京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第12763号 / 論農博第2786号 / 新制||農||1016(附属図書館) / 学位論文||H25||N4786(農学部図書室) / 30615 / (主査)教授 平竹 潤, 教授 植田 充美, 教授 小川 順 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第18177号 / 医博第3897号 / 新制||医||1004(附属図書館) / 31035 / 京都大学大学院医学研究科医学専攻 / (主査)教授 川口 義弥, 教授 中畑 龍俊, 教授 斎藤 通紀 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第19405号 / 医科博第65号 / 新制||医科||5(附属図書館) / 32430 / 京都大学大学院医学研究科医科学専攻 / (主査)教授 河本 宏, 教授 斎藤 通紀, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DGAM

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第21153号 / 農博第2279号 / 新制||農||1059(附属図書館) / 学位論文||H30||N5127(農学部図書室) / 京都大学大学院農学研究科食品生物科学専攻 / (主査)教授 入江 一浩, 教授 保川 清, 教授 橋本 渉 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Indiana University-Purdue University Indianapolis (IUPUI) / Cyclin Dependent Kinase regulation is often manipulated by cancer cells to promote unlimited proliferation. P27 is an important regulator of Cyclin E/CDK 2, which has been found in low amounts in many types of malignant cancers. Lovastatin has been shown to cause cell cycle arrest in the G1 phase of the cell cycle by increasing the P27 protein. There has been some question, however, if lovastatin regulates P27 at the transcriptional or translational level. Although it has been claimed that P27 expression regulation is due to an IRES located in its 5’UTR, other studies suggested that P27 expression is regulated at the level of transcription. To further investigate the regulation mechanism of P27 expression, the 5’-UTR of P27 and its deletion mutants were examined using a luciferase reporter gene in HeLa cells following exposure to lovastatin. It was found that lovastatin stimulated a significant 1.4 fold increase in its promoter activity of the full length 5’UTR (575). Deletion of 35 nucleotides from the 5’ end of the UTR eliminated the lovastatin-induced increase in promoter activity. Further mapping analyses of the first 35 bases showed that two regions, M1 (575-559) and M3 (543-527), were less sensitive to lovastatin than the other mutated constructs. Since M1 and M3 still showed some activity, a construct was created with deletions in both the M1 and M3 regions. This showed no increase in luciferase activity when exposed to lovastatin. Looking at RNA levels, there was a 1.5 fold increase in RNA when the full length 5’UTR was inserted into HeLa cells and exposed to 81 µM of lovastatin. In contrast, there was no increase in RNA when M1/M3 (575-559; 543-527) was inserted into HeLa cells and exposed to 81 µM of lovastatin. In addition, there was a 1.6 fold increase in endogenous P27 RNA levels after HeLa cells were exposed to 81 µM of lovastatin. In all of these experiments, there seems to be two promoters that work cooperatively: M1 (575-559) and M3 (543-527).

No description available.

Mosquitoes are notorious vectors for multiple diseases like malaria, yellow fever and dengue fever. To manipulate gene expression in mosquito and spread desired genes among natural population for vector control, a thorough understanding of mosquito development and gene regulation is critical. Early embryogenesis is a rapid, complex yet crucial process in the very beginning of development. Previous research in other species indicated genes transcribed that early evolved fast and played essential roles. The study of mosquito early zygotic genes (EZGs) would offer unique insights into mosquito gene evolution as well as potential targets for mosquito control. In this study, I identified 61 pure EZGs (pEZGs) in mosquito Aedes aegypti. These pEZGs were enriched in architectures adapting to the rapid embryonic cell cycles and were over represented by domains or functions related to maternal zygotic transition. Phylogenetic analysis showed that pEZGs originated mainly from duplication, retrotransposition and de novo emergence. The comparison of pEZGs in Ae. aegypti with those in Drosophila revealed an interesting evolutionary paradox where the early zygotic genes turned over fast but the regulatory motif was conserved in two species. Curiously, the motif binding protein in Drosophila (zelda) seemed unable to initiate the earliest zygotic transcription in Ae. aegypti due to late temporal expression. The regulatory motif (VBRGGTA) found in Ae. aegypti pEZGs was shown necessary and sufficient for driving early zygotic gene expression by transient reporter assays and one motif-bearing promoter was tested with success in driving gene expression as early as 2-4h after egg laying in transgenic Ae. aegypti. This was the first characterized promoter with early zygotic but no maternal expression in Ae. aegypti that can be used for future genetic studies and mosquito control strategies. As important gene regulators, miRNAs also play essential roles in early embryogenesis. The genome-wide predictions and systematic analysis of miRNAs in Ae. aegypti and Anopheles stephensi were conducted in this study. The first miRNA profiling in mosquito across all developmental stages was also performed to provide basis for future functional study. Several lineage-specific miRNAs were found highly expressed in embryos, indicating their special roles in the embryogenesis of mosquitoes. / Ph. D.

