With the critical role of aberrantly active Signal Transducer and Activator of Transcription (Stat) 3 protein in many human cancers, selective small-molecule inhibitors targeting the dimerization event which is required for stat3 activation, would be valuable as therapeutic agents. And the inhibitors will be useful chemical probes to clarify the complex biological functions of Stat3. By computational and structural analyses of the interaction between Stat3 and the lead dimerization disruptor, S3I-201, we have designed a diverse set of analogs. One of the most active analogs, S3I-201.1066 is derived to contain a cyclo-hexyl benzyl moiety on the amide nitrogen, which increases the binding to the Stat3 SH2 domain. Evidence is presented from in vitro biochemical and biophysical studies that S3I-201.1066 directly interacts with Stat3 or the SH2 domain, with an affinity (K[subscript D]) of 2.74 [micrometer], and disrupts the binding of Stat3 to the cognate pTyr-peptide, GpYLPQTV-NH2, with an IC₅₀ of 23 [micrometer]. Moreover, S3I-201.1066 selectively blocks the association of Stat3 with the epidermal growth factor receptor (EGFR), and inhibits Stat3 tyrosine phosphorylation and nuclear translocation in EGF-stimulated mouse fibroblasts. In cancer cells that harbor aberrant Stat3 activity, S3I-201.1066 inhibits constitutive Stat3 DNA-binding and transcriptional activities. By contrast, S3I-201.1066 has no effect on Src activation or the EGFR-mediated activation of the Erk1/2MAPK pathway. S3I-201.1066 selectively suppresses the viability, survival, and malignant transformation of the human breast and pancreatic cancer lines and the v-Src-transformed mouse fibroblasts harboring persistently active Stat3. Treatment with S3I-201.1066 on malignant cells harboring aberrantly active Stat3 down regulated the expression of c-Myc, Bcl-xL, Survivin, matrix metalloproteinase 9, and VEGF, which are known Stat3-regulated genes important in diverse tumor processes. The in vivo administration of S3I-201.1066 induced significant anti-tumor response in mouse models of human breast cancer, which correlates with the inhibition of constitutively active Stat3 and the suppression of known Stat3-regulated genes. Further computer-aided lead optimization derives higher-affinity (K[subscript D], 504 nM), orally bioavailable Stat3 SH2 domain-binding ligand, BP-1-102 as a structural analog of S3I-201.1066. The most significant modification is the pentafluorobenzene sulfonamide component of BP-1-102, which permits accessibility of a third sub-pocket of the Stat3 SH2 domain surface. BP-1-102-mediated inhibition of aberrantly-active Stat3 in human pancreatic cancer, Panc-1, breast cancer, MDA-MB-231, and prostate (DU145) cancer cells and in the mouse transformed fibroblasts harboring aberrantly-active Stat3. It also disrupts Stat3-NF[kappa]B cross-talk and suppresses the release of granulocyte colony-stimulating factor, soluble intercellular adhesion molecule-1, macrophage-migration-inhibitory factor/glycosylation-inhibiting factor, interleukin-1 receptor antagonist and the serine protease inhibitor (serpin) protein 1, and the expression of c-Myc, Cyclin D1, Bcl-xL, Survivin, and vascular endothelial growth factor expression in vitro and in vivo. Inhibition of tumor cell-associated constitutively-active Stat3 further suppresses focal adhesion kinase and paxillin induction, enhances E-cadherin expression, and down-regulates Kruüppel-like factor 8 expression. Consequently, BP-1-102 selectively suppresses anchorage-dependent and independent growth, survival, migration and invasion of Stat3-dependent tumor cells in vitro. Intravenous or oral gavage delivery of BP-1-102 furnishes micromolar or microgram levels in tumor tissues and inhibits growth of mouse xenografts of human breast and lung tumors. Computer-aided lead optimization has therefore derived a more suitable small-molecule inhibitor as a drug candidate. Our studies of the Stat3 SH2 protein surface and of the interactions between lead agents and the SH2 domain provided significant data to facilitate the structural optimization. From S2I-201 to S3I-201.1066 and to BP-1-102, we note the substantial gain in potency and efficacy, and the pharmacokinetic improvements. The oral bioavailability of BP-1-102 represents a substantial advancement in the discovery of small-molecule Stat3 inhibitors as novel anticancer agents.

