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<p>RNA-directed DNA methylation (RdDM) is a process by which epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on <i>Mediator of Paramutation 1 (Mop1</i>), a putative RNA dependent RNA polymerase. Here I show that although RdDM is essential for the maintenance of DNA methylation of a silenced <i>MuDR</i> transposon in maize, a loss of that methylation does not result in a restoration of activity of that element. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of the element, heritable silencing is mediated via H3K9 and H3K27 dimethylation, even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by H3K27 trimethylation, a mark normally associated with somatically inherited gene silencing. I find that a brief exposure of high temperature in a <i>mop1</i> mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in <i>mop1</i> wild type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once it has been reversed. To leverage the specificity of our observations made at bench, I also performed a transcriptome analysis in <i>mop1</i> mutants under heat. I found that a substantial number of genes as well as a subset of TEs are reactivated in <i>mop1</i> mutants under heat, which is consistent with the effects I observed on <i>MuDR</i>. Interestingly, I found that <i>mop1</i>-specific reactivation of TEs is closely correlated with changes in expression of nearby genes, most of which are involved in metabolic transportation and sensing. This suggests that one function of <i>MOP1</i> is to prevent inappropriate expression of genes in this pathway when they are close to TEs. Taken together, my work will provide an opportunity to better understand the causes and consequences of TE silencing and reactivation, as well as the effects of TEs on gene regulation under stress conditions.</p>

Obesity is the top risk factor for the development of type 2 diabetes mellitus in humans. Obese adipose tissue, particularly visceral depots, exhibits an increase in macrophage accumulation and is described as being in a state of chronic low-grade inflammation. It is characterized by the increased expression and secretion of inflammatory cytokines produced by both macrophages and adipocytes, and is associated with the development of insulin resistance. Based on these observations, we investigated the potential role of macrophage infiltration on whole body metabolism, using genetic and diet-induced mouse models of obesity. Using flow cytometry and immunofluorescence imaging we found that a significant percentage of macrophages proliferate locally in adipose tissue of obese mice. Importantly, we identified monocyte chemoattractant protein 1 (MCP-1) as the stimulating factor. We also found that ATMs can be targeted for specific gene silencing using glucan encapsulated siRNA particles (GeRPs). Knockdown of the cytokine osteopontin improved regulation of systemic glucose levels as well as insulin signaling in adipocytes. Conversely, targeting lipoprotein lipase (LPL) abrogated the buffering of lipid spillover from adipose tissue, resulting in increased hepatic glucose output. Finally, silencing of the master regulator of inflammation NF-κB in resident liver macrophages called Kupffer cells significantly improved hepatic insulin signaling. Thus this work demonstrates that macrophages can regulate whole body metabolism.

Transposons constitute much of the animal genome. While many transposons are ancient and inactivated, numerous others are intact and must be actively repressed. Uncontrolled transposons can cause genomic instability through DNA damage or mutations and must be carefully silenced in the germline or risk sterility or mutations that are passed on to offspring. In Drosophila melanogaster, 23–30 nt long piRNAs direct transposon silencing by serving as guides for Aubergine, Argonaute3, and Piwi, the three fly PIWI proteins. piRNAs derive from piRNA clusters—large heterochromatic DNA loci comprising transposons and transposon fragments. piRNAs are loaded into PIWI proteins via the ping-pong cycle which serves to amplify guide piRNAs. Loaded Piwi then enters the nucleus to transcriptionally repress transposons by establishing heterochromatin. Therefore, to silence transposons, transposon sequences must also be expressed. To bypass this paradox, the HP1 homolog Rhino (Rhi) allows non-canonical, promoter-independent, transcription of transposons embedded in heterochromatin. Transposon RNAs produced in this manner are “incoherent” and have little risk of being translated into transposon-encoded proteins required for transposition. This thesis focuses on understanding how piRNA clusters permit non-canonical transcription yet restrict canonical transcription. We found that although Rhi promotes non-canonical transcription in piRNA clusters, it also creates a transcriptionally permissive environment that is amenable to canonical transcription. In addition, we discovered that the conserved protein, Maelstrom, is required to repress promoter-driven transcription of individual, potentially active, transposons within piRNA clusters and allows Rhi to transcribe such transposon sequences into incoherent piRNA precursors.

