L'étape initiale dans l'expression génique est la transcription de l'ADN génomique du gène en ARN. La transcription être initiée par l'assemblage d'ARN pol II autour du site de début de transcription, qui est également connues comme promoteurs. Cependant, la transcription est nécessite un autre gène distal des régions, des amplificateurs, qui sont augmenté ainsi la probabilité de la transcription. Amplificateurs et les promoteurs sont généralement définis par leur éloigné des sites d'initiation de la transcription et souvent distingués par les modifications des histones. Récemment, des études, il a été de plus en plus ont révélé de grandes similitudes entre les amplificateurs et les promoteurs. Les résultats antérieurs ont suggéré la possibilité que certains promoteurs de gènes peuvent afficher les fonctions activatrices. Cependant l'étendue de ce type de promoteurs et si elles fonctionnent réellement réglementé l'expression des gènes distales sont restés insaisissable.Mon projet est réalisé en vue de répondre à ces questions. En exploitant un essai amplificateurs reporter à haut débit, je démêler une partie sous-estimée du promoteur de base présentant une activité d'activateur, définie comme Epromoters. Ils présentent des propriétés distinctes par rapport à des amplificateurs et des promoteurs classiques distales, sont associés à la réponse au stress et d'interagir plus souvent avec d'autres promoteurs. En utilisant CRISPR complète / cas9 approche de suppression I a démontré que Epromoters sont généralement impliqués dans l'activation des gènes distales. Nos résultats identifient d'abord une nouvelle catégorie de promoteurs avec activité in vivo dans amplificateurs. / The initial step of gene expression is the transcription of genomic DNA of the gene into RNA. The transcription can only be initiated by the assembly of RNAPII machinery around transcription start site of a gene, known as core promoter. However, transcription also requires other gene-distal regulatory DNA regions, known as enhancers. Enhancers and promoters are traditionally distinguished by their histone modifications. Recently, there has been increasing number of studies revealing broad similarities between enhancers and promoters. Previous findings have suggested the possibility that some gene promoters display enhancer activity. However, the questions of how can we identify this type of promoter in genome-wide and whether they actually function to regulated the expression of distal genes are remained elusive.My project has carried out aiming to answer these above questions. Firstly, I have optimized the technique that has developed in the lab, named CapStarr-seq, which used as an approach to exploiting a high-throughput enhancer activity. Performing CapStarr-seq in human cell lines, I unraveled an underestimated proportion of promoter displaying enhancer activity, defined as Epromoters. They display distinct properties as compared to distal enhancers and classical promoters, are associated with stress response genes and interact more frequently with other promoters. Moreover, by using comprehensive CRISPR/Cas9 genomic deletion approach, I demonstrated that Epromoters are generally involved in the activation of distal genes. Taken together, our results first identify a new category of promoters with dual promoter and enhancer functions.

