Insect herbivores often secrete glucose oxidase (GOX) onto plants to counteract plant defenses and potential pathogens. Whether generalist herbivores always have significantly higher GOX activities than their specialist counterparts at any comparable stage or conditions and how this is realized remain unknown. To address these two general questions, we subjected larvae of a pair of sister species differed mainly in host range, the generalist Helicoverpa armigera and its specialist counterpart Helicoverpa assulta, to the same sets of stage, protein to digestible carbohydrate (P:C) ratio, allelochemical or host plant treatments for simultaneous analyses of GOX transcripts and activities in their labial glands. GOX activity and transcripts are upregulated concurrently with food ingestion and body growth, downregulated with stopping ingestion and wandering for pupation in both species. The three tested host plants upregulated GOX transcripts, and to a lesser extent, GOX activity in both species. There were significant differences in both GOX transcripts and activity elicited by allelochemicals, but only in GOX transcripts by P:C ratios in both species. GOX activities were higher in H. armigera than H. assulta in all the comparable treatments, but GOX transcripts were significantly higher either in generalists or in specialists, depending on the developmental stages, host plants, P:C ratio and allelochemicals they encounter. These data indicate that the greater GOX activity in generalist herbivores is not achieved by greater transcription rate, but by greater transcript stability, greater translation rate, better enzyme stability and/or their combination.

Ce travail de thèse porte sur l’étude des régulations post-transcriptionnelles en réponse à la lumière chez A.thaliana. Nous avons étudié deux systèmes de réponse à la lumière, la régulation traductionnelle des antennes photosynthétiques (Lhc) et la régulation de la voie des anthocyanes par le RNA silencing permettant la photoprotection. Dans une première partie nôtre approche a permis de montrer que la lumière a un impact sur le niveau de traduction global. De plus nous avons pu mettre en évidence que certaines Lhc sont régulées de façon traductionnelles en réponse à la lumière. Cette régulation pourrait être une composante du signal rétrograde entre le chlorolaste et le noyau. En parrallèle dans une seconde partie nous avons caractérisé la voie TAS4 de RNA silencing chez les plantes. Cette voie est mise en place en réponse à la forte lumière et régule l’accumulation des anthocyanes. / This work concerned post-transcriptional regulations in response to light in Arabidopsis thaliana. We are interested in two light responsive systems, translational regulation of the photosynthetic antenna protein and the regulation of the anthocyanin pathway by RNA silencing.In a first part we have shown that light affects global translation level. Furthermore our data indicate that some Lhc proteins are regulated at translational level in response to light. It seems that transaltional regulation of Lhc is a part of retrograde signaling between chlroroplast and nuclear. In a second part we have characterized the TAS4 RNA silencing pathway in Arabidopsis. We show that TAS4 regulate the accumulation of anthocyanin pathway in respose to high light.

No description available.

We have previously shown that the insulin-like growth factor 1 receptor (IGF-1R) translocates to the cell nucleus, where it binds to enhancer-like regions and increases gene transcription. Further studies have demonstrated that nuclear IGF-1R (nIGF-1R) physically and functionally interacts with some nuclear proteins, i.e. the lymphoid enhancer-binding factor 1 (Lef1), histone H3, and Brahma-related gene-1 proteins. In this study, we identified the proliferating cell nuclear antigen (PCNA) as a nIGF-1R-binding partner. PCNA is a pivotal component of the replication fork machinery and a main regulator of the DNA damage tolerance (DDT) pathway. We found that IGF-1R interacts with and phosphorylates PCNA in human embryonic stem cells and other cell lines. In vitro MS analysis of PCNA co-incubated with the IGF-1R kinase indicated tyrosine residues 60, 133, and 250 in PCNA as IGF-1R targets, and PCNA phosphorylation was followed by mono- and polyubiquitination. Co-immunoprecipitation experiments suggested that these ubiquitination events may be mediated by DDT-dependent E2/E3 ligases (e.g. RAD18 and SHPRH/HLTF). Absence of IGF-1R or mutation of Tyr-60, Tyr-133, or Tyr-250 in PCNA abrogated its ubiquitination. Unlike in cells expressing IGF-1R, externally induced DNA damage in IGF-1R-negative cells caused G(1) cell cycle arrest and S phase fork stalling. Taken together, our results suggest a role of IGF-1R in DDT.

