The Drosophila genome is thought to have five recA like proteins: Rad51B, Rad51C, Rad51D, XRCC2 and XRCC3. In Drosophila Rad51/SpnA, XRCC3/SpnB, and Rad51 C/SpnD participate in homologous recombination repair. The function of DmRad51 D and DmXRCC2 are unknown. The goal of this project was to elucidate the function ofXRCC2 in Drosophila. RNA interference allowed us to knockdown the function XRCC2 and its possible binding partner Rad51D. It was seen the knocking down the function of either XRCC2 or Rad51D does not affect the viability of the fly. However, drug treatment data does not allow us to make any conclusions about how the knockdown ofXRCC2 affects the viability of the fly. RNA in-situ hybridization shows highly intricate and complex branching patterns for XRCC2, which resembles the embryonic tracheal system. Lastly, XRCC2 was purified to generate an antibody made to recognize the XRCC2 protein will help localize the XRCC2 protein in future studies as well as determine protein-protein interactions with XRCC2.

京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第21689号 / 医科博第93号 / 新制||医||1036(附属図書館) / 京都大学大学院医学研究科医科学専攻 / (主査)教授 斎藤 通紀, 教授 中川 一路, 教授 竹内 理 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

Clustered regularly interspaced short palindromic repeats (CRISPR) are derived from a bacterial and archaeal adaptive immune system. The core enzymes of CRISPR are RNA-guided endonucleases that sequence-specifically cleave foreign double-stranded DNA. Improving and controling the properties of the CRISPR system is a crucial step in advancing the therapeutic potential of CRISPR technology. Several classes of these enzymes exist and are being adapted for biotechnology, such as genome engineering. Cas12a (Cpf1) is a Type V CRISPR-associated (Cas) enzyme that naturally uses only one guide RNA, in contrast to Type II CRISPR-Cas9 enzymes. Thus, Cpf1 may represent a simpler, more practical tool for applications such as gene editing and therapeutics. This dissertation comprises four related studies in this area. To better understand the functional requirements for Cpf1-crRNA interaction and develop modified crRNAs suitable for synthetic biology and therapeutic applications, the first study performed nucleotide substitutions in the crRNA. It focused on the protein-interaction motif of the crRNA by incorporating base changes at the 2ʹ position that alter hydrogen-bonding capacity, sugar pucker, and flexibility. DNA substitutions in RNA can probe the importance of A-form structure, 2ʹ-hydroxyl contacts, and conformational constraints within RNA-guided enzymes. In addition, Chemical modifications include 2'-deoxy, 2'-fluoro, 2'- deoxy-arabinonucleic acid, and oxepane. Our study discovered that 2'-fluoro maintains the A-form structure and is compatible with AsCpf1 activity. Biochemical endonuclease activity, gene editing efficiency, Cpf1 binding affinity, and ribonucleoprotein stability were used to assess the tolerance and effects of modification. Characterizing structure-function requirements for Cpf1-crRNA interaction will facilitate better design and tuning of Cpf1 enzymes. The second study established a FRET-based assay in collaboration with a computational collaborator to identify small molecule inhibitors predicted by virtual docking and simulations. This study aims to lay the foundation for efficient, safe implementation of CRISPR-Cpf1. The third study used chemically modified Cas9-guide RNAs to offset known weaknesses of CRISPRi. It takes advantage of the high binding affinity and nuclease resistance of modified guides to potentially reduce the required components for CRISPRi.

No description available.

Clustered regularly interspaced palindromic repeats (CRISPR) and their associated (Cas) proteins co-opted as biotechnological tools have improved the simplicity and accessibility of gene editing for fields ranging from crop science to the treatment of human disease. These technologies, however, come with an inherent degree of risk associated with off-target events or direct misuse, accidental or intentional, leading to permanent genetic damage to ecosystems, livestock, or people. Naturally occurring anti-CRISPR proteins have been described, as well as synthetic small molecule inhibitors, but each of these approaches, while suitable for certain applications, leaves something to be desired in deliverability or efficacy in the face of many possible adverse CRISPR-related events. Inspired by strides in the field of oligonucleotide therapeutics, we developed the first reported anti-CRISPR nucleic acids for Streptococcus pyogenes (Sp)Cas9 to address the critical need for fail-safe inhibitors of Cas enzymes. These inhibitors, termed small nucleic acid-based inhibitors of Cas9 (SNuBs), comprise two modules which act in tandem to bind and disable the SpCas9 RNP. We have demonstrated that SNuBs inhibit Cas9 in vitro and in human cells. Successive rounds of optimization on our initial designs have yielded inhibitors capable of carrier-free uptake into human cells, high nuclease resistance, and robust inhibition at low stoichiometric concentrations relative to Cas9 and its RNA guide. In their current form, SNuBs quite possibly present the most tenable approach to inhibiting Cas9 in a variety of contexts including therapeutic applications in the near future.

