京都大学 / 0048 / 新制・課程博士 / 博士(生命科学) / 甲第21228号 / 生博第397号 / 新制||生||52(附属図書館) / 京都大学大学院生命科学研究科高次生命科学専攻 / (主査)教授 朝長 啓造, 教授 藤田 尚志, 教授 野田 岳志 / 学位規則第4条第1項該当 / Doctor of Philosophy in Life Sciences / Kyoto University / DFAM

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mRNA molecules form an intermediate in the transfer of sequences from DNA to ribosomes in order to guide protein production. Errors can be introduced into mRNA, producing aberrant proteins which place a strain on cellular regulatory machinery, causing increased risks of apoptosis, cancer, and decreased fitness. These errors may be introduced due to decreased transcriptional proofreading capabilities, exposure to chemicals, or mistakes in RNA editing machinery. It is important to investigate these causes of transcription errors to better understand the long-neglected area of mRNA fidelity which has such significant impacts on our cellular functions. In this paper, it was determined that addition of adenine opposite from abasic sites, not genomic uracil pairing with adenine, are a probable cause of G-to-A transcription errors. That exposure to Roundup causes increased levels of transcription errors, potentially due to oxidative stress. And finally, that off-target ADAR gene editing of transcripts occurs at high levels.

The development of super-resolution microscopy made it possible to surpass the diffraction limit of optical microscopy, enabling researchers to gain a nanometer scale understanding of cellular structures. While many applications have benefited from standard super-resolution microscopy, gaps remained making high-speed dynamic imaging in live cells impossible. To address this problem, single-point edge-excitation sub-diffraction (SPEED) microscopy was developed. This methodology enables the nanometer imaging of dynamic cell processes within live cells, the evaluation of subcellular structural information, the capacity to derive three-dimensional information from two-dimensional images within rotationally symmetric structures, and the interrogation of novel questions regarding the transport dynamics of macromolecules in a variety of cellular structures. Here, I have described the theory and method behind the current iteration of SPEED microscopy that we have developed and validated via Monte Carlo simulation. Further, a detailed description of how we have further developed SPEED microscopy to derive structural information within the nuclear pore complex as well as how SPEED has been applied to evaluate the export kinetics of mRNA. / Biology

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In this study we have focused on the codon usage bias in E. coli. In chapter 3, we use the population genetics model and the data available on the protein and mRNA levels of the E. coli genes to understand the pattern of codon usage in different genes with different expression levels and see which measure best explains the codon usage pattern. Besides codon bias, by testing for the over-parametrization of the model, we are able to test for the existence of context dependent mutation. We have also fitted the model for the codon usage patter in the Yeast and also tested for the context dependent mutation in this organism. In chapter 4, we focus on the first 10-15 codons in the genes of E. coli. Motivated by the fact that in this region we observe two phenomena, reduction in translation efficiency and suppression of mRNA secondary structures, we investigate whether the former is a side effect of selection for the latter. For this matter we have generated a set of synonymous randomized sequences, and then by selecting the ones which show weak secondary structures in the mentioned region, we would be able to test the theory. We will also look at the frequencies of the amino acids in E. coli genes and see whether the selection for weak secondary structures in the translation initiation region could be strong enough to not only affect the codon usage, but also the choice of amino acids. We would also provide information on the correlation between the strength of the mRNA secondary structure in the first 13 codons and the overall translation efficiency of the genes. / Thesis / Master of Science (MSc)