It is increasingly becoming clear in the post-genomic era that proteins in a cell do not work in isolation but rather work in the context of other proteins and cellular entities during their life time. This has lead to the notion that cellular components can be visualized as wiring diagrams composed of different molecules like proteins, DNA, RNA and metabolites. These systems-approaches for quantitatively and qualitatively studying the dynamic biological systems have provided us unprecedented insights at varying levels of detail into the cellular organization and the interplay between different processes. The work in this thesis attempts to use these systems or network-based approaches to understand the design principles governing different cellular processes and to elucidate the functional and evolutionary consequences of the observed principles. Chapter 1 is an introduction to the concepts of networks and graph theory summarizing the various properties which are frequently studied in biological networks along with an overview of different kinds of cellular networks that are amenable for graph-theoretical analysis, emphasizing in particular on transcriptional, post-transcriptional and functional networks. In Chapter 2, I address the questions, how and why are genes organized on a particular fashion on bacterial genomes and what are the constraints bacterial transcriptional regulatory networks impose on their genomic organization. I then extend this one step further to unravel the constraints imposed on the network of TF-TF interactions and relate it to the numerous phenotypes they can impart to growing bacterial populations. Chapter 3 presents an overview of our current understanding of eukaryotic gene regulation at different levels and then shows evidence for the existence of a higher-order organization of genes across and within chromosomes that is constrained by transcriptional regulation. The results emphasize that specific organization of genes across and within chromosomes that allowed for efficient control of transcription within the nuclear space has been selected during evolution. Chapter 4 first summarizes different computational approaches for inferring the function of uncharacterized genes and then discusses network-based approaches currently employed for predicting function. I then present an overview of a recent high-throughput study performed to provide a 'systems-wide' functional blueprint of the bacterial model, Escherichia coli K-12, with insights into the biological and evolutionary significance of previously uncharacterized proteins. In Chapter 5, I focus on post-transcriptional regulatory networks formed by RBPs. I discuss the sequence attributes and functional processes associated with RBPs, methods used for the construction of the networks formed by them and finally examine the structure and dynamics of these networks based on recent publicly available data. The results obtained here show that RBPs exhibit distinct gene expression dynamics compared to other class of proteins in a eukaryotic cell. Chapter 6 provides a summary of the important aspects of the findings presented in this thesis and their practical implications. Overall, this dissertation presents a framework which can be exploited for the investigation of interactions between different cellular entities to understand biological processes at different levels of resolution.

MicroRNAs (miRNAs) are a class of small, single-stranded, non-protein coding RNA molecules that regulate cellular messenger RNA (mRNA) and protein levels by binding to specific mRNAs. Aberrant miRNA expression has been shown to be implicated in several diseases, including cancer. Extracellular miRNAs have been found to circulate in the bloodstream and some of their levels have been associated with different diseases. Furthermore, they hold promise as tissue- and blood-based biomarkers for cancer classification and prognostication. Blood-based biomarkers are attractive for cancer screening due to their minimal invasiveness, relatively low cost and ease of reproducibility. New miRNA analysis techniques will add toward the understanding of their biological functions. In this thesis, I investigate the utility of capillary electrophoresis (CE) and mass spectrometry (MS) for analysis of miRNAs through proof-of-concept experiments. In the fi rst part of this work, we developed a Protein-Facilitated Affinity Capillary Electrophoresis (ProFACE) assay for rapid quantification of miRNA levels in blood serum (see List of publications (6)). We also implemented a capillary electrophoresis with laser induced fluorescence detection (CE-LIF) method with online sample pre-concentration for detection of endogenous microRNAs in human serum and cancer cells. 3' modification of miRNA is a physiological and common post-transcriptional event that shows selectivity for specific miRNAs and is observed across species. Recent studies have shown that post-transcriptional addition of nucleotides to the 3' end of miRNAs is a mechanism for miRNA activity regulation. For example, such modifications in plants and C. elegans influence miRNA stability. In humans, effects on miRNA stability and on mRNA target repression have both been observed. Thus, there is a need for miRNA detection techniques which are direct and multiplexed, require minimal sample preparation and provide qualitative information regarding these modifications. We developed a multiplexed miRNA detection technique based on capillary electrophoresis coupled on line with electrospray ionization mass spectrometry (CE-ESI-MS). This method allowed a label-free, direct detection of multiple miRNAs extracted from cancer serum as well as their post-transcriptional modifications with a high mass accuracy.