No description available.

Post-editing machine translation has become more common in recent years due to the increase in materials requiring translation and the effectiveness of machine translation systems. This project presents a system for formalizing structured translation specifications that facilitates the assessment of the performance of a post-editor. This report provides details concerning two software applications: the Ruqual Specifications Writer, which aids in the authoring of post-editing project specifications, and the Ruqual Rubric Viewer which provides a graphical user interface for filling out a machine readable rubric file. The project as a whole relies on a definition of translation quality based on the specification approach. In order to test whether potential evaluators are able to reliably assess the quality of post-edited translations, a user study was conducted that utilized the Specifications Writer and Rubric Viewer. The specifications developed for the project were based on actual post-editing data provided by Ray Flournoy of Adobe. The study involved simulating the work of five post-editors, which consisted of developing texts and scenarios. 17 non-expert graders rated the work of the five fictional post-editors, and an Intraclass Correlation of the graders responses shows that they are reliable to a high degree. The groundwork laid by this project should help in the development of other applications that assist in the assessment of translation projects in terms of a specification approach to quality.

It is widely assumed that current phylogenetic methods are fairly accurate at recovering the evolutionary relationships among different species, but evaluating the relative success of this enterprise is a difficult task. This study addresses some fundamental questions associated with generating phylogenetic trees. The complete genomes of five species of Steinernema were sequenced and assembled. Genes were predicted in AUGUSTUS and orthologous genes were found from those data using OrthoMCL. I aligned 3890 genes in MAFFT and eliminated poorly aligned positions with GBlocks. I created individual trees for each gene as well as a supermatrix tree in PAUP*, using a closely related taxon from another genus, Panagrellus redivivus. In the resulting gene trees, I found only a small subset of all the possible topologies. I discovered that the supermatrix tree has the same topology as the topology with the most gene trees in the gene-topology distribution. There are only a small number of histories for all of the genes and many of the genes have the same lineage. I bootstrapped the gene-topology distribution and found that the best-supported topology was sampled 22.1% of the time. I show that many genes must be sampled in order to converge on the topology with the most support from the gene trees in this dataset.

Though much work has been done on the definition of Standard English and on the standardization process, little attention has been paid to the role of copy editors in that process. Editors comprise a class of craft professionals employed to remove errors from texts and make them more consistent, but when editors speak about editors at all, they generally rely on anecdotes rather than hard data about what editors do. Since formal written English is often used as a baseline for determining what is standard, and since corpora of published writing are increasingly used to research questions of usage, it is important to understand the role of copy editors in shaping the text that we see on the printed page. This study examines the usage and grammar changes made by student editorial interns in twenty-three academic journal articles. Volunteer professional editors were then solicited to edit the same articles, and their changes were compared against the interns' changes. The changes were counted and categorized to determine which usage rules can be considered most important to copy editors and thus most essential to distinguishing Standard Edited English from standard unedited writing. It was found that the most frequent changes were several grammatical items and a few lexical items, including the that/which rule, avoidance of towards, increased parallelism, and standardization of s-genitive forms. These changes confirm the idea that editors play a role in standardization, particularly codifying certain forms by reducing optional variation. From this data we can conclude that educated written usage and edited usage are not necessarily the same and should not be conflated. These findings also have implications for the use of corpus data in usage studies by showing that the final version of a printed work does not necessarily show the usage of edited writers but likely has a substantial contribution from copy editors.

京都大学 / 0048 / 新制・課程博士 / 博士(農学) / 甲第22503号 / 農博第2407号 / 新制||農||1077(附属図書館) / 学位論文||R2||N5283(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 佐藤 健司, 教授 澤山 茂樹, 准教授 豊原 治彦 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM