doctoral thesis

本研究の目的は、皮膚潰瘍モデルマウスにおいて、凍結保存した他家線維芽細胞シートの治療効果を検討することである。他家細胞シートの凍結保存が可能であれば、様々な疾患への応用が可能であり、大幅なコストダウンによる再生医療の普及に貢献できると考えられる。本研究では、3Dフリーザーを用いて線維芽細胞シートの凍結を行った。凍結融解した線維芽細胞シートは、非凍結線維芽細胞シートと比較して、細胞生存率は約80であり、vascularendothelial growth factor VEGF ）、hepatocyte growth factor HGF）、stromalderived factor 1α SDF1αの培養上清中の濃度が50％以上で、transforminggrowth factor β1 TGFβ1の分泌能は同等であった。皮膚潰瘍モデルマウスにおいて、非凍結線維芽細胞シート群と凍結融解した線維芽細胞シート群の間で、自家細胞、他家細胞のどちらも創傷治癒率に差はなかった。また、血管新生の程度も同等であった。治癒組織におけるCD3陽性細胞数は、自家線維芽細胞シート群と比較して他家線維芽細胞シート群で多く見られた。しかし、病理組織学的には、凍結融解した他家線維芽細胞シート群の線維化、新生血管密度、創傷治癒速度は、非凍結他家線維芽細胞シート群に比べて凍結融解した自家線維芽細胞シート群に類似していた。これらの結果から、凍結融解した他家線維芽細胞シートが難治性皮膚潰瘍に対する有望な治療オプションとなる可能性が示唆された。

In recent years, many reports have been made of damage to reservoir embankments due to frequent heavy rains and earthquakes. “Internal erosion” is the phenomenon in which soil particles move through the pore spaces of embankments and flow out of them because of long-term seepage in the soil structures of reservoirs and river embankments. Internal erosion is classified according to the scale and form of the erosion. “Suffusion” is the phenomenon in which only the fine-grained soil particles move through the pore spaces formed by the coarse-grained soil particles. Suffusion is known to cause an increase in hydraulic conductivity and a decrease in soil bearing capacity. However, the mechanisms and causes of the occurrence and progression of this phenomenon, as well as its effect on soil structures, have not been fully elucidated. Therefore, the purpose of this study was to clarify the effect of suffusion on the strength properties of reservoir embankments as well as the mechanism of the progression of suffusion, focusing on the particle size of the eroded soil particles. In Chapter 1, the background of the study and the classification of internal erosion were provided. In Chapter 2, the mechanism of suffusion and its effect on geomaterials, which have been clarified in previous studies, were described. In Chapter 3, the effect of suffusion on the strength properties was firstly investigated by means of soil samples used for the reservoir embankments which were determined to be at risk of suffusion based on an internal stability evaluation. A triaxial compression testing system was established to simulate the suffusion inside each specimen. In the testing system’s apparatus, only the fine particles were able to be discharged from the specimen through a sieve by supplying water. The results showed that changes in the peak and residual strength occurred independent of changes in the void ratio due to suffusion, and that the strength properties differed depending on the presence or absence of suffusion, even when the void ratio was the same as that before shearing. However, since the amount of erosion was small (less than 0.7% of the total), it is considered that the change in strength was influenced by the fine soil particles that moved through the specimen but remained in the specimen without finally flowing out of it. In Chapter 4, in order to define the mechanism of small-scale internal erosion, a method was devised to examine the temporal changes in the size of the discharged soil particles, focusing on the relationship between the concentration and the turbidity of the drainage. The above method was also applied to the drainage obtained by seepage with suffusion in Chapter 3. As a result, the erosion rates were found to correlate with the particle size of the discharged particles. In Chapter 5, a one-dimensional water-passing experiment with suffusion was conducted to investigate in detail the relationship among the flow rate, soil particle discharge, and turbidity of the drainage. The particle size composition of the discharged soil, which varied with the progress of suffusion, was examined using the method described in Chapter 4. The results showed that, under all conditions, the soil particles with smaller diameters were selected from among those that were able to pass through the wire mesh at the outlet boundary and then discharged. It was also found that the particle size composition of the discharged soil changed with time as the suffusion progressed, and that this trend varied greatly depending on the saturation degree of each specimen before the start of permeation. After the completion of the experiment, the particle size composition of the soil samples showed that not only the distribution of the erodible component in the height direction, but also the particle size composition of the erodible component itself became heterogeneous, suggesting that soil particles with smaller particle sizes moved longer distances through the pores. Furthermore, the effect of overburden pressure on the behavior of suffusion under constant hydraulic gradient conditions was explored, and it was revealed that higher overburden pressure resulted in less frequent suffusion when the hydraulic gradient was large. The particle size composition of the discharged soil was examined, and no difference was found among the various levels of overburden pressure. In addition, the progression of suffusion was investigated under a fluctuating hydraulic gradient. The suffusion continued even after 30 cycles of hydraulic gradient variation. The particle size composition of the discharged soil particles showed that the small soil particles, that flow easily, had already been discharged when the specimen was first subjected to permeation force, and that the soil particles discharged by the change in hydraulic gradient were relatively large in diameter. The particle size of the soil particles discharged by suffusion were seen to depend on the conditions surrounding the occurrence of suffusion and the time of the progression. In studies on suffusion, the particle size composition of the soil discharged by suffusion is a significant factor that changes over time and can be used to evaluate the degree of the progression of suffusion.