Melatonin is an indoleamine hormone with neuromodulatory and neuroprotective properties. It mediates many of its effects by its two G protein-coupled receptors, MT1 and MT2. We have shown that valproic acid (VPA) induces melatonin receptor expression in cultured rat C6 glioma cells, and in the rat hippocampus. VPA is known to affect gene expression through several mechanisms, including the modulation of intracellular kinase pathways and/or transcription factors, as well as the inhibition of histone deacetylase (HDAC) activity. In this study, we show that HDAC inhibitors of distinct chemical classifications, including suberanilohydroxamic acid (SAHA) and 4-(dimethylamino)-n-[7-(hydroxyamino)-7-oxoheptyl] benzamide (M344), parallel the effects of VPA on MT1 induction in vitro. However valpromide, a VPA analogue that lacks the ability to inhibit HDAC activity, does not. The observed increase in MT1 expression by VPA is matched by an increase in global histone H3 acetylation. More importantly, an enrichment of histone H3 acetylation occurs along the rat MT1 promoter following treatment with VPA, indicating that histone acetylation and chromatin remodelling are a primary mechanism underlying this induction. Independent of VPA, the rat MT1 gene may be regulated by a number of intracellular kinase pathways and transcription factors, which are also targeted by VPA. KG501-mediated CREB inhibition did not block MT1 upregulation by VPA. Blockade experiments targeting the PKC, PI3K/AKT, or GSK3β signaling pathways suggest that VPA induces melatonin receptor expression independent of these intracellular signaling cascades as well. The relevance of melatonin receptor upregulation was assessed using in vivo VPA and melatonin combination treatments on neuroprotective gene expression. The results of this study provide evidence that expression of the melatonin receptor is epigenetically induced by VPA by means of promoter histone acetylation. Melatonin receptor upregulation by VPA, or other HDAC inhibitors, may represent a therapeutic strategy for the management of several nervous system disorders. / Dissertation / Doctor of Philosophy (PhD)

Our aim was to analyse (i) the presence of single nucleotide polymorphisms (SNPs) in the JUN and FOS core promoters in patients with rheumatoid arthritis (RA), knee-osteoarthritis (OA), and normal controls (NC); (ii) their functional influence on JUN/FOS transcription levels; and (iii) their associations with the occurrence of RA or knee-OA. JUN and FOS promoter SNPs were identified in an initial screening population using the Non-Isotopic RNase Cleavage Assay (NIRCA); their functional influence was analysed using reporter gene assays. Genotyping was done in RA (n = 298), knee-OA (n = 277), and NC (n = 484) samples. For replication, significant associations were validated in a Finnish cohort (OA: n = 72, NC: n = 548). Initially, two SNPs were detected in the JUN promoter and two additional SNPs in the FOS promoter in perfect linkage disequilibrium (LD). JUN promoter SNP rs4647009 caused significant downregulation of reporter gene expression, whereas reporter gene expression was significantly upregulated in the presence of the FOS promoter SNPs. The homozygous genotype of FOS promoter SNPs showed an association with the susceptibility for knee-OA (odds ratio (OR) 2.12, 95% confidence interval (CI) 1.2–3.7, p = 0.0086). This association was successfully replicated in the Finnish Health 2000 study cohort (allelic OR 1.72, 95% CI 1.2–2.5, p = 0.006). FOS Promoter variants may represent relevant susceptibility markers for knee-OA.

Promoter-enhancer interactions are essential for gene regulating, Capture Hi-C is a chromosome conformation capture method to map promoter-enhancer interactions at high resolution. We have Capture Hi-C data forGM12878 cells, immortalized primary B lymphocytes, in three replicates. Although Capture Hi-C maps enhancer elements together with the promoters they regulate, the overlap between enhancer datasets produced by other methods such as ChIP-seq and Capture Hi-C is lower than expected. In order to understand the reasons for lower overlap, we investigated the enhancer potential of replicated and non-replicated Capture Hi-C interactors, as well as enhancer overlapping and non-overlapping Capture Hi-C interactors. We performed a systematic comparison between our interactor and experimental regulatory and transcriptomic datasets to determine the extent of enhancer mapping. The results show replicated interactors have higher enhancer potential than non-replicated ones. However, there is evidence that interactors not overlapping with experimental validated regulatory datasets can also potentially be true enhancers.