Indiana University-Purdue University Indianapolis (IUPUI) / Alzheimer's disease (AD) is a neurodegenerative disorder characterized by the presence of neuritic plaques consisting of extracellular amyloid-beta (Aβ) and neurofibrillary tangles comprised of hyperphosphorylated microtubule associated tau. Aβ is produced following the cleavage of amyloid precursor protein (APP) by the enzyme BACE1. Transcription factors (TFs) are proteins involved in the regulation of gene transcription. Expression levels of some TFs are perturbed in AD. SP1 binding sites on both the APP and BACE1 promoters implicate its potential role in AD. Aβ peptide itself mediates activation of cyclindependent kinase 5 (CDK5), an enzyme which phosphorylates the FOXO (Forkhead Box) TFs. In order to study mechanisms of TF regulation of Aβ production in human models, neuronally differentiated cells as well as a primary human neurosphere culture were used to test the effects of TF-modulating compounds. Our hypothesis is that by targeting relevant TFs via pharmacological inhibitors in human cells, BACE1 activity or APP expression will decrease and Aβ production will be reduced as a result. To test the involvement of TFs in the regulation of APP, we treated several mammalian cells lines and post-mitotic human neuronal cells with roscovitine, mithramycin A (MTM), MTM analogs (MTM-SDK, MTM-SK), and tolfenamic acid (TA). MTM and TA treatment of neurons differentially activated several TFs implicated in AD. Treatment of differentiated neurospheres with MTM led to a significant decrease in APP and SP1 expression along with Aβ40 levels. Epigenetic mechanisms involve alteration of the binding affinity between DNA and transcription factors. We predict that modulation of these TFs may be influenced by epigenetic modifications. To test the effects of drugs on epigenetic markers, histone deacetylase (HDAC) and DNA methyltransferase (DNMT) activity was measured. MTM-SDK significantly decreased DNMT activity in differentiated neuroblastoma cells, this may enhance or decrease the ability of SP1 to bind to target DNA and affect transcription of BACE1 or APP. Targeting TF activity is a novel means to manipulate the amyloid pathway. Compounds modifying TF binding to sites on the BACE1 or APP promoters may provide a means to limit the production of amyloid-beta and slow the symptoms of AD.

Yukako Hattori, Tadao Usui, Daisuke Satoh, Sanefumi Moriyama, Kohei Shimono, Takehiko Itoh, Katsuhiko Shirahige, Tadashi Uemura, Sensory-Neuron Subtype-Specific Transcriptional Programs Controlling Dendrite Morphogenesis: Genome-wide Analysis of Abrupt and Knot/Collier, Developmental Cell, Volume 27, Issue 5, 9 December 2013, Pages 530-544, ISSN 1534-5807 / 京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第18418号 / 生博第298号 / 新制||生||39(附属図書館) / 31276 / 京都大学大学院生命科学研究科統合生命科学専攻 / (主査)教授 上村 匡, 教授 西田 栄介, 教授 荒木 崇 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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<p>Basic leucine zipper (bZIP) transcription factors can have an oncogenic role in cancer development. Nuclear factor erythroid related 2-factor 2 (Nrf2) is a bZIP transcription factor that traditionally is thought of as a cellular protector. In normal cells, Nrf2 is only activated after exposure to reactive oxygen species or electrophiles and induces expression of antioxidant and detoxification genes. However, in many cancers (<i>e.g.,</i> lung, pancreatic, and breast) Nrf2 is constitutively activated and is associated with poor overall survival and intrinsic resistance to anticancer therapies. Nrf2 heterodimerizes with transcription factors small musculoaponeurotic fibrosarcoma Maf (sMAF) proteins (e.g., MafG) in the nucleus and binds DNA, inducing transcription of Nrf2 target genes, conferring chemotherapeutic resistance to cancer cells. c-Myc another bZIP transcription factor is often overexpressed in a variety of cancers and acts like a protooncogenic transcriptional regulator. Mutations that drive abnormal MYC expression are the most common cariogenic event in tumor progression. c-Myc heterodimerizes with Max, its obligate bHLH-LZ heterodimerization partner, to form an active transcriptional state and induces DNA transcription. Hence inhibiting the interaction between c-Myc-Max and Nrf2-MafG will not only prevent bZIP heterodimerization but also DNA binding and downstream functions of c-Myc and Nrf2 that promote carcinogenesis. Stapled peptides, with their ability to target large surface area interactions, have shown promise for specifically inhibiting protein-protein interactions. Stapled peptides have improved cell permeability and oral bioavailability when compared to biologics. We have designed and synthesized stapled peptide for Nrf2/MafG interaction inhibition and stapled peptides for c-Myc/Max heterodimerization inhibition. Nrf2/MafG inhibition using the synthesized stapled peptide N1S, was demonstrated by luciferase and fluorescence polarization assays. Overall, we hypothesize that stapled peptides will be an effective therapeutic strategy resulting in decreased chemotherapeutic resistance and cancer cell proliferation.</p>

