Die Berlin Fett Maus Inzuchtlinie (BFMI) ist ein Modell für jugendliche Fettleibigkeit, die natürliche Mutationen aufweist, welche uns helfen können, die genetischen Mechanismen zu verstehen, die zu Fettleibigkeit führen. Bei früheren Untersuchungen, von Kreuzungen zwischen BFMI und B6N, wurde ein rezessiver Defekt auf Chromosom 3 (jObes1) identifiziert, der juvenile Fettleibigkeit verursacht. Die Feinkartierung engte den jObes1-Lokus, mit Hilfe einer fortgeschrittenen Kreuzungslinie auf das Bardet-Biedl-Syndrom 7 - Gen (Bbs7), das wahrscheinlichste Kandidatengen für den Phänotyp der juvenilen Fettleibigkeit in BFMI ein. Die Genexpressionsanalyse des gesamten Gehirns von BFMI- und B6N-Mäusen zeigte, dass die Expression des Gens Bbs7 bei BFMI-Mäusen im Vergleich zu den B6N-Referenzmäusen deutlich reduziert war. Sequenzvergleiche zwischen den beiden Linien BFMI und B6N zeigten zwei wesentliche Unterschiede zwischen ihnen: (1) eine 1.578 bp Deletion im Intron 8 von Bbs7 in BFMI-Mäusen, die einen CCCTC-bindenden Faktor (CTCF-Element) enthält (2) 16 Sequenzvarianten, die in der Bbs7-Promotorregion (36. 613.319 - 36.614.267, Ensembl release 102) in den BFMI-Mäusen, im Vergleich zur B6N-DNA-Sequenz, für die Unterschiede in der Bbs7-Genregulation verantwortlich sein könnten. Mit dieser Studie sollten zwei Hauptfragen beantwortet werden: (1) Hat die gelöschte Intron-8-Region von Bbs7, die ein CTCF-Element enthält, einen teilweisen oder vollständigen Einfluss auf die Entwicklung von juveniler Adipositas bei BFMI-Mäusen? (2) Was ist die ursächliche Variante in der Promotorregion des Bbs7-Gens, die zu Expressionsunterschieden des Bbs7-Gens zwischen BFMI und B6N führt? Sowohl der einzelne 5'-UTR-SNP, als auch die Deletion im Intron 8 von Bbs7, können zu dem beobachteten Phänotyp der BFMI-Mäuse beitragen, indem sie höchstwahrscheinlich die Bbs7-Expression verringern und den Fettanteil erhöhen. / The Berlin Fat Mouse Inbred (BFMI) line is a model for juvenile obesity which harbors natural mutations that can help us understand the genetic mechanisms leading to obesity. Previous research on crosses between BFMI and B6N identified a recessive defect causing juvenile obesity on chromosome 3 (jObes1). This explains around 40% of the bodyweight differences in an F2 cross between BFMI and the reference B6N mouse. Fine mapping, using an advanced intercross line, of the jObes1 locus, revealed the Bardet-Biedl syndrome 7 (Bbs7) gene as the most likely candidate gene for the juvenile obesity phenotype in the BFMI. Gene expression analysis on the whole brain of BFMI and B6N mice showed that the expression of the Bbs7 gene in BFMI mice was reduced significantly compared to the B6N reference mice. Sequence comparisons between the two lines BFMI and B6N showed two major differences between them: (1) a 1,578 bp deletion in intron 8 of Bbs7 in BFMI mice harboring a CCCTC-binding factor (CTCF-element) which works as a transcriptional activator, a repressor, or an insulator, located within the deletion, and (2) 16 sequence variants identified in the Bbs7 promoter region (36.613.319 – 36.614.267, Ensembl release 102) in the BFMI mice compared to the B6N DNA sequence which can be responsible for differences in Bbs7 gene regulation. This study aimed to answer two main questions: (1) Does the deleted intron 8 region of Bbs7 which includes a CTCF-element have any partial or complete impact on the development of juvenile obesity in BFMI mice and what is the explanation for that? (2) What is the causal variant in the promoter region of the Bbs7 gene that leads to expression differences of the Bbs7 gene between BFMI and B6N? Both, the single 5’ UTR-SNP and the deletion in intron 8 of Bbs7 can contribute to the observed phenotype in the BFMI mice most likely by reducing Bbs7 expression and increasing fat accumulation.

Regulation of expression DLX1 gene, whose elevated levels are detected in patients with acute myeloid leukemia with FLT3-ITD mutations, is not still completely explored topic. The first aim of this study was to determine which selected signaling pathways regulate gene expression of DLX1. ERK a JNK pathways were selected by using qRT-PCR and western blot. These pathways cause activation of the transcription factor AP-1 subunits, the AP-1 putative promoter binding site was identified also in the promoter of the DLX1 gene. The second aim of this study was to test the hypothesis on the regulation of gene expression of DLX1 (via ERK/JNK pathway) through AP-1 binding site on the promoter. Dual luciferase assay using luminescent luciferase activity was performed to test this hypothesis. Gene of the luciferase is contained in the used luciferase vector. The short and the long part of the DLX1 promoter (around AP-1 site) were inserted before the gene of the luciferase in the constructs used in this method. The results of this study indicate that the regulation of gene expression through AP-1 promoter binding site is important but not sufficient part of the regulatory cascade running through ERK and JNK pathway. There must be another transcription factors activated by ERK1/2 kinase which are probably also involved in...

