verstärkend und einige hemmend wirken. Eine zu beobachtende Synergiewirkung von Dex und IL-6 wird durch die Aufregulation des IL-6-Rezeptors durch Dex verursacht. Die LBP-Expression kann durch TGF (Transforming Growth Factor)-beta gehemmt werden. Der TGF-beta-Signalweg über Smads ist in den Hepatomzellen aktiv, vermittelt aber nicht den TGF-beta-Hemmeffekt, sondern eine geringe stimulierende Wirkung, die bei alleiniger TGF-beta-Inkubation auftritt. Die inhibierende Wirkung von TGF-beta wird durch Gfi-1- und AP-1-Bindungsstellen vermittelt. Die Gfi-1-Bindungsstelle nimmt dabei, wie hier erstmals gezeigt werden konnte, eine herausragende Stellung ein. Die Aufklärung der LBP-Regulation und dabei besonders die Hemmung der LBP-Expression kann mittelfristig dazu beitragen, den klinischen Verlauf von inflammatorischen und infektiösen Erkrankungen zu beeinflussen und bietet daher Potenzial für neue Therapieansätze. / Lipopolysaccharide (LPS) binding protein (LBP) is an acute phase protein with the ability to bind and transfer LPS of Gram-negative bacteria. This soluble pattern recognition molecule represents an important defense principle of the host. Regulation of the hepatic acute phase response and its termination are important mechanisms for limiting systemic inflammatory activity of the host. Here were analyze the cooperation of Interleukin (IL)-1, IL-6, and Dexamethasone (Dex) at LBP expression in the hepatoma cell lines HuH-7 and Hep G2. The major inducer of LBP expression is IL-6. Within the LBP promoter numerously highly consensus binding sites such as AP-1, C/EBP-beta? and STAT3 are present, that confer transcriptional activity as shown by truncation and mutation experiments. Additionally, activate NF-kappaB sites activated by IL-1 were detected at the LBP promoter. By mutation experiments of the promoter furthermore were found differentially active glucocorticoid response elements (GREs). The promoter contains GREs enhancing the activity as well as inhibitory ones. The enhancing effect towards LBP expression by Dex was mediated by IL-6. Dex stimulated the expression of the IL-6 receptor and therefore upregulated the IL-6 pathway. Transforming Growth Factor (TGF)-beta is able to inhibit LBP expression in stimulated cells. An AP-1 binding site was identified mediating inhibitory TGF-beta effects towards LBP promoter activity. Furthermore it was shown that a growth factor independence (Gfi)-1 binding site localized near the AP-1 site is essential for mediating the TGF-beta inhibitory effect. The relevancy of the Gfi-1 site fore mediating TGF-beta effects indicates a novel mechanism for understanding inhibitory TGF-beta effects at the transcriptional level. In summary the complex regulation of LBP were elucidate which may help to eventually develop novel intervention strategies for acute phase, sepsis, and septic shock.

The development of shape in multicellular organisms has intrigued human minds for millenia. Empowered by modern genetic techniques, molecular biologists are now striving to not only dissect developmental processes, but to exploit their modularity for the design of custom living systems used in bioproduction, remediation, and regenerative medicine. Currently, our capacity to harness this potential is fundamentally limited by a lack of spatiotemporal control over gene expression in multicellular systems. While several synthetic genetic circuits for control of multicellular patterning have been reported, hierarchical induction of gene expression domains has received little attention from synthetic biologists, despite its fundamental role in biological self-organization. In this thesis, I introduce the first synthetic genetic system implementing population-based AND logic for programmed hierarchical patterning of bacterial populations of Escherichia coli, and address fundamental prerequisites for implementation of an analogous genetic circuit into the emergent multicellular plant model Marchantia polymorpha. In both model systems, I explore the use of bacteriophage T7 RNA polymerase as a gene expression engine to control synthetic patterning across populations of cells. In E. coli, I developed a ratiometric assay of bacteriophage T7 RNA polymerase activity, which I used to systematically characterize different intact and split enzyme variants. I utilized the best-performing variant to build a