細胞シート移植治療の普及には利便性の高い細胞シートの保存方法の開発が不可欠である。細胞シートから分泌される成長因子によって創傷治癒は促進されると考えられてきたため、その保存には細胞生存率が重要視されてきた。そのため、積層線維芽細胞シートの乾燥保存に関する検討はこれまでにない。本研究では、乾燥保存した積層線維芽細胞シート（Dryシート）を開発し、糖尿病マウス全層皮膚欠損モデルでの創傷治癒促進効果を検証した。 マウスの尾から単離した線維芽細胞を用いて、積層線維芽細胞シート（Livingシート）を作製した。Livingシートを風乾させ、Dryシートとした。Livingシートに凍結解凍操作を繰り返してFreeze-thaw（FT）シートとした。各シートを培地に浸漬して溶出液とし、各溶出液中の成長因子を測定した。Vascular endothelial growth factor（VEGF）とhepatocyte growth factor（HGF）はDryシートとLivingシートで検出したのに対し、fibroblast growth factor-2（FGF-2）とhigh mobility group box 1（HMGB1）はDryシートのみで検出した。FTシートではこれらの成長因子をほとんど検出しなかった。細胞シートの溶出液を添加して線維芽細胞を培養し、溶出液の生理活性を線維芽細胞増殖試験で検討した。DryシートはLivingシートと比べ、細胞増殖と成長因子産生量を有意に促進し、FGF-2中和抗体で阻害すると、この細胞増殖反応は抑制された。糖尿病マウス全層皮膚欠損モデル（C57BL/6N）において、自家及び他家（C3H/He）の線維芽細胞から作製したDryシート貼付群は無治療群に比べ、創の閉鎖を有意に促進した。Dryシートは常温（23℃）よりも冷蔵（4℃）での保存安定性に優れ、少なくとも4週間の冷蔵保存ではDryシートのFGF-2の減少を認めなかった。 以上から、DryシートはFGF-2の放出という新たな作用機序で創傷治癒を促進することが明らかになった。他家細胞から作製されたDryシートは、創傷治癒を促す再生医療において新たな扱いやすい被覆材であることが示唆された。

背景：UGT1A1*28および*6遺伝子多型は、イリノテカンに関連する毒性の危険因子として知られている。しかし、UGT1A1*28および*6に遺伝子変異を持たない患者においても、イリノテカンによる重篤な副作用が認められている。我々は、UGT1A以外のイリノテカン毒性の有用なバイオマーカーを同定するために、全エクソームにおける遺伝子多型を調査した。 方法：FOLFIRI療法、FOLFOX療法、FOLFOXIRI療法を投与された転移性大腸癌（mCRC）患者178例とmodified FOLFIRINOX療法、ゲムシタビン＋ナブパクリタキセル療法を投与された膵臓癌患者87例を対象とした。ゲノムワイドスクリーニングは全エクソームシーケンス（WES）を用いて行い、バリデーション解析は加水分解プローブを用いたqPCRを用いて実施した。 結果：FOLFIRI療法症例のWES（n = 15）により、7つの一塩基多型（SNP）がイリノテカン関連毒性である好中球減少のバイオマーカー候補として同定された。7つのSNPのうち、R3H domain and coiled-coil containing 1（R3HCC1; c.919G>A, rs2272761）のSNPは、検証サンプル症例のグレード3以上の好中球減少と有意な関連性を示した。mCRC患者に対するFOLFOXIRI療法（n = 23）または膵臓癌に対するmodified FOLFIRINOX療法（n = 40）といったイリノテカン含有の3剤併用化学療法患者でも、R3HCC1多型と好中球減少との間に有意な線形傾向がみられた（それぞれP = 0.017 および0.046 ）。一方で、イリノテカンを含まないレジメン（mCRC患者に対するFOLFOX療法（n = 66）、膵臓癌に対するゲムシタビン＋ナブパクリタキセル療法（n = 47））患者では、有意な関連は認められなかった。 結論：R3HCC1多型は、mCRCと膵臓癌に対するイリノテカンを含む化学療法の毒性に関する有用なバイオマーカーとなる可能性がある。

目的：アルコール使用障害（Alcohol use disorder: AUD）は治療後も再発率が高く再発を予測する患者の要因は確立されていない。AUDでは衝動制御障害、実行機能障害および意思決定障害が報告されているため、本研究ではそれらを用いてAUDの予後予測因子を前方視的に明らかにすることを目的とした。 方法：20歳から70歳までの入院中のAUD患者41名を対象とした。入院中に、①情動顔go/no-go課題（衝動制御課題）および言語流暢性課題（実行機能課題）中の脳血流活性化の指標である酸素化ヘモグロビン積分値を機能的近赤外分光法（functional nearinfrared spectroscopy: fNIRS）を用いて測定し、さらに②不確実な状況下でのリスク選好課題（意思決定）を行った。退院後6ヶ月の再発の有無を主要アウトカムとした。 結果：退院後、24名（58.5％）が断酒を継続し、17名（41.5％）が再飲酒した。断酒群に比べて再飲酒群では、①情動顔go/no go課題における右前頭側頭領域の活性化が有意に低下し、②意思決定課題では有意にリスク追及的であった。断酒群でのみ、右前頭側頭領域の活性と渇望尺度の間に負の相関が観察された。さらに、退院後6ヶ月の飲酒の有無を従属変数とし、年齢、AUD重症度、発症年齢、右前頭葉領域の酸素化ヘモグロビン積分値及び意思決定課題でのリスク選好の５つの独立変数を用いた2項ロジスティック回帰分析を行い、各独立因子の再発への影響を検討した結果、右前頭側頭領域の酸素化ヘモグロビン積分値が小さいほど（オッズ比＝0.161、p＝0.013）、またリスク追及傾向が強いほど（オッズ比＝7.04、p＝0.033）再発リスクが増加することが示された。 結論：右前頭側頭領域の情動刺激に対する脳血流活性化の低下と、リスク追及傾向が、AUDにおける退院6か月後の再発を予測し得る可能性が示された。