The Piwi interacting RNA pathway (piRNA) transcriptionally and post-transcriptionally silences transposons in the germline to maintain host genome integrity and faithful transmission of the genetic materials. In Drosophilaovaries, maternally loaded piRNAs kick-start piRNA biogenesis and convert precursor transcripts into piRNAs to replenish the piRNA pool during oogenesis. piRNA clusters are the genomic source of piRNA precursors, which are determined by the HP1 homolog Rhino and accessary factors. Rhino specifically binds to piRNA cluster chromatin. I was intrigued by how Rhino localizes to piRNA clusters to specify piRNA precursors. TREX is a conserved mRNA biogenesis complex composed of UAP56 and the THO complex. Identification of UAP56 as a cluster transcript-processing factor established the link between piRNA biogenesis and the general mRNA processing machinery. In my thesis, I investigated the functions of UAP56 and THO in piRNA cluster transcript processing. I characterized an RNP specific to cluster transcripts, defined by binding with both factors, which is distinct from RNP of bulk mRNA transcripts, and found that assembly of these RNPs depends on Rhino. These findings imply that piRNA precursors are specified co-transcriptionally. Additionally, I found that TREX mutants lead to a loss of Rhino binding specificity. I propose that Rhino and TREX co-transcriptionally scan for cluster and transposon sequences to establish loci that produce piRNA precursors. Surprisingly, I also discovered a piRNA-independent function for TREX in transposon silencing. I showed that TREX mutants lead to transcriptionally activation of a number of transposon families without affecting their piRNA biogenesis and piRNA mediated repressive histone modifications. I propose that TREX could mediate a conserved transposon silencing mechanism.

Sveriges befolkning ökar och det föds allt fler barn. Detta skapar ett stort tryck på förskoleplatser och kommunerna tvingas starta nya avdelningar och förskolor i lokaler som inte är anpassade till antalet barn. Hörselskador, stress, kommunikationssvårigheter och sömnsvårigheter är några av de följder som både barn och vuxna kan få av att exponeras i för höga bullernivåer.Studien syftar till att undersöka hur produktdesign kan användas för att minska bullret på förskolor. Studien har använt sig av en användarcentrerad designprocess där målgruppen, barnen och pedagogerna på förskolan, varit involverade genom hela arbetet.Det teoretiska ramverket innehåller buller, buller inom förskolan, förskolans inomhusmiljö och användarcentrerad design. Till det empiriska arbetet gjordes observationer, frågeformulär och intervjuer med målgruppen. Resultatet visar att de högsta bullernivåerna kommer från barnen själva och lokalerna som de vistas i, vid tillfällen mellan aktiviteter. Det slutliga produktförslaget är ett ljuddämpande pusselmoln som har flera funktioner och syftar till att underhålla barnen vid bland annat väntetid mellan aktiviteter. Studiens slutsats visar att det finns utvecklingsmöjligheter kring ljuddämpande produkter. / Sweden’s population is increasing, and more children are born. This creates great pressure on preschool places and the municipalities are forced to start new departments. Preschools in premises that are not adopted to the number of children. Hearing damage, stress, communication difficulties and difficulty sleeping are some of the consequences that both children and adults can get from being exposed to excessive noise levels. The study aims to investigate how product design can be used to reduce the noise in preschools. The study has used a user-centered design process in which the target group, the children and the educators, have been involved throughout the work.The theoretical framework contains backgroundnoise, within the preschool, the preschool’s physical environment and user-centered design. The empirical work consisted of observations, questionnaires and interviews with the target group. The result shows that the highest noise levels come from the children themselves and the premises in which they are staying, on occasions between activities. The final product proposal is a sound-absorbing puzzle cloud that has several functions and aims to entertain the children during, among other things, waiting time between activities. The study’s conclusion shows that there are development opportunities for sound-absorbing products.

RNA provides the framework for the assembly of some of the most intricate macromolecular complexes within the cell, including the spliceosome and the mature ribosome. The assembly of these complexes relies on the coordinated association of RNA with hundreds of trans-acting protein factors. While some of these trans-acting factors are RNA binding proteins (RBPs), others are adaptor proteins, and others still, function as both. Defects in the assembly of these complexes results in a number of human pathologies including neurodegeneration and cancer. Here, we demonstrate that Silencing Defective 2 (SDE2) is both an RNA binding protein and also a trans-acting adaptor protein that functions to regulate RNA splicing and ribosome biogenesis. SDE2 depletion leads to widespread changes in alternative splicing, defects in ribosomal biogenesis, and ultimately complete loss of cell viability. Our data highlight SDE2 as a previously uncharacterized essential gene required for the assembly and maturation of some of the most fundamental processes in mammalian cells.

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

No description available.

Weeds and soil-borne diseases can cause large yield losses in field-grown tomato (Solanum lycopersicum) production. Techniques have been developed to reduce soil-based problems. In this study, we evaluated the impacts of cover crops, soil steaming, and plastic mulch to reduce weed and disease pressure in field-grown tomatoes. Four cover crop treatments were grown in the fall and winter before spring planting. Soils were steamed to a target temperature of 71.1 °C for 0, 5, or 20 minutes. Plastic mulch was also used on half of the rows. Yield, weed densities, and disease incidence were recorded. Reduced flowering time and stringent flowering requirements may reduce the ability to conduct crosses in many plants. Many factors control flowering. Terminal Flowering Locus 1 (TFL1) inhibits flower development. In this study, we attempted to transiently downregulate TFL1 via virus-induced gene silencing (VIGS) in Antirrhinum, Penstemon, Petunia, Rosa, and Rudbeckia.