three-color patterning system responsive to two different homoserine lactones. I validated the AND gate-like behavior of this system both in cell suspension and in surface culture. Then, I used the synthetic circuit in a membrane-based spatial assay to demonstrate programmed hierarchical patterning of gene expression across bacterial populations. To prepare the adaption of bacteriophage T7 RNA polymerase-driven synthetic patterning from the prokaryote E. coli to the eukaryote M. polymorpha, I developed a toolbox of genetic elements for spatial gene expression control in the liverwort: I analyzed codon usage across the transcriptome of M. polymorpha, and used insights gained to design codon-optimized fluorescent reporters successfully expressed from its nuclear and chloroplast genomes. For targeting of bacteriophage T7 RNA polymerase to these cellular compartments, I functionally validated nuclear localization signals and chloroplast transit peptides. For spatiotemporal control of bacteriophage T7 RNA polymerase in M. polymorpha, I characterized spatially restricted and inducible promoters. For facilitated posttranscriptional processing of target transcripts, I functionally validated viral enhancer sequences in M. polymorpha. Taking advantage of this genetic toolbox, I introduced inducible nuclear-targeted bacteriophage T7 RNA polymerase into M. polymorpha. I showed implementation of the bacteriophage T7 RNA polymerase/PT7 expression system accompanied by hypermethylation of its target nuclear transgene. My observations suggest operation of efficient epigenetic gene silencing in M. polymorpha, and guide future efforts in chassis engineering of this multicellular plant model. Furthermore, my results encourage utilization of spatiotemporally controlled bacteriophage T7 RNA polymerase as a targeted silencing system for functional genomic studies and morphogenetic engineering in the liverwort. Taken together, the work presented enhances our capacity for spatiotemporal gene expression control in bacterial populations and plants, facilitating future efforts in synthetic morphogenesis for applications in synthetic biology and metabolic engineering.

Obesidade é fator de risco para a hipertensão arterial e os mecanismos envolvidos nesta doença não são totalmente esclarecidos. Camundongos C57BL/6J e transgênicos com com deleção em neurônios e glia da via inflamatória do receptor toll-like-NFκB foram submetidos à dieta hiperlipídica (HL) por 8 e 15 semanas e avaliados parâmetros metabólicos, pressão arterial, frequência cardíaca, atividade do sistema nervoso autônomo, fatores inflamatórios e neuropeptídeos no hipotálamo e no tronco encefálico. Os camundongos expostos HL desenvolveram hipertensão arterial acompanhada de disfunção autonômica e aumento de CART no DMH. Os animais transgênicos quando submetidos à dieta HL desenvolveram um quadro de obesidade, porém não apresentaram hipertensão arterial e disfunção autonômica. Além disso, o grupo de animais HL aumentou o RNAm de CCL5 no hipotálamo e de CD86 no tronco-encefálico e a densidade de microglia no NTS caudal. Os resultados sugerem novos mecanismos para a hipertensão e disfunção autonômica secundárias à ingestão de dieta hiperlipídica mostrando o papel do CART o DMH e o envolvimento da via inflamatória do TLR-NFκB em neurônios e glia nos mecanismos desta patologia. / Obesity is a risk factor for high blood pressure and the mechanisms involved in this disease are not fully clarified. C57BL/6J and transgenic mice with toll-like-NFκB receptor inflammatory- pathway deletion in neurons and glia were fed with high-fat diet (HL) for 8 or 15 weeks and assessed metabolic parameters, blood pressure, heart rate, autonomic nervous system activity, inflammatory factors and neuropeptides in the hypothalamus and brainstem. The HL mice developed hypertension accompanied with autonomic dysfunction and increased CART in DMH. Transgenic animals when submitted to HL diet developed obesity, but not showed high blood pressure and autonomic dysfunction. In addition, HL animals had increased CCL5 mRNA in hypothalamus, CD86 mRNA in brainstem and micróglia density in caudal NTS. The results suggest new mechanisms for hypertension and autonomic dysfunction secondary to intake of high-fat diet by showing CART role in DMH and the involvement of the inflammatory pathway TLR-NFκB in neurons and glia.