Huntington’s disease (HD) is a neurodegenerative disorder caused by the inheritance of one mutant copy of the huntingtin gene. Mutant huntingtin protein (mHtt) contains an expanded polyglutamine repeat region near the N-terminus. Cleavage of mHtt releases an N-terminal fragment (N-mHtt) which translocates, and accumulates in the nucleus. Nuclear accumulation of N-mHtt has been directly associated with cellular toxicity. Decreased transcription is among the earliest detected changes that occur in the brains of HD patients and is consistently observed in all animal and cellular models of HD. Transcriptional dysregulation may trigger many of the perturbations that occur later in disease progression and an understanding of the effects of mHtt may lead to strategies to slow the progression of the disease. Current models of N-mHtt-mediated transcriptional dysregulation suggest that abnormal interactions between N-mHtt and transcription factors impair the ability of these transcription factors to associate at N-mHtt-affected promoters and properly regulate gene expression. We tested various aspects of these models using two N-mHtt-affected promoters in in vitro transcription assays and in two cell models of HD using techniques including overexpression of known N-mHtt-interacting transcription factors, chromatin immunoprecipitation, promoter deletion and mutation analyses and in vitro promoter binding assays. Based on our results and those in the literature, we proposed a new model of N-mHtt-mediated transcriptional dysregulation centered on the presence of N-mHtt at affected promoters. We concluded that simultaneous interaction of N-mHtt with multiple binding partners within the transcriptional machinery would explain the gene-specificity of N-mHtt-mediated transcriptional dysregulation, as well as the observation that some genes are affected early in disease progression while others are affected later. Our model explains why alleviating N-mHtt-mediated transcriptional dysregulation through overexpression of N-mHtt-interacting proteins has proven to be difficult and suggests that the most realistic strategy for restoring gene expression across the spectrum of N-mHtt affected genes is by reducing the amount of soluble nuclear N-mHtt.

La sénescence est considérée comme un mécanisme de suppression tumorale puisque les cellules potentiellement dangereuses, activent leurs protéines de sauvegarde pour arrêter leur prolifération. Les protéines de sauvegarde telles que RB et p53 sont activées suite à différents stress comme des dommages à l’ADN, le raccourcissement des télomères ou l’induction oncogénique. Les cellules sénescentes restent métaboliquement actives, subissent des modifications dans leur expression génique, et sécrètent des cytokines et des chimiokines qui ont des effets paracrines pro-oncogéniques, mais peuvent également contribuer à la stabilité de l’arrêt du cycle cellulaire dans la sénescence de façon autocrine. Une des particularités du phénotype sénescent est la dégradation sélective des protéines dépendante de l’ubiquitination et du protéasome. Parmi les cibles de dégradation se trouvent des protéines impliquées dans la biogenèse du ribosome, ainsi que celles d’autres voies cellulaires requises pour la croissance de cellules cancéreuses. Ceci est lié à un stress nucléolaire qui affecte la biogenèse du ribosome, menant à l’accumulation, dans le nucléoplasme ou le nucléole, de protéines ribosomiques. Ce comportement suggère que les ribosomes des cellules sénescentes seraient structurellement différents. Par conséquent, ceci pourrait entrainer des effets sur leurs capacités à réguler l’initiation, l’élongation et/ou la terminaison de la traduction des ARN messagers (ARNm). Par ailleurs, la déplétion de certaines protéines impliquées dans la ribogenèse, ainsi que la surexpression de protéines ribosomiques telles que RPS14/uS11 amènent à la sénescence. Malgré le stress nucléolaire et les défauts de ribogenèse associés à la sénescence, les cellules sénescentes présentent des niveaux de translecture du codon d’arrêt très diminué, suggérant l’existence de défauts de production de protéines allongées en C-terminal. Nous émettons l’hypothèse que les défauts de la ribogenèse affecteraient la fonction des protéines ribosomiques et des ribosomes. Cette perturbation aurait un impact sur le rôle de suppresseur tumoral de la sénescence. Le premier objectif de cette thèse consiste à démontrer le rôle de RPL22/eL22 en tant que régulateur du cycle cellulaire et inducteur de la sénescence. Le deuxième but est de démontrer que, malgré la perturbation nucléolaire, les ribosomes des fibroblastes sénescents reconnaissent les codons d’arrêt de façon plus efficace que les ribosomes des cellules transformées, ou des cellules normales en prolifération. Nous avons démontré que le phénotype de sénescence peut être induit quand l’expression de RPL22/eL22 est augmentée. RPL22/eL22 s’accumule principalement dans le nucléole, de manière différente de RPS14/uS11, dont l’accumulation est nucléoplasmique. En effectuant des essais kinases in vitro, nous avons montré que RPL22/eL22, tout comme RPS14/uS11, peuvent interagir et inhiber le complexe CDK4-Cycline D1 afin d’activer la voie de RB et établir l’arrêt du cycle cellulaire et la sénescence. Afin de démontrer la fidélité de la terminaison de la traduction dans les cellules sénescentes, nous avons utilisé un système de rapporteurs de luciférases, pour détecter les erreurs de translecture