人工股関節全置換術(Total hip arthroplasty, 以下THA）においてカップ設置角度、カップ設置位置は脱臼の予防、外転筋レバーアームの再建、腸腰筋インピンジメント予防などの点で重要性はますます高くなっている。これまで後方アプローチでのComputed Tomography（CT）-based navigation使用によるcup 設置の正確性に関する報告は多数あるが、仰臥位前方アプローチ（Direct anterior approach、以下DAA）での報告は少なく、また術中イメージを使用した設置精度との比較をした報告はない。今回DAAでのTHAにおけるCT-based navigationを使用したカップ設置精度を検討した。DAAによるTHAを施行した156例171股における、カップ設置精度について、術中mechanical cup alignment guide使用群（MG群63股）、術中fluoroscopy使用群（FS群58股）、CT-based navigation使用群（CTN群50股）の3群について比較した。検討項目は、Lewinneckのsafe zone内のカップ設置割合（％）、カップ外転角(radiographic inclination、以下RI)、カップ前捻角（radiographic anteversion、以下RA）、カップの骨頭中心位置（上下、前後、内外）に関して、術前計画と術後設置角度、位置を比較し、その絶対値誤差をそれぞれ3群にて検討した。術後2週で全例CT検査を実施し、三次元解析ソフトにて誤差を抽出した。Lewinneckのsafe zone内の設置割合は、MG群80.9％（55/63）、FS群81％（47/58）、CTN 群100％（50/50）で、CTN群はMG群（p=0.005）、FS群（p=0.005）よりも有意に高かった。RIはMG群4.4±3.2°、FS群3.6±3.1°、CTN群2.8±2.5°であり、CTN群はMG群（p=0.01）よりも有意に誤差が小さかった。RAはMG群5.8±4.7°、FS群4.8±4.1°、CTN群2.8±1.9°で、CTN群はMG群（p=0.0001）、FS群（p=0.02）より有意に誤差が小さかった。RI、RAに対して絶対値誤差3°以上をoutlierとし各群間で比較した。RI についてMG群63.5％（40/63）、FS群43.1％（25/58）、CTN群36％（18/50）で、CTN群はMG群（p=0.03）より有意にoutlierが少なかった。RAについて、MG群66.7％（42/63）、FS群58.6％（34/58）、CTN群48％（24/50）でCTN群はMG群（p=0.04)よりも有意にoutlierが少なかった。カップの骨頭中心座標（内外:X軸、前後:Y軸、上下:Z軸）については、X軸、Y軸では3群とも有意差は認めなかった。しかしZ軸ではMG群3.3±3.2mm、FS群3.2±3.0mm、CTN群1.8±1.4mmで、CTN群がMG群（p=0.02）、FS群（p=0.007）に対して有意に誤差が少なかった。絶対値誤差より2mm以上をoutlierとし各群間で比較を行った。X軸、Y軸において有意差は認められなかった。一方Z軸に関してはCTN群がMG群（p=0.03)、FS群（p=0.04)よりも有意にoutlierが少なかった。今回の結果から、DAAでのTHAにおいて、CT-basedNavigation使用することでmechanical cup alignment guide使用や、fluoroscopyを使用するよりもカップ設置精度が向上し、有用であることが確認された。

背景・目的：大腸癌（CRC）は、世界的に癌関連死亡の第2位の原因となっており、転移を伴う大腸癌（mCRC）患者の予後不良は緊急の課題である。以前、我々はSOMAscanアッセイを用いて、予後を予測する10種類のバイオマーカー候補を得ることができた。本研究の目的は、得られた候補タンパクの一つであるC-C motif chemokine ligand 7（CCL7）について、mCRC患者における治療前の血清CCL7濃度の予後予測性能を明らかにすることであった。 材料と方法：mCRC患者の血清（n=110）および手術標本（n=85）について、それぞれCCL7のタンパク濃度をELISA法および免疫組織化学法で検討した。また、Cox回帰分析、受信者動作特性曲線(ROC)分析、Kaplan-Meier法を用いて、タンパクの濃度と予後との関係を検討した。結果：血清CCL7濃度が高い患者の全生存期間（OS）は、低い患者と比較し、有意に不良であった。間質中のCCL7発現レベルが高い患者は、低い患者に比べ、有意に予後不良であった。 Carcino embryonic antigen（CEA）および糖鎖抗原19-9（CA19-9）の濃度は、CCL7低値群に比べ、高CCL7群で有意に高値であった。単変量解析および多変量解析により、血清CCL7濃度はmCRCの有意な予後因子であることが明らかになった。また、血清CCL濃度とCEA濃度の組み合わせにおいて、血清CCL7濃度、CEAの両方が高値である患者は、両方が低値である群と比較し、有意に予後不良であった。 結論：治療前の血清CCL7濃度および血清CCL7濃度とCEAとの組み合わせは、mCRCの予後を予測する有用なバイオマーカーである。

This dissertation focuses on the experiences of a group of Japanese university students who participated in a short-term study abroad program in the Philippines. While the main objective of the four-week sojourn was to improve the participants’ English language skills, I explored the effects of the trip holistically as a qualitative case study to examine the unique learning context of studying English in the Philippines, a relatively new phenomenon that has