Obesidade é fator de risco para a hipertensão arterial e os mecanismos envolvidos nesta doença não são totalmente esclarecidos. Camundongos C57BL/6J e transgênicos com com deleção em neurônios e glia da via inflamatória do receptor toll-like-NFκB foram submetidos à dieta hiperlipídica (HL) por 8 e 15 semanas e avaliados parâmetros metabólicos, pressão arterial, frequência cardíaca, atividade do sistema nervoso autônomo, fatores inflamatórios e neuropeptídeos no hipotálamo e no tronco encefálico. Os camundongos expostos HL desenvolveram hipertensão arterial acompanhada de disfunção autonômica e aumento de CART no DMH. Os animais transgênicos quando submetidos à dieta HL desenvolveram um quadro de obesidade, porém não apresentaram hipertensão arterial e disfunção autonômica. Além disso, o grupo de animais HL aumentou o RNAm de CCL5 no hipotálamo e de CD86 no tronco-encefálico e a densidade de microglia no NTS caudal. Os resultados sugerem novos mecanismos para a hipertensão e disfunção autonômica secundárias à ingestão de dieta hiperlipídica mostrando o papel do CART o DMH e o envolvimento da via inflamatória do TLR-NFκB em neurônios e glia nos mecanismos desta patologia. / Obesity is a risk factor for high blood pressure and the mechanisms involved in this disease are not fully clarified. C57BL/6J and transgenic mice with toll-like-NFκB receptor inflammatory- pathway deletion in neurons and glia were fed with high-fat diet (HL) for 8 or 15 weeks and assessed metabolic parameters, blood pressure, heart rate, autonomic nervous system activity, inflammatory factors and neuropeptides in the hypothalamus and brainstem. The HL mice developed hypertension accompanied with autonomic dysfunction and increased CART in DMH. Transgenic animals when submitted to HL diet developed obesity, but not showed high blood pressure and autonomic dysfunction. In addition, HL animals had increased CCL5 mRNA in hypothalamus, CD86 mRNA in brainstem and micróglia density in caudal NTS. The results suggest new mechanisms for hypertension and autonomic dysfunction secondary to intake of high-fat diet by showing CART role in DMH and the involvement of the inflammatory pathway TLR-NFκB in neurons and glia.

L’ostéonécrose (ON) et les fractures (FR) sont des complications qui prennent de plus en plus place dans le traitement pédiatrique de la leucémie aiguë lymphoblastique (LAL). L’ON peut être causée par différents facteurs, dont principalement l’utilisation de glucocorticoïdes. Les glucocorticoïdes sont administrés lors du traitement de la leucémie dans le but d’initier l’apoptose des cellules malignes tout en ayant un effet anti-inflammatoire. Cependant, l’utilisation de ces corticostéroïdes comprend des effets secondaires sérieux, notamment le développement d’ostéonécrose. Des variantes génétiques peuvent mettre certains patients plus à risque que d’autres. Plusieurs gènes ont déjà été signalés comme régulés par les actions glucocorticoïdes (GC). Les variations génétiques présentes dans les régions régulatrices de ces gènes peuvent affecter leur fonctionnement normal et, en fin de compte, de déterminer un risque accru de développer l’ON associé au traitement contre la leucémie. Pour cette raison, plusieurs polymorphismes ont été identifiés et étudiés dans la cohorte QcALL de Ste-Justine, concernant les gènes suivants : ABCB1, ACP1, BCL2L11, NFKB1, PARP1, et SHMT1. Ces gènes jouent majoritairement un rôle dans les mécanismes d’action des glucocorticoïdes, mais quelques-uns ont plutôt un effet direct sur le développement d’ostéonécrose. Nos recherches ont démontré une corrélation entre ces polymorphismes et l’apparition d’ostéonécrose chez les patients de la cohorte QcALL, traités aux glucocorticoïdes. L'incidence cumulative de l'ostéonécrose a été évaluée rétrospectivement chez 305 enfants atteints de la leucémie qui ont subi un traitement à l’hôpital Ste-Justine selon les protocoles DFCI de Boston (87-01, 91-01, 95-01 et 2000-01). Parmi les huit polymorphismes de BCL2L11 étudiés, les 891T> G (rs2241843) et 29201C> T (rs724710) ont été significativement associés à ON (p = 0.01 et p = 0.03, respectivement). L'association du polymorphisme 891T> G a été modulée par le type de corticostéroïde (CS), l’âge, le sexe et le groupe à risque (p ≤ 0,05). Le polymorphisme 29201C> T était particulièrement apparent chez les patients à haut risque (p = 0,003). La même étude était conduite en parallèle sur des patients de la cohorte DFCI de Boston (N = 192), et montrait des résultats significatifs pour les polymorphismes étudiés. En conclusion, les