When exposed to either abiotic or biotic stressors, plants release chemical compounds that can serve as defense mechanisms. For example, plants of the mustard and cabbage family produce a class of anti-herbivory compounds called glucosinolates. In the model mustard plant Arabidopsis thaliana, some glucosinolates are produced from the amino acid tryptophan and are called indole glucosinolates (IGs). Expression of IG synthesis genes is positively regulated by the partially redundant transcription factors, MYB34 and MYB51. Recent studies have shown that these two transcription factors have distinct roles in regulating IG production in different tissues and in mediating responses to different environmental cues. To understand the distinct roles of these transcription factors at a more detailed temporal and spatial level, reporters for CYP79B2, a transcriptional target of both MYB34 and MYB51, were used. CYP79B2-GFP and CYP79B2-GUS reporter expression was analyzed in wild-type and MYB34 and MYB51 mutant plants in response to increased ambient temperature, increased light intensity, ATP exposure, and chitin exposure. Reverse-phase HPLC quantification of IGs was also performed to determine how these transcription factors are mediating the synthesis of IGs in response stressors. Overall, it was found that MYB51 is responsible for the temperature induction of IG production, while increased light intensity has no impact on IG synthesis. Furthermore, ATP appears to induce IG production independently of both MYB34 and MYB51, while chitin does not increase IG synthesis. Taken together, these studies allow us to better understand how plants respond to and defend themselves from different abiotic and biotic stress / 2025-02-26T00:00:00Z

