付記する学位プログラム名: 充実した健康長寿社会を築く総合医療開発リーダー育成プログラム / 京都大学 / 0048 / 新制・課程博士 / 博士(医科学) / 甲第22697号 / 医科博第112号 / 新制||医科||8(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 竹内 理, 教授 髙折 晃史, 教授 河本 宏 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM

The ability to precisely regulate gene expression patterns and to modify genome sequence in a site-specific manner holds much promise in determining gene function and linking genotype to phenotype. DNA-binding modules have been harnessed to generate customizable and programmable chimeric proteins capable of binding to site-specific DNA sequences and regulating the genome and epigenome. Modular DNA-binding domains from zinc fingers (ZFs) and transcriptional activator-like effectors (TALEs) are amenable to engineering to bind any DNA target sequence of interest. Deciphering the code of TALE repeat binding to DNA has helped to engineer customizable TALE proteins capable of binding to any sequence of interest. Therefore TALE repeats provide a rich resource for bioengineering applications. However, the TALE system is limited by the requirement to re-engineer one or two proteins for each new target sequence. Recently, the clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated 9 (Cas9) has been used as a versatile genome editing tool. This machinery has been also repurposed for targeted transcriptional regulation. Due to the facile engineering, simplicity and precision, the CRISPR/Cas9 system is poised to revolutionize the functional genomics studies across diverse eukaryotic species. In this dissertation I employed transcription activator-like effectors and CRISPR/Cas9 systems for targeted genome regulation and editing and my achievements include: 1) I deciphered and extended the DNA-binding code of Ralstonia TAL effectors providing new opportunities for bioengineering of customizable proteins; 2) I repurposed the CRISPR/Cas9 system for site-specific regulation of genes in plant genome; 3) I harnessed the power of CRISPR/Cas9 gene editing tool to study the function of the serine/arginine-rich (SR) proteins.

Purpose: The purpose of this study was to determine the effect of word processing on students' composition when used as a tool in the revision/editing stages of the writing process. Procedure: Two experimental groups were established; Group A Computer, Group B No Computer. For each group, the generic method of teaching writing remained the same. Group A, however, was able to use the microcomputer in editing their work; Group B was not. Final essays were scored by independent raters and the data were analyzed by the use of the independent 1-test. A Pearson product -moment correlation coefficient was computed to assess interrater reliability. Conclusion: This study suggests there is a significant positive difference in student writings when the microcomputer is used as a word processor in the revision/editing stages of writing.

Please note: Editorial Studies works are permanently embargoed in OpenBU. No public access is forecasted for this item. To request private access, please click on the locked Download file link, and fill out the appropriate web form. / This dissertation considers the life and art of Osip Mandelstam in the 1930s, under the aspect of a disjunction between Mandelstam's posthumous image and the biographical evidence that emerged between 1993 and 2010. It traces this disjunction not solely to prior lack of information but also to the moral ambiguities that complicate the reception of this biographical material. Among the chief difficulties of Mandelstam's biography is his testimony to the OGPU, in which Mandelstam gave the names of those among his friends to whom he had recited his "Stalin Epigram." Close analysis of the exact words of the interrogation protocols, along with memoir evidence, is used to establish that the protocols constitute digests of information elicited previously by coercion. This conjecture is supported by reading the relevant parts of Nadezhda Mandelstam's memoirs under the aspect of the double bind--a pathogenic social situation studied by Gregory Bateson and described in structure and in its potential for inducing psychosis. Mandelstam's composition of the "Ode" to Stalin is considered in the light of new evidence about his exile and its effects on the poet's state of mind. The dissertation proceeds largely by scrutinizing the language of witnesses and their interpreters, of poets, understood as witnesses of truths available to the creative imagination, and of critics, the interpreters of poets and witnesses of the workings of poems and language. The idea of witness literature is considered in relation to the concept of textual witnesses, in the editorial sense, and to a specific instance of the latter in the marginalia of Nadezhda Mandelstam. Because this study must find a footing in the English language while attending closely to the Russian, it makes recourse to poets and critics who wrote in English, whose judgments and sensibilities help establish a broader frame of reference for a discussion focused on Stalin's Russia. Geoffrey Hill's particular artistic engagement with Mandelstam is contemplated as an instance of a special kind of bearing witness--the witness of imagination. / 2031-01-01

