Currently, most of the RNAs used in lab research are prepared by in vitro transcription or chemical synthesis, which can be costly. In vivo expression in bacterial cells is another approach to RNA preparation that allows large scale production at a lower cost. However, there are some obstacles in bacterial expression, including RNA degradation in host cell, as well as RNA extraction and purification. tRNA and 5S RNA have been reported as scaffolds to circumvent the degradation problem. These scaffolds can not only make the RNA product survive in the cell but also increase the stability after extraction. The packaging RNA (pRNA) of bacteriophage phi29 is a small non-coding RNA with a compact structure. The three-way junction (3WJ) region from pRNA is a thermodynamically stable RNA motif good for constructing therapeutic RNA nanoparticles. The 3WJ can not only integrate multiple RNA modules, but also stabilize them. Here I report a series of approaches made to express recombinant RNAs based on pRNA or 3WJ in bacteria, including 1) Investigating the mechanism of RNA folding in vitro and in vivo using 3WJ. 3WJ-based RNAs were expressed in E. coli using pET system. The results show that the folding of RNA is affected by both overall and regional energy landscape. 2) Expression of an RNA nanoparticle harboring multiple functional modules, a model of therapeutic RNA, in E. coli using a combination of tRNA scaffold and pRNA-3WJ. The expression was successful and all of the RNA modules were functional. 3) Expression of pRNA-based recombinant RNAs in B. subtilis. This is a novel system of expressing recombinant RNAs in Gram-positive bacteria.

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Cancer is increasingly prevalent globally and influenced by factors such as obesity and smoking. Cancer itself and chemotherapies, like cisplatin, can affect our reproductive organs and increase the risk of involuntary childlessness. In the ovaries there are oocytes that are surrounded by cumulus cells (CC) and the CC provides the oocyte with nutrients like pyruvate, which is important for the oocytes ability to mature. If the oocyte does not receive sufficient amount of nutrients the ovulation may be compromised. The CC are not well studied, and few prior studies have been done specifically to observe the impact of chemotherapies on CC. Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PPARGC1A) is important for the mitochondrial biogenesis in cells. The purpose of the project was to evaluate a method for RNA extraction in CC and observe the impact of cisplatin exposure on the PPARGC1A expression. CCs were aspirated from bovine ovaries and exposed to cisplatin during in vitro maturation. RNA was eluted using the QIAcube and then quality assured. The primer β2 microglobulin (B2M) was used as an endogenous control in the qPCR. The results showed that PPARGC1A was minimally expressed in bovine CC and was inadequate for use when evaluating if cisplatin changes the RNA expression in CC. However, the QIAcube proved to be a suitable method for RNA extraction from bovine CC and B2M showed to be suitable as an endogenous control in bovine CC.

Introduction: The epigenetic modifications can significantly affect and alter the gene activity by regulating their expression, having direct impact on various processes in human body. Epigenetic processes are involved in ethiopathogenesis of many diseases. From this point of view, MHC genes are very important as they were linked to many autoimmune disorders, for example type 1 diabetes mellitus. In general autoimmune diseases appear to be connected to certain MHC class II genes. Aims: The aim of this thesis is to determine the relationship between expression levels and histone modifications present in the promoter area of MHC class II gene, DQA1. Moreover, we also analyze and compare the DQA1 gene mRNA expression depending on the QAP promoter allele. Methods: We isolated both nucleic acids (DNA and RNA) and leukocytes from peripheral blood samples collected from voluntary donors. DNA was utilized for genotypization of individuals. RNA was subjected to reverse transcription and the quantitative PCR was performed in order to determine the level of expression. Leukocytes were used for chromatin immunoprecipitation, which was evaluated using quantitative PCR. Results: The expression level of QAP allele 3.1 was found to be higher than for the rest of the alleles Allele 4.1A showed, on the other hand,...

京都大学 / 新制・課程博士 / 博士(農学) / 甲第24240号 / 農博第2519号 / 新制||農||1094(附属図書館) / 学位論文||R4||N5411(農学部図書室) / 京都大学大学院農学研究科農学専攻 / (主査)教授 白岩 立彦, 教授 土井 元章, 教授 那須田 周平 / 学位規則第4条第1項該当 / Doctor of Agricultural Science / Kyoto University / DFAM

Human endogenous retroviruses (HERVs) constitute about 8% of the human genome. There are around 4000 pol-containing retroviral integrations in the human genome, which makes it impractical to measure each of them separately. Therefore we developed a set of degenerate real time PCRs to detect major groups bearing sequence similarities to gammaretroviruses, one of the largest groups of human endogenous retrovirus, and betaretroviruses, some of which have integrated into the human genome most recently and which remain the most intact. It was found that, although both gammaretroviral and betaretroviral RNAs were broadly expressed in various healthy tissues including reproductive tissues and brain, a differential expression pattern was observed. My work further revealed that HERVE and HERVW, two gammaretroviral sequences, were ubiquitously and highly expressed in pathologic and normal female reproductive tissues with tissue specific patterns. Expression of HERVE was higher in endometriotic tissue than in normal endometrium. HERVE and HERVW RNAs were higher in normal ovarian tissue than in ovarian cancer. Besides these tissue- and neoplasia-related differences, there were wide differences in HERV expression among individuals. Next, a selective pattern of HERVW upregulation was demonstrated in SK-N-DZ, a neuroblastoma cell line, upon re-oxygenation after a period of hypoxia or with 5-azacytidine, a demethylating agent. Furthermore, broad and high expressions of gammaretrovirus-like transcripts in different brain areas analyzed were identified. The expression levels were variable among different donors. In conclusion a ubiquitous HERV expression was observed in tissues and cell lines, with various patterns. At this stage the data are not sufficient to conclude whether HERV has any physiological or pathological roles in humans. However, their differential expression patterns are compatible with functional roles of HERV in humans.