Skeletal muscle development and growth involve significant changes in gene expression. The overall objective of this dissertation project was to identify transcription factors, enhancers, and mechanisms that control gene expression during skeletal muscle development and growth on a genome-wide scale. Three independent studies were conducted in this project. The objective of the first study was to identify potentially novel mechanisms that mediate myoblast differentiation, a process whereby the mononuclear muscle precursor cells myoblasts express skeletal muscle-specific genes and fuse with each other to form multinucleated myotubes. Comparing gene expression profiles in C2C12 cells, a widely used model of myoblasts, before and 6 days after induced myogenic differentiation by RNA sequencing (RNA-seq) revealed 11,046 differentially expressed genes, of which 5,615 and 5,431 were upregulated and downregulated, respectively. Functional enrichment analyses revealed that the upregulated genes were associated with biological processes or cellular components such as skeletal muscle contraction, autophagy, and sarcomere. In contrast, the downregulated genes were associated with biological processes or cellular components such as ribonucleoprotein complex biogenesis, mRNA processing, and ribosome. Western blot analyses showed an increased conversion of LC3-I to LC3-II protein during myoblast differentiation, further demonstrating the upregulation of autophagy during myoblast differentiation. Blocking the autophagic flux in C2C12 cells with chloroquine inhibited the expression of skeletal muscle-specific genes and the formation of myotubes, confirming a positive role of autophagy in myoblast differentiation and fusion. The aim of the second study was to identify enhancers and transcription factors that regulate gene expression during the differentiation of bovine satellite cells, which are the myogenic precursor cells in adult skeletal muscle, into myotubes. In this study chromatin immunoprecipitation followed by sequencing (ChIP-seq) was used to identify active enhancers, i.e., genomic regions marked with histone modification H3K27ac (acetylation of lysine 27 of H3 histone protein). 19,027 and 47,669 H3K27ac-marked enhancers were identified from undifferentiated and differentiating bovine satellite cells, respectively. Of these enhancers, 5,882 and 35,723were specific to undifferentiated and differentiating bovine satellite cells, respectively while 13,199 were shared by both undifferentiated and differentiating bovine satellite cells. Many of the H3K27ac-marked enhancers specific to differentiating bovine satellite cells were associated with muscle structure and development genes and were enriched with binding sites for MyoD, AP-1, AP-4, KLF, TEAD, and MEF2 transcription factors. Through siRNA-mediated knockdown, AP-4 was found to be essential for differentiation of bovine satellite cells into myotubes. The objective of the third study was to identify enhancers and transcription factors that control differential gene expression in skeletal muscle between neonatal and adult cattle. First, RNA-seq was performed to compare gene expression profiles in skeletal muscle between neonatal calves and adult steers. This analysis identified 924 genes downregulated and 1,021 upregulated from calf to steer muscle. Among genes downregulated in steer muscle were myosin heavy chain3 (MYH3) and MYH8, and among genes upregulated in steer muscle were MYH7 and myoglobin. Surprisingly, many so-called adult muscle genes, such as MYH1 and MYH2, were not differentially expressed between calf and steer muscle. Gene ontology analyses showed that many genes downregulated in steer muscle are involved in protein synthesis and glycolysis and that many genes upregulated in steer muscle function in blood vessel development and immune cell activation. Next, ChIP-seq was performed to identify genomic regions marked with H3K27ac, i.e., active enhancers, in the skeletal muscle of neonatal calves and adult steers. This experiment led to the finding of 20,163 enhancers specifically active in the calf muscle, 14,909 enhancers specifically active in the steer muscle, and 27,002 enhancers active in both the calf and steer muscle. Motif enrichment analyses revealed the enrichment of binding sites for the KLF family and TEAD family transcription factors in enhancers active specifically in the calf muscle, the enrichment of binding sites for the FOXO family and the SMAD family transcription factors in enhancers specifically active in the steer muscle, and the enrichment of binding sites for the MRF family and MEF2 family transcription factors in enhancers active in both the calf and steer muscle. . These results shed light on the differences in gene expression and biology between newborn calf and adult steer skeletal muscle. These results also shed light on the enhancers and transcription factors that control these differences. / Doctor of Philosophy / Muscle is the central part of meat. So, to improve meat yield, it is essential to know how muscle development is controlled. Muscle development, also called myogenesis, starts with muscle progenitor cells developing into myoblasts. Myoblasts then differentiate and fuse with each other to form myotubes. Myotubes undergo hypertrophy and form functional muscle fibers. During myogenesis, each step involves significant changes in gene expression. Gene expression is controlled mainly by proteins called transcription factors. The overall goal of this project was to identify transcription factors and DNA sequences bound by these factors that control gene expression during muscle development. This project consisted of three studies. In the first study, we used the RNA sequencing (RNA-seq) technique to find genes differentially expressed in myoblasts between before and after terminal differentiation. Analyzing the RNA-seq data led to the discovery that autophagy, a 'self-eating' biological process, is required for myoblast differentiation. In the second study, we used a technique called chromatin immunoprecipitation followed by sequencing (ChIP-seq) to identify genomic regions called active enhancers in differentiating bovine myoblasts. This work led to the identification of thousands of active enhancers and dozens of transcription factors binding to these genomic regions that control the differentiation of bovine myoblasts. In the third study, we combined RNA-seq and ChIP-seq to explore the genes and genomic regions controlling muscle transition from newborn calves to adult cattle. This part of the project led to the finding of thousands of genes differentially expressed and thousands of genomic regions differentially activated between newborn calf and adult steer muscle.

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Thesis (Ph.D. in Molecular Biology) -- University of Colorado Denver, 2007. / Typescript. Includes bibliographical references (leaves 160-175). Free to UCD affiliates. Online version available via ProQuest Digital Dissertations;

京都大学 / 0048 / 新制・課程博士 / 博士(理学) / 甲第21946号 / 理博第4524号 / 新制||理||1650(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)准教授 佐藤 ゆたか, 教授 高橋 淑子, 教授 中務 真人 / 学位規則第4条第1項該当 / Doctor of Science / Kyoto University / DGAM

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