<p>  </p> <p>Adenosine-to-Inosine (A-to-I) mRNA editing is a post-transcriptional modification of specific sites within the mRNA that has only recently been observed in filamentous fungi. In the wheat scab fungus <em>Fusarium graminearum,</em> this phenomenon has shown to be facilitated by FgTad2 and FgTad3, homologs of Adenine Deaminase Acting on tRNA (ADAT). Interestingly, these two proteins are constitutively expressed in all different life stages<em>, </em>in contrast to only the sexual stage-specific nature of A-to-I mRNA editing in <em>F. graminearum</em>. To understand the molecular mechanisms regulating this process, six candidate co-factor genes were identified which interact with FgTad2 and/or FgTad3, specifically during sexual reproduction. Deletion mutants of four candidate co-factor genes were successfully generated. All four mutants displayed normal asexual development of <em>F. graminearum</em>, but four mutants also altered sexual function. Those four mutant led to formation of morphologically normal perithecia and ascospores, but the perithecia failed to discharge ascospores. More interestingly, in <em>FGSG_10943 </em>deletion mutant, most of these ascospores germinated precociously within the perithecium. I also observed, that among the candidate co-factor genes which are specifically expressed during sexual reproduction, <em>FGSG_10943</em> was significantly upregulated during the later stage of sexual development. This gene is restricted in nature to only a few orders of fungi in the class Sordariomycetes that form dark pigmented ascocarps, particularly Hypocreales and Glomerellales. Taken together, these results indicate that the four candidate co-factor genes are dispensable for vegetative growth of the fungus and involved in ascospore discharge. <em>FGSG_10943</em> appears to be involved in autoinhibition of ascospores inside the perithecia and interact with FgTad2 during sexual reproduction to mediate A-to-I mRNA editing in <em>F. graminearum</em>.</p>

In a world that is moving towards more mobile viewing, and shorter ad formats –the filmmaker must adapt. A lot of advertisers now ask for video which is originally created for a longer format, to be adapted into a shorter format. But how much of the emotional impact is lost when adapting an emotionally based narrative commercial? This study aims to find out what the difference in emotional response is regarding different lengths of the same emotionally based narrative video. The author edited an already finished emotionally based narrative commercial video into two new, shorter versions. The process is documented and presented. These three videos were then shown to volunteers, alongside questions regarding their emotional state before and after. When analyzing the data gathered from the questionnaire it was clear that the emotional response differed from the various videos. The original video omitted the largest emotional response, and the shortest video omitted the lowest amount of emotional response. It seems that when adapting an emotionally based narrative video into a shorter format, some aspects of the video get lost. In turn, the emotional response of the viewers will be impacted.

The development of a drug able to distinguish between tumor and host cells has been long sought, but the solid tumor microenvironment (TME) confounds many current therapeutics. Solid tumors present several challenges for oncotherapeutics, primarily, (1) aberrant vascularization, resulting in hypoxia, necrosis, abnormally high pH, and (2) tumor immune suppression. Oncolytic microbes are drawn to this microenvironment by an innate ability to selectively penetrate, colonize, and eradicate solid tumors as well as reactivate tumor associated immune components. To consider oncolytic bacteria deployment into this microenvironment, Chapter 1 dives into the background of oncolytic microbes. A discussion of the oncolytic bacterial field state, identifying Clostridium novyi? as a promising species, and details genetic engineering techniques to develop customized bacteria. Despite the promise of C.novyi in preclinical/clinical trials when administered intratumorally, the genetic and biochemical uniqueness of C.novyi necessitated the development of new methodologies to facilitate more widespread acceptance. Chapter 2 reports the development of methods that facilitate experimental work and therapeutic translation of C.novyi, including the ability to work with this obligate micro-anaerobe aerobically on the benchtop. While methods development is a necessary step in the clinical translation of C.novyi so too is choosing the correct model of the TME within which to test a potential anti-cancer therapy. While the typical solid TME includes both phenotypic and genotypic heterogeneity, the methods used to model this disease state often do not reflect this complexity. This simplistic approach may have contributed to stagnant five-year survival rates over the past four decades. Nevertheless, simplistic models are a necessary first step in clinical translation. Chapter 3 explores the impact of cancer cell lines co-cultured with C. novyi to establish the efficacy of this oncolytic bacteria in a monolayer culture. Chapter 4 extends this analysis adding not only a level of complexity by using an in vivo model, but also using CRISPR/Cas9 to modify the genome of C.novyi to encode a tumor targeting peptide, RGD, for expression within the spore coat. The combination of these studies indicates that C. novyi is uniquely poised to accomplish the long sought after selective tumor localization via intravenous delivery.

We propose an e cient algorithm to embed a given image into the latent space of StyleGAN. This embedding enables semantic image editing operations that can be applied to existing photographs. Taking the StyleGAN trained on the FFHQ dataset as an example, we show results for image morphing, style transfer, and expression transfer. Studying the results of the embedding algorithm provides valuable insights into the structure of the StyleGAN latent space. We propose a set of experiments to test what class of images can be embedded, how they are embedded, what latent space is suitable for embedding, and if the embedding is semantically meaningful.