developed from the turn of the 21st century. The primary form of data I used was interview responses obtained from 14 focal participants among a larger cohort of 103 students who traveled together in the summer of 2018. The target students were mostly low to intermediate level (CEFR A2 to B1) before departure and returned at a marginally higher benchmark (CEFR B1 average) according to standardized testing scores. More importantly, most students did not have extensive international experience prior to the sojourn, and overall they expressed a more open stance to using English for communication after the trip. In this case and context, positive affective development occurred due to intensive one-on-one instruction with Filipino teachers who interacted with the Japanese students in a warm, friendly, and supportive manner. This learning situation is characteristically different from North American and other Western contexts typically associated with English learning, and with its relative ease of access geographically and financially, offers a practical alternative to Japanese students unable to study abroad otherwise. The results of this study indicate how Japanese learners of English adopt subtle changes in outlook through short-term study abroad programs in diversifying contexts. This research contributes significantly to the field of language learning in study abroad by documenting the perspectives of learners from Japan traveling within Asia. Study abroad itself is a relatively new area of inquiry which has focused predominantly on Western learners and contexts. Existing research on Japanese learners tends to concern privileged, advanced-level learners whose experiences may seem removed from the ordinary lives of students attending university in rural areas or those who do not consider themselves academically or socioeconomically exceptional. Furthermore, the amount of existing research on short-term study abroad is severely limited and does not thoroughly account for the experiences of Japanese learners of English in the Philippines, a study abroad destination which has gained substantial popularity but remains underrepresented. Accounting for the social, cultural, and ideological circumstances of learning in a non-native English speaking context in Asia allows for a more nuanced interpretation of how monolingual Japanese speakers first encounter English as an international language. I collected data in multiple forms to document the complexity of the case. As the participants’ English teacher and chaperone to the Philippines, I was immersed in the learning contexts in the students’ home country as well as abroad. Semi-structured interviews before and after the study