résultats de cette étude permettront de confirmer l’association de ces polymorphismes au développement d’ON chez les patients de LLA traités aux GC. / Osteonecrosis (ON) and fractures (FR) are complications that take place in the treatment of children acute lymphoblastic leukemia (cALL). They can be caused by various factors, mainly using glucocorticoids. The corticosteroids, dexamethasone (DXM) and prednisone (PDN) are administered during the treatment of leukemia to initiate apoptosis of malignant cells; while having an anti-inflammatory effect. However, the use of these corticosteroids has severe side effects, including the development of osteonecrosis. Moreover, some patients develop resistance to treatment, and are at risk of developing side effects. The genetic variants predispose some patients at higher risk than others. Several genes have been previously reported as up- or down regulated by the GCs actions. The genetic variations present in gene coding or regulatory regions can affect their function and ultimately determine an increased risk of developing ON associated to ALL therapy. Therefore, we investigated the association between several single nucleotide polymorphisms (SNPs) in six candidate genes: BCL2L11, NFKB1, PARP1, ABCB1, ACP1, and SHMT1. These genes play a role in the mechanisms of action of glucocorticoids, but some have more of a direct effect on the development of osteonecrosis. Our research has shown a correlation between these polymorphisms and the occurrence of osteonecrosis in patients in the QCALL cohort, treated with glucocorticoids. Cumulative incidence of osteonecrosis was assessed retrospectively in 305 children with ALL who underwent treatment with DFCI protocols (87-01, 91-01, 95-01 and 2000-01) in childhood ALL cohort from Quebec (QcALL). Among the eight tag BCL2L11 polymorphisms studied the 891T>G (rs2241843) and 29201C>T (rs724710) were significantly associated with ON (p = 0.01 and p = 0.03, respectively). Association of 891T>G polymorphism was modulated by type of corticosteroid (CS), age, sex and risk group (p ≤ 0.05 and that of 29201C>T was particularly apparent among high risk (p = 0.003) patients. These polymorphisms have shown significant ON association in several QcALL risk groups, mainly in corticosteroid groups, age < 10 years, and high risk (HR) group. Furthermore, the same study was conducted in parallel with patients in the replication (DFCI) cohort (N = 192), and we showed significant genetic association results for all studied polymorphisms. In conclusion, this study identifies that some ALL children have a high incidence of ON during the treatment that is highly associated with polymorphisms in different genes regulated by corticosteroids and ALL prognostic factors.

L'activació sostinguda de Hog1 porta a una inhibició del creixement cel·lular. En aquest treball, hem observat que el fenotip de letalitat causat per l'activació sostinguda de Hog1 és parcialment inhibida per la mutació del complexe SCFCDC4. La inhibició de la mort causada per l'activació sostinguda de Hog1 depèn de la via d'extensió de la vida. Quan Hog1 s'activa de manera sostinguda, la mutació al complexe SCFCDC4 fa que augmenti l'expressió gènica depenent de Msn2 i Msn4 que condueix a una sobreexpressió del gen PNC1 i a una hiperactivació de la deacetilassa Sir2. La hiperactivació de Sir2 és capaç d'inhibir la mort causada per l'activació sostinguda de Hog1. També hem observat que la mort cel·lular causada per l'activació sostinguda de Hog1 és deguda a una inducció d'apoptosi. L'apoptosi induïda per Hog1 és inhibida per la mutació al complexe SCFCDC4. Per tant, la via d'extensió de la vida és capaç de prevenir l'apoptosi a través d'un mecanisme desconegut. / Sustained Hog1 activation leads to an inhibition of cell growth. In this work, we have observed that the lethal phenotype caused by sustained Hog1 activation is prevented by SCFCDC4 mutants. The prevention of Hog1-induced cell death by SCFCDC4 mutation depends on the lifespan extension pathway. Upon sustained Hog1 activation, SCFCDC4 mutation increases Msn2 and Msn4 dependent gene expression that leads to a PNC1 overexpression and a Sir2 deacetylase hyperactivation. Then, hyperactivation of Sir2 is able to prevent cell death caused by sustained Hog1 activation. We have also observed that cell death upon sustained Hog1 activation is due to an induction of apoptosis. The apoptosis induced by Hog1 is decreased by SCFCDC4 mutation. Therefore, lifespan extension pathway is able to prevent apoptosis by an unknown mechanism.