Mucosa associated lymphoid tissue (MALT) lymphoma is characterized by t(11;18)(q21;q21)/API2-MALT1, t(1;14)(p22;q32)/BCL10-IGH andt(14;18)(q32;q21)/IGH-MALT1, which commonly activate the NF-κB pathway. Gastric MALT lymphomas harbouring such translocation do not respond to Helicobacter pylori eradication, while those without translocation can be cured by antibiotics. To understand the molecular mechanism of MALT lymphoma with and without chromosome translocation, 24 cases (15 translocation-positive and 9 translocation-negative) of MALT lymphomas together with 7 follicular lymphomas and 7 mantle cell lymphomas were analysed by Affymetrix gene expression microarray platform. Unsupervised clustering showed that cases of MALT lymphoma were clustered as a single branch. However, within the MALT lymphoma group, translocation-positive cases were intermingled with translocation-negative cases. Gene set enrichment analysis (GSEA) of the NF-κB target genes and 4394 additional gene sets covering various cellular pathways, biological processes and molecular functions showed that translocation-positive MALT lymphomas were characterized by an enhanced expression of NF-κB target genes, particularly TLR6, CCR2, CD69 and BCL2, while translocation-negative cases were featured by active inflammatory and immune responses, such as IL8, CD86, CD28 and ICOS. Separate analyses of the genes differentially expressed between translocation-positive and negative cases and measurement of gene ontology term in these differentially expressed genes by hypergeometric test reinforced the above findings by GSEA. The differential expression of these NF-κB target genes between MALT lymphoma with and without translocation was confirmed by quantitative RT-PCR and immunohistochemistry or Western blot. Expression of TLR6, in the presence of TLR2, enhanced both API2-MALT1 and BCL10 mediated NF-κB activation in vitro. In addition, there was cooperation between expression of BCL10, MALT1 or API2-MALT1, and stimulation of the antigen receptor or CD40 or TLR in NF-κB activation as shown by both reporter assay and IκBα degradation. Interestingly, expression of BCL10 but not API2-MALT1 and MALT1, in the presence of LPS stimulation, also triggered IκBβ degradation, suggesting activation of different NF-κB dimers between these oncogenic products. Study by co-immunoprecipitation showed that BCL10 directly interacts with MALT1. Sub-cellular localisation experiments in BJAB B-cells, showed that BCL10 localisation was affected by MALT1. When BCL10 was over-expressed, the protein was predominantly expressed in the nuclei, but when MALT1 was over-expressed, BCL10 was mainly localised in the cytoplasm. When both BCL10 and MALT1 were over-expressed, BCL10 was expressed in the cytoplasm in the early hours when the protein level was low, but in both the cytoplasm and nuclei after 9 hours when the protein level was high. Over-expression of API2-MALT1 did not shown any apparent effect on BCL10 sub-cellular localisation in vitro. Finally, comparison of MALT lymphoma expression microarray with other lymphomas showed lactoferrin to be highly expressed in MALT lymphoma. This was confirmed by qRT-PCR, showing lactoferrin to be significantly over-expressed in MALT lymphoma compared to FL and MCL. Thus lactoferrin may be a potential marker for MALT lymphoma.

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第20953号 / 理博第4405号 / 新制||理||1633(附属図書館) / 京都大学大学院理学研究科生物科学専攻 / (主査)教授 中野 伸一, 教授 木庭 啓介, 教授 中川 尚史 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DFAM

No description available.

The Hedgehog (Hh) signaling pathway plays many important roles throughout embryonic development, including the regulation of tissue patterning, cell differentiation, proliferation, and apoptosis. The loss of SHH signaling in human development has been shown to cause holoprosencephaly. Conversely, inappropriately activated Shh signaling in adults has been implicated in many cancers. Furthermore, Shh has been found to be a key regulator of neural stem cells in the mammalian brain. To further study the roles of Hh, I have developed a transgenic zebrafish line as a tool to monitor tissues that respond to Hh signaling throughout the vertebrate life-cycle. A number of genes have been identified that are transcriptionally up-regulated by Hh signaling. Transcription of these genes is initiated through binding of activated Gli transcription factors to an identified Gli binding site (GBS) in the cis-regulatory region. This Gli binding site is largely conserved across vertebrate species. I have generated transgene constructs in which 12 GBSs have been placed upstream of a minimum promoter that drives GFP, RFP, or Kaede fluorescent proteins. These plasmid constructs are activated in embryonic regions known to be Hh responsive, such as the ventral CNS. Treatment with cyclopamine eliminates this expression, confirming that these transgenes accurately report an active Hh response. These transgenic lines will be extremely powerful tools for research into the mechanisms by which Hh signaling regulates adult cell types such as neural stem cells. These lines will also be important tools that will help understand how misregulation of Hh signaling can lead to cancer.

No description available.

The radiation-induced bystander effect is an important phenomenon in the field of radiation biology. It has been shown that cells, after exposure to radiation, can communicate with surrounding cells and affect their physiology. Otherwise-healthy recipient cells can be influenced to undergo cellular senescence or apoptosis through this process. This has potential utilizations for radiation oncology and as well as our understanding of radiation safety. The radiation-induced bystander effect has been extensively investigated since the 1990s, but the scientific community struggles to come to a unanimous decision on how strongly these signals impact the survival of bystander cells. Results show various degrees of impact on cell survival whereas certain studies refute the existence of a radiation-induced bystander effect. This may be due to the fact that there is a great deal of study heterogeneity within the radiation-induced bystander effect community. Most experiments follow a similar general bystander protocol but often use different donor and reporter cell lines that vary in sex, organ of origin, and p53 status. The type of radiation and dose rate also typically differ between experimental designs. In this analysis, 67 in vitro, medium-transfer, radiation-induced, bystander effect studies were retrospectively graphed and analyzed to determine which intrinsic and external factors contributed significantly to the overall survival percentage change observed in reporter cells. A Two-Way ANOVA was conducted on each variable and showed that the reporter cell line, p53 status, and radiation type had a statistically significant effect on survival percentage change. These findings may explain the variation in results seen in past experiments and may help standardize future research allowing for more direct comparisons. / Thesis / Master of Science (MSc)