abroad experience were conducted in the informants’ native language. Written questionnaires were administered to the larger cohort of students who traveled together to obtain a more general assessment of students’ impressions regarding the study abroad experience. Standardized test scores were accessed for reference. These multiple data sources were drawn on to illustrate the changes observed in the perspectives of the focal informants. While the learning experiences of each informant were unique, I identified common themes among the ways students collectively changed their attitudes and beliefs toward English and language learning through their participation in the short-term study abroad program. First, the learners in this study increased their self-confidence and motivation to learn English and overcame their fear, anxiety, and inhibition toward using English to varying degrees. Second, they expanded their interests in learning about other languages and cultures while becoming more aware of the boundaries of their own culture. Third, their perceived communicative ability in English improved, which they found was not the same as having to perform well on standardized English tests. Fourth, participants’ sense of success in improving their English language proficiency was less certain, reflecting their divergent test score gains on standardized exams. These reported changes in perspective were not lifechanging transformations of learners’ social identities, but their previously constructed views of language, culture, communication, and learning were evidently affected. The qualitative effects of study abroad experienced by the informants of this study cannot be assessed adequately with the language proficiency tests highly regarded in mainstream Japanese society. Language ideologies prevalent in the participants’ home and host contexts for learning need to be considered in order to explain why the changes occurred. The learners in this study initially had an additional affective barrier toward communication in intercultural contexts because they could not take advantage of the widespread use of English as a lingua franca without first making efforts to use English itself. Since they were used to monolingual discourses in Japan, the idea of using English as a lingua franca, not only as a “perfect” native language, had to be presented to them in a reallife situation. The study abroad experience nudged learners toward using English in ways that reflect the Global Englishes paradigm. These slight shifts in mindset are indicative of attitudinal changes gradually taking place within Japan and are not restricted to individuals who identify with international communities.