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Transcription factors (TFs) control gene expression by binding to highly specific DNA sequences in gene regulatory regions. This TF binding is central to control myriad biological processes. Indeed, transcriptional dysregulation has been associated with many diseases such as autoimmune diseases and cancer. In this thesis, I studied the transcriptional regulation of cytokines and gene transcriptional dysregulation in cancer. Cytokines are small proteins produced by immune cells that play a key role in the development of the immune system and response to pathogens and inflammation. I mined three decades of research and developed a user-friendly database, CytReg, containing 843 human and 647 mouse interactions between TFs and cytokines. I analyzed CytReg and integrated it with phenotypic and functional datasets to provide novel insights into the general principles that govern cytokine regulation. I also predicted novel cytokine promoter-TF interactions based on cytokine co-expression patterns and motif analysis, and studied the association of cytokine transcriptional dysregulation with disease. Transcriptional dysregulation can be caused by single nucleotide variants (SNVs) affecting TF binding sites (TFBS). Therefore, I created a database of altered TFBS (aTFBS-DB) by calculating the effect (gain/loss) of all possible SNVs across the human genome for 741 TFs. I showed how the probabilities to gain or disrupt TFBSs in regulatory regions differ between the major TF families, and that cis-eQTL SNVs are more likely to perturb TFBSs than common SNVs in the human population. To further study the effect of somatic SNVs in TFBS, I used the aTFBS-DB to develop TF-aware burden test (TFABT), a novel algorithm to predict cancer driver SNVs in gene promoters. I applied the TFABT to the Pan-Cancer Analysis of Whole Genomes (PCAWG) cohort and identified 2,555 candidate driver SNVs across 20 cancer types. Further, I characterized these cancer drivers using functional and biophysical assay data from three cancer cell lines, demonstrating that most SNVs alter transcriptional activity and differentially recruit cofactors. Taken together, these studies can be used as a blueprint to study transcriptional mechanisms in specific cellular processes (i.e. cytokine expression) and the effect of transcriptional dysregulation in disease (i.e. cancer).

As Arabidopsis thaliana ages it becomes more resistant to virulent Pseudomonas syringae pv. tomato (Pst) bacteria. This is known as age-related resistance (ARR). ARR is associated with flowering and intercellular accumulation of salicylic acid (SA). A microarray experiment identified a number of jasmonic acid/ethylene (JA/ET)-associated genes whose expression was up-regulated during ARR. This thesis explores the role of JA/ET signaling during ARR by characterizing the role of two JA/ET-associated No Apical Meristem Cup-shaped Cotyledons (NAC) transcription factors: ANAC055 and ANAC092. Analysis of nae single and double mutants suggests that the NACs play nonredundant roles during ARR. The partial ARR defect of anac092 is followed one week later by an enhanced ARR response, and this is associated with a delay in flowering. Furthermore, mature 35S:ANAC092 exhibits increased susceptibility to Pst. Collectively, this data suggests that ANAC092 is a negative regulator of ARR and it contributes to positive regulation of flowering and the onset of ARR. The late flowering mutant luminidependens1 also exhibits a partial ARR defect, suggesting that the autonomous flowering pathway contributes to ARR. Gene expression data suggests that ANAC055 and ANAC092 regulate expression of some JA/ET-associated genes during ARR. The JA/ET signaling mutant ethylene insensitive2 (ein2) exhibits a partial ARR defect and reduced expression of ANAC055 and ANAC092, suggesting that EIN2 is a positive regulator of expression of ANAC055 and ANAC092 during ARR. Phytohormone analyses reveal that JA accumulates to similar levels in young and mature wild-type plants after inoculation with Pst, suggesting that increased expression of some JA/ET-associated genes in mature compared to young plants after inoculation with Pst is not associated with elevated levels of JA. This thesis contributes to our understanding of ARR by identifying some components of the NAC pathway, exploring the relationship between flowering and ARR, and conducting some phytohormone analysis. / Thesis / Doctor of Philosophy (PhD)

Goblet cells in the mucosal layer of the gastrointestinal tract are the primary source of gel-forming mucins, representing front-line defense. Sterile alpha motif-pointed domain ETS family transcription factor (SPDEF) has a crucial role in terminal differentiation, proliferation and maturation of goblet cells. Gut microbiota is an integral part of our internal environment. In a murine model of intestinal helminthic infection Trichuris muris, the interaction between host microbiota and parasite was seen to play critical roles in immune defense. This interaction is mediated through various mechanisms, including Toll-like receptor (TLR) and nucleotide-binding oligomerization domain (NOD)-like receptor signaling cascades. However, the precise role of intestinal microbiota and NOD/TLR signaling in regulating SPDEF is not yet understood. Hence, we investigated the role of SPDEF in intestinal goblet cell response, the role of helminth-microbiota axis and NOD/TLR signaling in modulating SPDEF during T. muris infection. Experiments were conducted in wild-type (SPDEF+/+) and SPDEF-deficient (SPDEF-/-) mice on BALB/c background at different timepoints of T. muris infection. We observed increased PAS+ goblet cells and higher expression of SPDEF and Muc2 in SPDEF+/+ mice following infection with elevated levels of IL-4 and IL-13. SPDEF+/+ mice showed decreased worm burden from day 14 to 21 post-infection. Microbial analysis revealed altered composition in SPDEF+/+ and SPDEF-/- after infection. Microbiota was transplanted from naïve and T. muris infected mice to separate groups of antibiotic-treated (ABX-treated) mice. Increased PAS+ goblet cells and higher expression of SPDEF and Muc2 were observed in ABX-treated mice after receiving naive and T. muris-altered microbiota. Goblet cell number, the expression of SPDEF and Muc2 were higher in ABX-treated mice who received T. muris-altered microbiota. Microbial analysis revealed differences in T. muris-altered microbiota compared to naïve microbiota. In vitro experiment was conducted in human colonic mucin secreting LS174T cells where we observed stimulated mRNA expression of SPDEF and MUC2 by T. muris excretory-secretory products. These findings reveal new information about major interactions among parasites, microbiota and SPDEF-mediated intestinal goblet cell response in the context of host defense. / Thesis / Master of Health Sciences (MSc)

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24242号 / 農博第2521号 / 新制||農||1094(附属図書館) / 学位論文||R4||N5413(農学部図書室) / 京都大学大学院農学研究科応用生物科学専攻 / (主査)教授 髙野 義孝, 教授 寺内 良平, 教授 吉田 健太郎 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DGAM