ainsi que pour avoir un contrôle interne du système. L’inactivation de la voie du suppresseur tumoral RB par surexpression de CDK4 ou de l’oncoprotéine virale E7, nous a permis d’observer l’augmentation de la translecture dans les cellules sénescentes. Tandis que l’activation de la voie de suppression tumorale RB, à l’aide du suppresseur de tumeur PML, de la surexpression de RPL22/eL22 et de RPS14/uS11, ainsi que de l’utilisation de Palbociclib (PD-0332991), un inhibiteur des kinases CDK4/6, a montré une réduction des erreurs de translecture. Ces résultats indiquent une nouvelle fonction des protéines du ribosome en tant que suppresseurs de tumeur, permettant d’inhiber les erreurs de translecture du codon d’arrêt de façon dépendante de la voie de RB. Ces travaux suggèrent que de petites molécules ou peptides pourraient simuler les fonctions inhibitrices de ces protéines ribosomiques afin de traiter certains cancers où la voie de RB est activable. / Senescence is considered a mechanism for tumor suppression since potentially dangerous cells activate their protective proteins to stop their proliferation. Safeguard proteins such as RB and p53 are activated as a result of stress such as DNA damage, telomere shortening or oncogenic induction. Senescent cells are metabolically active, they undergo changes in their gene expression and secrete cytokines and chemokines with pro-oncogenic paracrine effects, but which can also contribute to the stability of the senescent cell cycle arrest in an autocrine way. One of the peculiarities of the senescent phenotype is the selective ubiquitination and proteasome dependent-degradation of proteins involved in ribosome biogenesis and other cellular pathways required for cancer cell growth, leading to the accumulation, in the nucleoplasm or nucleolus, of ribosomal proteins. This behavior suggests that the ribosomes of senescent cells are structurally different. Therefore, this could have effects on their ability to regulate the initiation, elongation and/or translation termination of messenger RNAs (mRNAs). Moreover, the depletion of some proteins involved in ribogenesis, as well as the overexpression of ribosomal proteins such as RPS14/uS11 lead to senescence. Despite nucleolar stress and ribogenesis defects associated to senescence, global translation does not seem to be affected in senescence. Strikingly, senescent cells have reduced translational readthrough suggesting that they have defects in the production of C-terminal extended proteins. We hypothesize that defects in ribogenesis would affect the function of ribosomal proteins and ribosomes influencing the tumor suppressor role of senescence. The first aim of this thesis is to demonstrate the role of RPL22/eL22 as a regulator of the cell cycle and senescence inducer. The second aim of this thesis is to demonstrate that, despite the nucleolar disruption, the ribosomes of senescent fibroblasts recognize stop codons more efficiently than ribosomes from transformed cells, but also than ribosomes from proliferating normal cells. We found that the senescent phenotype can be induced by enhancing the expression of RPL22/eL22. RPL22/eL22 accumulates mainly in the nucleolus, unlike RPS14/uS11, whose accumulation is nucleoplasmic. By performing an in vitro kinase assay, we showed that RPL22/eL22, just like RPS14/uS11, can interact and inhibit the CDK4-Cyclin D1 complex in order to activate the RB pathway and establish cellular arrest and senescence. To assess translation termination accuracy in senescent cells, we used a system of luciferase reporters to measure the fidelity of translation termination. Inactivation of the RB tumor suppressor pathway using CDK4 or the viral oncoprotein E7 also increased readthrough in senescent cells while overexpression of PML, a tumor suppressor that activates the RB pathway, overexpression of RPL22/eL22 and RPS14/uS11, as well as the use of Palbociclib (PD-0332991), a CDK4/6 inhibitor, reduce readthrough errors. These results indicate a novel function of ribosomal proteins as tumor suppressors, making it possible to inhibit translational readthrough errors, in a RB-dependent pathway. This work suggests that small molecules or peptides could mimic the inhibitory functions of these ribosomal proteins in order to treat cancers where the RB pathway is activatable.

Tick-borne encephalitis is a serious disease of the central nervous system. It is caused by tick-borne encephalitis virus, which is transmitted by ticks. The Czech Republic is one of the countries with the highest prevalence of this disease. Tick-borne encephalitis virus is able to replicate in several cell types. In this work we focused on macrophage line IC-21 and dendritic cells, because these cells are the first, which encounter the virus and support its spreading in the host at early stage of infection. So far there is not known any specific receptor for virus entry into cells or which signaling pathways activates. Therefore, we decided to investigate the activation of selected signaling pathways after infection with tick-borne encephalitis virus and influence of tick saliva on this activation. We employed methods of dual luciferase reporter assay, immunosandwich assay and western blot. The obtained results showed that in virus infected IC-21 cells are activated phosphatidyl-inositol pathway, NF-κB pathway, signaling molecule Erk1/2 and others. Testing of tick saliva effect revealed significantly decreased activity of NF-κB, AP-1 and CREB.