細胞は熱ストレスなどのタンパク質毒性ストレスにさらされると、熱ショックタンパク質群（HSPs）を誘導することで適応する。この適応機構は熱ショック応答と呼ばれ、熱ショック転写因子HSF1によって主に転写レベルで制御される。活性化されたHSF1はHSP遺伝子プロモーターに存在する熱ショック応答配列（HSE）に結合し、メディエーターを含む転写開始前複合体を集積させることで転写を促進する。一般に、転写因子及びその調節因子は液―液相分離によってプロモーター上に凝縮体を形成すると考えられている。しかし、HSP遺伝子プロモーター上でも同様かどうかについては、凝縮体が微小であるために十分な解析ができていない。本研究では、ヒトHSP72プロモーター由来のHSEを多数連結したレポーター遺伝子をマウス細胞に導入した。このHSEレポーター遺伝子を持つ細胞に蛍光タンパク質mEGFPを融合したHSF1を発現させることで、熱ストレス条件下でHSF1凝縮体を可視化することに成功した。この人工的なHSF1凝縮体は部分的に液体様の性質を持つ、すなわち液―液相分離により形成されていた。また、大腸菌から精製したタンパク質を用いた実験から、HSF1の天然変性領域IDR)が相分離に寄与することも分かった。さらにこの実験系を用いて、HSF1凝縮体の形成が転写調節因子によって制御されるかを調べた。特に、熱ショック応答を促進するメディエーターの一つであるMED12に着目して解析したところ、MED12のIDRはHSF1凝縮体に集積すること、そしてMED12の発現抑制はHSF1の凝縮体形成を著しく抑制することが明らかとなった。本研究は、HSP72プロモーター上のHSF1凝縮体を解析する実験系を提示するとともに、それが転写調節因子によって制御されることを示唆する。

Population growth drives the demand for energy, which is the most pressing human need today. It puts pressure on other related sectors and increases wastewater production, which is a big problem in some countries that is inaccessible to available wastewater treatment facilities. Concerning the SDGs 2030 target, which mentions integrated sectors to deal with environmental issues, energy and water as a part of the water-energy-food nexus have a high linkage to build substantial economic and ecological benefits. Therefore, they have become the core attention of the world at this moment. To deal with the problem solving mentioned above, nowadays, an alternative energy converter integrated wastewater treatment has been massively developed to achieve green label production of products. Many countries, favoured by their scientists and practitioners, compete to find technology that is a user-friendly, eco-friendly, back-to-nature concept, inexpensive and can be accepted in any society stratifications. However, there is an available standalone converter technology known as conventional technology, which is a disintegrated system with no added value. This challenge opens an opportunity to develop a fully integrated system with any advantages. One of the promising technologies needed for solving the environmental problem and simultaneously producing other benefits for human living is a part of the bioelectrochemistry system (BES), which is microbial fuel cell (MFC). It could be easily assisted with the available conventional wastewater treatment, and it gives more benefits not only in energy production but also for remediating the environment through the superior biocatalyst, named electroactive bacteria (EAB), which has the availability to reduce organic and inorganic matter and generating electricity. However, factors affecting MFC have been a drawback in their field application that must be concerned extensively. Therefore, this study accommodates to investigation more in order to get an optimum condition in operation so that the technology could be widely used properly on the full scale. The disadvantages of the air–cathode single-chamber microbial fuel cell (AC-SCMFC) performance can be caused by numerous factors, and retention time (RT) is one such factor. It is difficult to conclude the ideal RT run for the specific tests under the same conditions. To determine the optimum RT for various types of microbial fuel cell (MFC), an AC-SCMFC batch-mode reactor was carried out by comparing different types and concentrations of substrates based on the main parameters of organic removal and power generation. The AC-SCMFC reactor was designed for the effective working volume of 500 mL and operated for 52 d in batch mode with factors being significantly correlated with the performance of the MFC reactor, which were two different substrates, sucrose and acetate, and three different chemical oxygen demand (COD) levels of 400; 1000, and 2500 mg/L (low, medium, and high, respectively) equipped with two graphene nanoplatelets (GNPs)-based electrodes connected to 100 Ω resistance and plugged onto a ii data logger. The results of this study indicated a significant pattern at the medium level, at which the optimum RT of sucrose was achieved at 24 h and that of acetate at 48 h. In comparison, the performances pattern at low and high levels of both substrates was insignificant to determine the optimum RT. For further application, the recommended RT for both substrates at any concentration is 24 h due to high overall performance, and the optimum RT established in this study could be applied to all types of MFC research, particularly in oxidizable or biodegradable organic ranges, which ensures high performance. One of the important factors in enhancing the performance of microbial fuel cells (MFCs) is reactor design and configuration. Therefore, this study was conducted to evaluate the regressors and their operating parameters affecting the double anode chamber–designed dual-chamber microbial fuel cell (DAC-DCMFC) performance. Its primary design consists of two anode chamber compartments equipped with a separator and cathode chamber. The DAC-DCMFCs were parallelly operated over 8 days (60 days after the acclimation period). They were intermittently pump-fed with the different organic loading rates (OLRs), using chemically enriched sucrose as artificial wastewater. The applied OLRs were adjusted at low, medium, and high ranges from 0.4 kg.m-3.d-1 to 2.5 kg.m-3.d-1. The reactor types were type 1 and type 2 with different cathode materials. The pH, temperature, oxidation-reduction potential (ORP), optical density 600 (OD600), chemical oxygen demand (COD), and total organic carbon (TOC) were measured, using standard analytical instruments. In general, the power production achieved a maximum of 866 ± 44 mW/m2, with a volumetric power density of 5.15 ± 0.26 W/m3 and coulombic efficiency of 84%. Two-stage COD and TOC removal at medium OLR achieved a range of 60–80%. Medium OLR is the recommended