Rare gene-disrupting variants and common regulatory variants play key roles in rare and common disease, respectively. These variants are of great interest for investigation into genetic contributions to disease, but experimental methods to validate their impact on gene expression levels are lacking. In this study, we utilized CRISPR/Cas9 genome editing to validate regulatory variants including cis-eQTLs, rare stop-gained variants in healthy and disease cases and one immune-response trans-eQTL master regulator. For investigation into common and rare regulatory variants within transcribed regions, we developed a scalable CRISPR-based polyclonal assay for experimental assessment. First, we applied this assay to nine rare stop-gained variants found in the general population, in GTEx. After editing, the stop-gained variants show a significant allele-specific depletion in transcript abundance, as expected. Next, we utilized the assay to validate 33 common eQTLs found in GTEx. After editing, the eQTL variants show higher variance in effect size than control variants, indicating a regulatory effect. Finally, we applied the polyclonal editing approach to clinical and new stop-gained variants in two disease-associated genes. The results follow the expected trend, with NMD being triggered by variants upstream of the NMD threshold but not by those beyond. This method demonstrates scalable experimental confirmation of putative causal regulatory variants, and improved interpretation of regulatory variation in humans. Next, we sought to experimentally validate an immune-response eQTL for IRF1 in cis and many genes in trans under LPS stimulation. We used CRISPRi to repress the enhancer locus and found that the enhancer is active in our immune cell system. Next, we used CRISPR-Cas9 genome editing and isolation of monoclonal cell lines to target this variant locus. After LPS stimulation, we performed RNA-sequencing on wild type and edited clones, showing that the effect size of the genes which are associated with the trans-eQTL are correlated with differential expression between the edited and wild type cell lines for the same genes. Additionally, we find that the differential expression between edited clones is correlated with CRISPRi repression of the IRF1 promoter and enhancer. In this way, we were able to identify a common genetic variant which modifies the transcriptomic immune response to LPS and validate the trans-eQTL signal.

Maintenance of genomic integrity is essential for the survival of an organism and its ability to pass genetic information to its progeny. However, DNA is constantly exposed to exogenous and endogenous sources of damage, which demands cells to possess DNA repair mechanisms. Of the many forms of DNA damage, double-strand breaks (DSBs) are particularly cytotoxic DNA lesions that cause genome instability and cell lethality, but also provide opportunities to manipulate the genome via repair. One of the major DSB repair pathways shared between single-celled yeast and humans is homologous recombination (HR). HR is initiated by the evolutionarily conserved Mre11-Rad50-Xrs2/Nbs1 (MRX in yeast, MRN in mammals) complex. The MRX complex has a multitude of functions such as damage sensing, adduct removal from DSB ends, and end tethering – a process to maintain the two ends of a DSB in close proximity. The role of the MRX complex has been uncovered by studying the repair of DSBs generated from meganucleases such as HO and I-SceI. However, it is unclear if this knowledge translates to the repair of DSBs from genome editing nucleases such as TALEN and CRISPR-Cas9 (Cas9), as these nucleases create DSBs with different end polarities. While the repair efficiencies and outcomes of TALEN and Cas9 are actively studied, less is known about the earlier stages of repair. The objective of this thesis is to examine the role of the MRX complex in repair processes at both ends of a DSB after cleavage with I-SceI, TALEN, and Cas9 in vivo using the model organism Saccharomyces cerevisiae. In Chapter 1, I describe the importance of DSB repair, a summary of HR and its sub-pathways, the functions of the MRX complex, and properties of I-SceI, TALEN, and Cas9. The materials and methods used in this thesis are detailed in Chapter 2. The work described in Chapter 3 focuses on end tethering and recruitment of downstream repair proteins in haploid cells. I find that DSB ends from the three nucleases all depend on the MRX complex for end tethering, and that initial end polarity does not affect tethering. DSBs created by Cas9 show greater dependence on the Mre11 nuclease of the MRX complex for Rad52 recruitment compared to DSBs from I-SceI and TALEN. Despite Mre11-dependent end processing and Rad52 recruitment at Cas9-induced DSBs, Cas9 stays bound to one DNA end after cleavage, irrespective of the MRX complex. These results suggest that Mre11 exonuclease activity required for adduct removal from DSB ends is not critical for Rad52 recruitment, and that Mre11 endonuclease activity may be driving processing of Cas9-bound DSBs. I also find that MRX tethers DSB ends even after Rad52 recruitment, and unexpectedly, untethered ends are processed asymmetrically in the absence of MRX for all three nucleases. In Chapter 4, I explore the interaction of DSB ends with their repair template, the intact homologous chromosome, in diploid cells. The primary goal is to monitor interhomolog contact in real time from homology search to completion of HR. Although technical limitations make it difficult to capture the entire HR program from DSB formation to repair, I show that untethered ends interact with the homolog separately in the absence of the MRX complex. Similar to haploids, diploid cells display defects in end tethering and end processing without the MRX complex. Repair outcomes of WT cells show an even distribution of G2 crossovers and non-crossovers, while pre-replication crossovers and break-induced replication are undetected. Overall, the results in this thesis provide insight into the functions of the MRX complex in repairing different DSB ends created by I-SceI, TALEN, and Cas9. In Chapter 5, I summarize all of these findings and discuss the motivation for future cell biology studies of HR.