Telomeres are specialized nucleoprotein structures found at the end of eukaryotic chromosomes. The telomere DNA in humans is composed of the sequence "5'-TTAGGG-3'" tandemly repeated in a stretch of 5-30kb of double stranded DNA. TTAGGG Repeat Factor 2 (TRF2) is a telomere DNA binding protein that has a critical role in telomere end protection. The current model for telomere protection by TRF2 is through its ability to remodel telomeres into looped higher-order structures, called the t-loop, which sequesters the end from DNA damage sensors. Since telomeres are known to be comprised of nucleosomal chromatin, it is important to determine how TRF2 binds to and affects the structure of nucleosomal arrays. The ability of TRF2 to bind to unusual DNA structures such as the t-loop and the single stranded/double (ss/ds) stranded telomere DNA junction may facilitate its binding to DNA in the form of nucleosomal arrays and promote higher-order chromatin structures. In this study, we have reconstituted a 2kb DNA fragment containing 550bp of telomere DNA into nucleosomal arrays and tested the binding of full-lengthTRF2 and four truncation mutants to telomeric nucleosomal arrays. Our data indicates that TRF2 and its truncation mutants bind to telomere nucleosomal arrays as well as it binds to telomere DNA. We used a novel electrophoretic technique, Analytical Agarose Gel Electrophoresis (AAGE), to measure changes in surface charge density, hydrodynamic radius, and conformational flexibility of DNA and nucleosomal arrays upon protein binding. Our results indicate that the C-terminal DNA binding Myb/SANT domain of TRF2 might be rearranging nucleosomal structure through either nucleosome sliding, unwrapping, or changing the arrangement of the linker DNA, while the N-terminal basic DNA binding region is causing nucleosomal arrays compaction. Instead of significant compaction, histone-free DNA undergoes DNA condensation and self-association. This activity is observed with the full-length protein and all regions of the protein, with the exception of TRF2-DBD, participate in the process. We speculate that the ability of TRF2-DBD to rearrange nucleosomal structure and N-terminal basic region to cause nucleosomal fiber compaction may allow TRF2 to promote t-loop formation in the context of chromatin. We propose that TRF2, possessing all the features, has a new role at telomeres as a chromatin architectural protein.

京都大学 / 新制・課程博士 / 博士(工学) / 甲第23921号 / 工博第5008号 / 新制||工||1782(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 古賀 毅, 教授 中村 洋, 教授 竹中 幹人 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DGAM

DNA中の二本鎖切断(DSB)に対するアスコルビン酸(AA)およびDMSOの保護効果を、蛍光顕微鏡による巨大DNA(T4 DNA; 166kbp)の単分子観察によって評価した。凍結／解凍の状態に対して3つの異なる形態の放射源、可視光、γ線、および超音波の環境下にさらした。1‐プロパノールと2‐プロパノールの間で異なる効果が表れた。ゲノムDNA分子の高次構造の変化は、1−プロパノールを用いると、長軸長が濃度60％で最小を示し、次にアルコール含有量の増加と共に増加する傾向があることを見出した。一方、2−プロパノールを用いると、長軸長はアルコール含有量の増加と共にほぼ単調な減少を示した。 / The protective effect of ascorbic acid (AA) and DMSO against double-strand breaks (DSBs) in DNA was evaluated by single-molecule observation of giant DNA (T4 DNA; 166kbp) through fluorescence microscopy. Samples were exposed to three different forms of radiation: visible light, γ-ray, and ultrasound or freeze/thawing. The change of the higher-order structure of genomic DNA molecules in the presence of alcohols by use of single DNA observation with fluorescence microscopy, by focusing our attention to unveil the different effect between 1-propanol and 2-propanol. / 博士(工学) / Doctor of Philosophy in Engineering / 同志社大学 / Doshisha University