level to enhance power production and organic removal in DAC-DCMFC. The separated anode chambers into two parts in a dual anode chamber microbial fuel cell adjusted by various organic loadings expressed a preferable comprehension of the integrated MFCs for wastewater treatment. With respect to both studies, RT influences the design and configuration of MFCs, particularly in this regard, modified anode compartment of DCMFCs adapted to the range of oxidizable or biodegradable organics and reactor components towards control and dependent variables provide the simultaneous performance of DCMFCs in organic removal and power generation. In addition, DAC-DCMFC offers an opportunity to achieve optimal conditions in concurrent MFC-assisted wastewater treatment. Therefore, this study is one step closer to understanding the operating conditions comprehensively, which are the dominant factors affecting performance.

Indonesia has extreme risk of natural disasters because of its position at the confluence of four tectonic plates: the Asian Continent, the Australian Continent, the Indian Ocean, and the Pacific Ocean. The volcano eruption is one of the geological disasters that frequently occur in this country. Mount Merapi is Indonesia’s most active volcano and is famous worldwide. The evacuation crisis in 2010, the last major eruption, led to many fatalities for humans and cattle. Nowadays, the local government developed the "sister village" strategy for mitigation. That means cooperation within or between the local community has been constructed to provide shelter, logistics, and other disaster-related services. In this scenario, the meeting area and shelter have been coordinated. However, people's behavior has not been fully considered yet in the vulnerability assessment and government's contingency plan. On the other hand, evacuation issues in a volcanic disaster such as difficulty in expecting evacuation period, aging population, missed communication and risk perception, limited private vehicles in the community, and limited evacuation transport and supporters by the government need to be addressed for better mitigation. This situation led some people to walk to the meeting area, and low walking speeds by vulnerable persons may increase the risk and delay during an emergency. The objective of this study is to find the effectiveness of the evacuation process, especially for the vulnerable community in the Mount Merapi area. Especially, this study purposes to develop a “mutual assistance” model for vulnerable people in the affected regencies with the people’s behavior and vehicle ownership as a viewpoint. The first goal is an assessment of the mutual assistance strategy and social vulnerability index (SoVI) of pedestrian evacuation. I conducted the surveys and then analyzed the data using a multicriteria method to obtain the SoVI values for communities. The second goal is the development of the assembly model to support safer and faster evacuation for vulnerable people. The AnyLogic software was selected for model simulation using input parameters from field surveys. In conducting a survey, I measured the walking speed directly of the evacuation drills in four affected regencies. I also investigated the people’s behavior and eruption characteristics using an interview process with stakeholders and group discussions with local communities. After that, I used the multicriteria method and focused on two factors, social and age structure (young, vulnerable, and mutual assistance between them), and risk perception (work, rain, night, alert, and evacuation map). The index reflects the distribution of actual walking speed, mutual assistance, and the government's plan. The result showed that mutual assistance groups have a higher walking speed than vulnerable people but lower than young people. Mutual assistance coordination is crucial to support the vulnerable in shorter evacuation times. The social and age structure of the social vulnerability index has a stronger risk influence than the perception factor in the evacuation process. The successful evacuation of vulnerable people during emergencies is a significant challenge. In this study, a mutual assistance strategy is proposed to support vulnerable people by evacuating them with young people. This strategy was simulated using AnyLogic software with the agent-based model concept. Pedestrians and vehicles played the roles of significant agents in this experiment. Evacuation departure rate, actual walking speed, group size, route, and coordination were crucial agent parameters. Residents’ attitudes, distribution of each agent, and actual walking speed were obtained from surveys. Then, I developed three scenarios and three models for the evacuation process. Scenarios considered traffic conditions of evacuation routes and models represented behavior approach. The results revealed that this mutual assistance model is effective for the rapid evacuation and risk reduction of vulnerable communities where successful evacuation rates have improved. As for mutual assistance behavior, Model 3, where young people are matched with vulnerable people in advance, has shown better results than Model 2. Additionally, Scenario C, where pedestrians have separate lanes from vehicles during the evacuation process, has resulted in more number of vulnerable people reaching the shelter than Scenario B in Model 3. The highest arrival rate was obtained by the combination of scenario C and Model 3. These findings are a novelty in the volcano context and reflect all categories of vulnerable behavior involving the elderly, disabled, children, and pregnant mothers. The model will benefit disaster management studies and authorities’ policies for sustainable evacuation planning and aging population mitigation.