Human rotavirus (HRV) is a major causative agent of acute gastroenteritis (AGE), which causes severe dehydrating diarrhea in children under the age of five and results in up to 215,000 deaths worldwide each year. There are two live oral attenuated vaccines licensed for use in the United States that are highly effective in high-income countries but much less so in low-and middle-income countries (LMICs). Several factors contributing to decreased efficacy in these areas include chronic malnutrition, gut dysbiosis, and concurrent viral infection. Along with this, currently used vaccines require constant cold-chain storage to maintain vaccine stability, and those resources can be scarce in LMICs. These areas continue to maintain a high burden of HRV morbidity and mortality, and more efficacious vaccines are needed. The gnotobiotic (Gn) pig model of HRV infection and diarrhea has long been used in the evaluation of novel HRV vaccines due to Gn pigs' susceptibility to HRV infection, development of clinical signs, histopathological changes in the intestine, and the infection kinetics that mimic those seen in human infants. The first project in this dissertation used the Gn pig model to evaluate a thermostable live oral attenuated vaccine administered as a dissolvable film. Two doses of the tetravalent dissolvable film vaccine conferred significant protection from virus shedding by delaying its onset and reducing peak titers in feces. It also significantly delayed the onset of diarrhea and reduced the duration and area under the curve (AUC) of diarrhea. The dissolvable film was highly immunogenic, inducing high titers of serum virus neutralizing (VN) antibodies specific to each of the four G-types included in the vaccine formulation, HRV-specific serum IgA and IgG, and intestinal IgA. These data confirm the thermostable platform as a useful alternative to liquid vaccines that require cold-chain. The second project evaluated three mRNA-based nonreplicating vaccine candidates in the Gn pig model. All three mRNA candidates encoded a universal CD4+ T cell epitope, P2, derived from tetanus toxoid, fused with the encoded VP8* from P[4], P[6], and P[8] HRVs. Two candidates also encoded for a lumazine synthase (LS) domain fused with the P2-VP8*. A dose response study of the LS-P2-VP8* candidates was conducted simultaneously. Significant protection against virus shedding was induced by all three candidates, with LS-P2-VP8* candidates inducing significantly higher VP8*-specific serum IgG. All three candidates induced significantly higher numbers of P[8]-VP8*-specific IgG antibody-secreting cells (ASCs) and IFN-γ-producing T cells in the ileum, spleen and blood. These data provide guidance for further development of the relatively new mRNA-based technology for use in HRV vaccine development. In the final study of this dissertation, we used the Gn pig model of both P[8] and P[6] HRV infection to evaluate a cocktail nanoparticle-based HRV vaccine. This vaccine was made up of an S60 nanoparticle, self-assembled from the S domain of the human norovirus capsid protein. The exposed C-termini on the S60 nanoparticle were utilized as an antigen display platform, where VP8* from P[4], P[6] and P[8] HRVs was fused. This vaccine was tested as both a two-dose intramuscular (IM) regimen, or as an IM booster preceded by an oral priming immunization with commercial monovalent Rotarix®. Pigs were challenged with either P[6] or P[8] HRV to evaluate cross-protection of the nanoparticle vaccine. Both regimens were highly immunogenic, inducing high titers of serum VN, IgG and IgA antibodies. Furthermore, the prime-boost regimen conferred significant protection against virus shedding in P[8] HRV-challenged pigs as evidenced by the shortened duration of fecal virus shedding. There was also significant protection in P[6] HRV-challenged pigs vaccinated with the prime-boost regimen, as evidenced by the shortened duration, reduced mean peak titer and AUC of virus shedding. Prime-boost-vaccinated pigs challenged with P[8] HRV had significantly higher P[8]-specific IgG ASCs in the spleen post-challenge. Prime-boost-vaccinated pigs challenged with P[6] HRV had significantly higher numbers of P[6] and P[8]-specific IgG ASCs in the ileum, as well as significantly higher numbers of P[8]-specific IgA ASCs in the spleen post-challenge. Oral priming followed by parenteral boosting appears to be a promising vaccination strategy for HRV and these data warrant further investigation into this regimen. Through these studies, we improved our understanding of the effect of different vaccination routes and formulations in the effectiveness of conferring protection against an enteric virus. The knowledge will facilitate the development of more effective vaccination strategies against HRV, the leading cause of infantile diarrhea in LMICs, as well as other enteric viruses. / Doctor of Philosophy / Human rotavirus (HRV) is a major causative agent of acute gastroenteritis (AGE) in children under the age of five. Acute gastroenteritis is characterized by nausea, vomiting, and potentially deadly dehydrating diarrhea. There are two highly effective vaccines licensed for use in the United States; however, these vaccines are much less effective in low- and middle-income countries (LMICs), where HRV disease burden is the highest. There are several reasons thought to be responsible for the decrease in effectiveness seen in these areas, including chronic malnutrition and gut dysbiosis. Non-biological reasons for decreased efficacy may include the breakdown of cold-chain storage for these vaccines, which require constant low temperature storage that is often unavailable in LMICs. Thermostable vaccines are necessary for increasing vaccine distribution and efficacy in these areas. Because many of the biologic factors thought to interfere with the effectiveness of these vaccines appear to be confined to the gastrointestinal tract, development of next generation HRV vaccines has focused on the parenteral route of administration. The gnotobiotic (Gn) pig model is a highly relevant animal model that has been used for decades to evaluate novel HRV vaccine efficacy. Our first study evaluated a thermostable, dissolvable live oral vaccine administered as a dissolvable film in our Gn pig model. Two doses of this vaccine significantly reduced the severity of diarrhea and virus shedding in the stool. Our second study evaluated three mRNA-based intramuscular (IM) vaccines in the Gn pig model. Three doses of all mRNA candidates provided significant protection from virus shedding in the stool, as well as inducing the production of strong HRV-specific antibodies in the serum and high numbers of virus-specific T cells in the tissues. In our final study, we evaluated a nanoparticle-based vaccine as a two-dose IM regimen or as an IM booster preceded by an oral immunization using the commercially available Rotarix® vaccine. The prime-boost regimen significantly shortened the duration and severity of virus shedding in the stool. We also detected more cross-strain HRV-specific antibody-secreting cells in the tissues. All three vaccines evaluated in this dissertation offer differing novelty in the field of HRV vaccine development, and the Gn pig model has been instrumental in the evaluation of these vaccines.