Opportunistic bacterial pathogens are present in the intestines of all mammals. These bacteria are symbionts to a certain extent, but under certain conditions these organisms can be deadly. Intestinal opportunistic pathogens encompass many genera and include organisms such as those in the Bacteroides fragilis group (i.e. B. fragilis and B. thetaiotaomicron), Escherichia coli, and Clostridium perfringens, resulting in an array of diseases and serious health risks. Typically these diseases affect individuals in poor or weakened health (elderly, immuno-compromised, neonates, etc.) but can affect healthy individuals as well. The intestinal tract is the main area of infection for these bacteria, however some of these organisms can be involved in wound infections, septicemia, urinary tract infections, and meningitis. This study focused on three areas: 1) Analysis of differences in gene expression between Bacteroides and Escherichia coli, in order to learn more about promoter structure, 2) Establishment of a diabetic mouse model for use in examining bacterial synergy during a polymicrobial infection, and 3) Characterization of Escherichia coli 360A and evaluation of the role of several virulence factors and environmental modulators in the pathogenesis of this strain. We used a newly developed lux gene reporter to evaluate gene expression in Bacteroides. We observed that there are barriers in both transcription and translation initiation that appear to limit the expression of foreign genes in Bacteroides. We were able to establish a mouse model for studying synergy during a polymicrobial infection and observed that E. coli 360A provided synergy towards B. fragilis NCTC 9343. These experiments also showed that the longer a mouse is afflicted with the complications of diabetes the more susceptible it is to polymicrobial infections. Systemic infections were used to evaluate the contribution of several virulence factors and environmental modulators in the pathogenesis of E. coli 360A. The results showed that a strain lacking both virulence factors CNF1 and HlyA, the terminal oxidase cytochrome o, or a double cyo/cyd mutant were, deficient in survival in the spleen, but not the liver of BALB/c mice. / Ph. D.

No description available.

In order to maintain a multicellular organism cells need to interact and communicate with each other. Signalling cascades such as the Bone Morphogenic Protein (BMP) and Hedgehog (Hh) signalling pathways therefore play essential roles in development and disease. Intercellular signalling also underlies the function of stem cell niches, signalling microenvironments that regulate behaviour of associated stem cells. Range and intensity of the niche signal controls stem cell proliferation and differentation and must therefore be strictly regulated. The testis and ovary of the fruit fly Drosophila melanogaster are established models of stem cell niche biology. In the apical tip of the testis, germ line stem cell (GSCs) and somatic cyst stem cells (CySCs) are arranged around a group of postmitotic somatic cells termed hub. While it is clear which signals regulate GSC maintenance it is unclear how these signals are spatially regulated. Here I show that BMP signalling is specifically activated at the interface of niche and stem cells. This local activation is possible because the transport of signalling and adhesion molecules is coupled and directed towards contact sites between niche and stem cells. I further show that the generation of the BMP signal in the wing disc follows the same mechanism. Hh signalling controls somatic stem cell populations in the Drosophila ovary and the mammalian testis. However, it was unknown what role Hh might play in the fly testis, where the components of this signalling cascade are also expressed. Here I show that overactivation of Hh signalling leads to an increased proliferation and an expansion of the cyst stem cell compartment. Finally, while the major components of the Hh signalling pathway are known, detailed knowledge of how signal transduction is implemented at the cell biological level is still lacking. Here, I show that localisation of the key signal transducer Smo to the plasma membrane is sufficient for phosphorylation of its cytoplasmic tail and downstream pathway activation. Using advanced, microscopy based biophysical methods I further demonstrate that Smo clustering is, in contrast to the textbook model, independent of phosphorylation.