As an important posttranslational modification, protein acetylation plays critical roles in many biological processes such as gene transcription, DNA damage repair, apoptosis and metabolism. The acetylation occurs on the ε-amino group of specific lysine residues, and is catalyzed by histone acetyltransferases (HATs). In cellular contexts, HATs are found to target hundreds and thousands of substrates including histone and nonhistone proteins. Lysine acetylation changes the microenvironment of protein and may potentially alter protein activity and protein-protein interaction. The goal of this dissertation project is to investigate the impact of lysine acetylation on the catalysis of MYST HATs, and to establish the strategy for labeling substrates of the MYST HATs at cellular level. To understand the regulatory mechanism of MYST HATs, a detailed study was carried out to investigate the active site lysine acetylation of two MYST HATs (MOF and Tip60). Autoradiography and immunoblotting data shows that mutation of active site lysine differentially affects the enzyme autoacetylation activity and the cognate substrate acetylation activity. In addition, deacetylated MOF and Tip60 were prepared by using the nonspecific lysine deacetylase Sirt1. Kinetic study demonstrated that the acetylation of the active site lysine on MYST HATs marginally modulates the HAT catalysis. This work provides new insights into the regulatory mechanism of MYST catalysis. In the second part of my work, we designed and synthesized a series of Ac-CoA analogs conjugated with alkynyl or azido functional groups. Meanwhile, the active site of the MOF was engineered to expand the cofactor binding capability. Fluorescence screening was carried out to characterize the enzyme activity to Ac-CoA analogs. MOF-I317A with all analogs and MOF-I317A/H273A–5HYCoA were identified and further applied in the labeling of the cognate histone H4 protein and HAT substrates in 293T cell lysate. Visualizing of the labeled substrate was achieved using the alkynyl or azido-tagged fluorescent reporters through the copper-catalyzed azide−alkyne cycloaddition. As expected, the histone H4 protein was successfully labeled by the active enzyme-cofactor pairs. More intriguingly, multiple protein bands in cell lysate were labeled and observed. This work provides a new versatile strategy in exploring the substrates of MYST HATs at the proteomic level.

The histone acetyltransferase protein, Tip60, has many important functions in epigenetic regulation of gene expression and DNA repair. Our objective is to design, synthesize, and evaluate potent inhibitors for Tip60. Full-length Tip60 (fl-Tip60) and catalytic domain of Tip60 (cat-Tip60) were expressed in E. coli and purified with nickel affinity chromatography. Quantitative analysis of enzyme activities demonstrated that both enzyme forms had very high activity with the substrate H4. To create new Tip60 inhibitors, various histone H3 peptides conjugated with CoA were synthesized using the Fmoc solid-phase peptide synthesis and solution phase synthesis protocols. The results from the inhibition radioactive assay showed that the synthetic H3-CoA conjugates inhibited effectively the enzymatic activity of both fl-Tip60 and cat-Tip60; and the addition of methyl groups to the Lys-4 or Lys-9 residue of H3 aided in a 7-9 fold enhancement in potency in comparison to nascent H3-CoA inhibitor.

Tip60 is a 60 kDa nuclear protein which exists in three isoforms, belongs to the MYST/HAT family of proteins and was discovered after its interaction with the Human HIV-1 Tat. As a nuclear protein, Tip60 can act as a coactivator or repressor. To understand the HAT action of Tip60, two possible catalytic models exist; the ping-pong and the ternary complex formation models. In correlation with the exploration of HAT catalytic action, mutations of a Cys to Ala and a Glu to Gln on Esa1 (yeast homolog of Tip60 and MYST/HAT prototype), was reported to show wild type-like and decreased acetylating properties, respectively. In this work, Tip60 HAT action was explored. In Tip60, the Cys in the active site is important for acetylation of the H4(1-20) substrate and the Glu showed semi loss in acetylating the H4(1-20) peptide substrate. These data highlight a unique mechanism of Tip60 catalysis.

This research considers how the culture of game developer Cyan Worlds influences the gameplay environment and the culture of fans in Myst Online: Uru Live. The game has gone through two commercial releases and in both cases it was cancelled after a short time. Fans have attempted to salvage the game by producing their own server software and content creation tools. Recently, Cyan released their own source code and development tools to the fan community, giving fans an official channel for creating new content. This work builds off of Pearce's (2009b) study of the culture of Uru players and emergent play, but adds the dimension of considering the culture of developers themselves. A primary goal of this study was to determine how the culture of a game developer like Cyan shapes the constraints of the designed "play ecosystem" (Pearce 2009b: 7), and how it shapes the processes by which fans can salvage aspects of the game to create new content. One finding is that the design of Uru's gameplay environment is rooted in the cultural practices, personal philosophical goals and individual personality traits of its developers. Fans were able to assert ownership over the Uru story-world and the means of production of new content by proactively applying technical and problem-solving skills--the same sorts of skills that players must apply to solving puzzles in Myst games. This fan action, coupled with Cyan's goal of making an open-ended world, has helped to propel the initiative to provide open-source tools for creating new content. When fans produce new content, they draw significantly from an existing shared cultural repertoire of cues and conventions. These conventions are supported both by the software affordances of the development environment and by cultural precedent--they are readily adapted to Myst-like narratives and are easily "read" by experienced players.

Darstellung des Augustusbildes in der französischen Literatur des 9.-16. Jhs. (lateinische und französische Quellen, hauptsächlich Chroniken und religiöse Theaterstücke) in Abhängigkeit von Ort und Zeit der Entstehung (Frankreich bzw. Deutsches Reich). Hinführend eine Darstellung der Wandlung des Augustusbildes in der lateinischen Literatur seit der augusteischen Zeit als Voraussetzung für die Rezeptionssituation.

京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第20779号 / 生博第385号 / 新制||生||51(附属図書館) / 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 西田 栄介, 教授 米原 伸, 教授 松本 智裕 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

Darstellung des Augustusbildes in der französischen Literatur des 9.-16. Jhs. (lateinische und französische Quellen, hauptsächlich Chroniken und religiöse Theaterstücke) in Abhängigkeit von Ort und Zeit der Entstehung (Frankreich bzw. Deutsches Reich). Hinführend eine Darstellung der Wandlung des Augustusbildes in der lateinischen Literatur seit der augusteischen Zeit als Voraussetzung für die Rezeptionssituation.