The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...

The oocyte-to-zygote transition represents the only physiological event in mammalian life cycle, during which a differentiated cell is reprogrammed to become pluripotent. For its most part, the reprogramming relies on the accurate post-transcriptional control of maternally deposited mRNAs. Therefore, understanding the mechanisms of post-transcriptional regulation in the oocyte will help improve our knowledge of cell reprogramming. Short non- coding microRNAs have recently emerged as an important class of post-transcriptional regulators in a wide range of cellular and developmental processes. MicroRNAs repress their mRNA targets via recruitment of deadenylation and decapping complexes, which typically accumulate in cytoplasmic Processing bodies (P-bodies). The presented work uncovers an unexpected feature of the microRNA pathway which is found to be suppressed in fully-grown mouse oocytes and through the entire process of oocyte-to-zygote transition. This finding is consistent with the observation that microRNA-related P-bodies disassemble early during oocyte growth and are absent in fully-grown oocytes. Some of the proteins normally associated with P-bodies localize to the oocyte cortex. At the final stage of oocyte growth, these proteins, together with other RNA-binding factors, form subcortical...

京都大学 / 0048 / 新制・論文博士 / 博士(農学) / 乙第13017号 / 論農博第2827号 / 新制||農||1042(附属図書館) / 学位論文||H28||N4964(農学部図書室) / 32945 / 京都大学大学院農学研究科農学専攻 / (主査)教授 土井 元章, 教授 裏出 令子, 教授 奥本 裕 / 学位規則第4条第2項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

The physiological responses of angiotensin II (Ang II) are mediated across the cell membrane through the angiotensin II type 1 receptor (AT1R), a heptahelical membrane protein coupled to trimeric G-proteins on the cytosolic side. AT1R on binding its ligand, Ang II, leads to downregulation of cell-surface receptor and also its mRNA. We have investigated whether the 3'- and 5'-untranslated regions of the human AT1R mRNA mediate the degradation of hAT1R mRNA by post-transcriptional mechanisms in human adrenocortical carcinoma cell line (H295R cells). Protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA), showed that the downregulation of hAT1R mRNA is mediated by the PKC pathway. Experiments performed in the presence of cycloheximide and/or Ang II demonstrated that protein translation is essential for hAT1R mRNA downregulation. In vitro cell-free RNA degradation assays did not show any increase in the rate of degradation of in vitro transcribed RNA in the presence of cytoplasmic extract from cells treated with Ang II, which suggested that hAT1R steady state mRNA levels may not be mediated by changes in mRNA degradation rates. Luciferase assay after transient transfection of chimeric plasmids of luciferase and hAT1R-3'-UTR showed that Ang II stabilizes the mRNA rather than increase the rate of degradation. Similar results were observed in Northern blot experiments utilizing beta-globin fusion with 3'-UTR that led to stabilization of the chimeric mRNA. Luciferase fusion constructs with both 5'- and 3'-UTRs demonstrated that UTRs are not involved in the Ang II-mediated degradation of hAT1R mRNA. Experiments using transcriptional inhibitor actinomycin D demonstrated that the hAT1R mRNA is not destabilized in response to Ang II activation in H295R cells. Nuclear run-on assay performed in the adrenocortical carcinoma cells demonstrated that the Ang II-stimulated downregulation of hAT1R is mediated by transcriptional inhibition. The transcription of hAT1R mRNA was reduced by 44 and 70% after Ang II treatment for 1 and 2 hours, respectively. Taken together, these findings suggest that the Ang II-induced downregulation of hAT1R steady state mRNA levels is transcriptionally controlled and is not mediated by post-transcriptional mechanisms.