Even after their discovery more than 60 years ago, little is known about how messenger RNAs (mRNAs) are packaged inside the cells. To ensure efficient and accurate delivery of the intended message to its proper destination, it is important to package the informational molecule in a way that protects it from premature degradation but also proper decoding at the destination. However, very little is known about the this fundamentally important step of mRNA packaging inside eukaryotic cells. To this end, we developed a novel approach, RIPPLiT (RNA ImmunoPrecipitation and Proximity Ligation in Tandem), to capture the 3D architecture of the ribonucleoprotein particles (RNPs) of interest transcriptome-wide. To begin with, we applied RIPPLiT to the exon-junction complex (EJC), a set of proteins stably bound to a spliced RNA. EJCs have been shown to interact with other proteins like SR- and SR-like to form megadalton sized complexes and help protect large regions of mRNAs. Thus, we hypothesized that these RNPs would provide an ideal system to elucidate the higher order organization of mRNPs. Preliminary analysis of data obtained from RIPPLiT consisted of “chimeric reads”, reads with multiple RNA fragments ligated together, which could not be analyzed with any of the existing bioinformatics tools. Thus, we developed a new bioinformatics suite, ChimeraTie, to map, analyze and visualize chimeric reads. Performing polymer analysis on chimeric reads obtained for hundreds of mRNAs, we were able to predict that mRNPs are linearly and densely packed into flexible rod-like structures before they undergo translation. In this thesis, along with the detailed biological conclusion, I have also provided a step-wise manual to perform RIPPLiT experiment and analyze the ensuing data using ChimeraTie.

京都大学 / 0048 / 新制・課程博士 / 博士(工学) / 甲第21124号 / 工博第4488号 / 新制||工||1697(附属図書館) / 京都大学大学院工学研究科高分子化学専攻 / (主査)教授 辻井 敬亘, 教授 山子 茂, 教授 竹中 幹人 / 学位規則第4条第1項該当 / Doctor of Philosophy (Engineering) / Kyoto University / DFAM

No description available.

Cuando los proyectos o tareas pendientes que existen en una empresa son de mediana o gran envergadura, no siempre es posible realizarlos todos a la vez, por limitaciones de tiempo, de recursos, de personal, porque hay una dependencia de orden entre algunos proyectos, etc. Si son varias áreas o varias personas las que deben de decidir en qué orden se deben ejecutar los proyectos, no siempre hay un consenso, y cuando no lo hay se impone la propuesta del CEO, del gerente de mayor experiencia o cualquier otra que generalmente no satisface a todos. Es decir, la decisión final recae solo en una persona o en pocas personas quedando fuera las propuestas de la mayoría. El presente trabajo de investigación propone un algoritmo que permita determinar en qué orden se deben ejecutar los proyectos pendientes considerando la jerarquía de los proyectos y la opinión de todas las áreas o personas responsables. Se aplicó el algoritmo, como un piloto, en siete empresas de diferentes rubros, y la opinión de todos los gerentes generales y gerentes de diversas áreas fue que es muy relevante la propuesta de este algoritmo porque de alguna manera considera la opinión de todos los responsables y esto compromete, hace responsable a todo el equipo por el éxito o fracaso de la decisión tomada. Con las recomendaciones dadas por los gerentes de las empresas visitadas, se va a potenciar la estructura del algoritmo para que sus resultados sean lo más objetivos posibles. / When the projects or pending tasks that exist in a company are medium or large, it is not always possible to do them all at the same time, due to time, resource, and personnel limitations, because there is a dependency of order between some projects, etc. If several areas or several people must decide in which order the projects should executed, there is not always a consensus, and when there is not, the proposal of the CEO, the most experienced manager or any other that generally prevails. it does not satisfy everyone. In other words, the final decision falls on only one person or on a few people, leaving out the proposals of the majority. This research work proposes an algorithm that allows determining in what order the pending projects should executed, considering the hierarchy of the projects and the opinion of all the areas or responsible persons. The algorithm was applied, as a pilot, in seven companies of different areas, and the opinion of all the general managers and managers of different areas was that the proposal of this algorithm is very relevant because in some way it considers the opinion of all those responsible and these commits, makes the entire team responsible for the success or failure of the decision made. With the recommendations given by the managers of the companies visited, the structure of the algorithm will be strengthened so that its results are as objective as possible. / Trabajo de investigación

博士(工学) / Doctor of Philosophy in Engineering / 同志社大学 / Doshisha University