H1 and core histone mRNA levels have been examined in the presence of portein synthesis inhibitors with different mechanisms of action. Total HeLa cell RNAs were analyzed by Northern Blot hybridization using cloned human histone genes as probes. Inhibition of DNA replication resulted in a rapid decline in histone mRNA levels. However, in the presence of cycloheximide or puromycin, H1 and core mRNAs did not decrease in parallel with DNA synthesis, but were stabilized and accumulated. Inhibition of DNA synthesis with hydroxyurea after the inhibition of protein synthesis did not lead to a decline in histone mRNA levels. These results suggest that synthesis of a protein(s) - perhaps a histone protein(s) - is required for the coordination of DNA synthesis and histone mRNA levels.

Hepatitis B virus (HBV) represents one of the hot topics of current basic and pharmaceutical research. Although an effective vaccine against HBV exists since 1982, the world prevalence of chronic infection is still alarming. The infection can lead to significant liver damage, often resulting in hepatocellular carcinoma. Chronic HBV infection cannot be cured due to the fact that the viral genome persists in the infected hepatocyte hidden from the host immune response as well as from the antiviral treatment. One of the novel approaches aiming for HBV cure suggests that this cccDNA pool could be destroyed using gene editing tools such as CRISPR/Cas9 system. In order to shift this gene editing system to possible medicinal application, CRISPR/Cas9 has to be specifically delivered into the target cell in order to minimize its putative off-target activity. This thesis focuses at first on the design and efficacy testing of new sgRNAs targeting HBV cccDNA and secondly, it describes modular lipid nanoparticles developed specially for delivery of the CRISPR/Cas9 system in the form of RNA. Keywords: hepatitis B virus, CRISPR/Cas9, gene editing, lipid nanoparticles, mRNA delivery, targeted delivery