本研究では、特定の遺伝子型と表現型との関連を明らかにするために、EDAR遺伝子の潜性（劣性）変異に着目し、その特徴を詳細に検討した。具体的には、過去にEDARのDD内に同定された潜性（劣性）遺伝形式を示す4 種類のミスセンス変異（p.R358Q、p.G382S、p.I388T、p.T403M）について、培養細胞での過剰発現系で一連の解析を実施した。これらの変異の中で、p.R358QはEDARADDとの結合能を失い、下流のNF-κB活性を低下させることが知られており、機能喪失の陽性対照として用いた。 まず、細胞溶解液を用いたwestern blot法では、p.R358Qおよびp.T403M変異型EDAR蛋白は野生型EDAR蛋白よりも発現量が減衰し、より大きい分子量を示した。一方で、p.G382Sおよびp.I388T変異型EDAR蛋白は野生型EDAR蛋白と同様の発現パターンを示した。また、各EDAR蛋白の細胞内での局在を解析するために実施した蛍光免疫染色法では、野生型EDAR蛋白と同様にp.G382Sおよびp.I388T変異型EDAR蛋白は細胞質内に局在が認められたが、p.R358Qとp.T403M変異型EDAR蛋白は細胞膜に発現していた。これらの結果から、変異型蛋白間で発現パターンが異なることが示された。続いて行ったNF-κBレポーターアッセイでは、すべての変異型EDAR蛋白がNF-κBの活性化を抑制したが、p.R358Qとp.T403M変異型EDAR蛋白に比べ、p.G382Sとp.I388T変異型EDAR蛋白による抑制効果は軽微であった。EDARとEDARADDの結合を検討した共免疫沈降法では、p.R358Qとp.T403M変異型EDAR蛋白はEDARADDとの結合能を完全に喪失していたが、p.G382Sとp.I388T変異型EDAR蛋白は、ある程度結合能を維持した。これらの解析で、p.G382S変異型EDARの機能喪失の程度は最も軽度と考えられた。 過去の研究で、野生型EDARはTRAF6とは直接結合しないことが報告されており、本研究で実施した野生型EDARとTRAF6間の共免疫沈降法でも同様の結果が得られた。しかしながら、驚くべきことに、本研究で解析した全ての変異型EDAR蛋白はTRAF6と直接結合する性質を示した。 培養細胞での過剰発現系においては、機序は不明だがEDAR蛋白を含む種々のTNF受容体が細胞質内に発現する傾向を示すことが知られていたことから、p.R358Qおよびp.T403M変異型EDAR蛋白の細胞膜への局在は異常な発現パターンと考えられる。NF-κBレポーターアッセイおよび共免疫沈降法の結果から、各変異型EDAR蛋白とEDARADDの親和性はNF-κB活性低下の程度と強く相関することが示唆された。今回解析した4 種類のミスセンス変異は、いずれもEDARの機能や構造に重大な影響を与えると複数のデータベースで推測されていたが、各データベースのスコアは4つの変異の間で非常に類似していた。すなわち、現在の予測ツールの解析能力には限界があり、本研究のように実際に発現・機能解析を行う重要性がハイライトされたといえる。 4種類の変異型EDAR蛋白に共通する唯一の現象は、野生型EDAR蛋白がEDARADDを介して間接的にTRAF6と相互作用するのに対し、TRAF6と直接結合することである。これは、変異型EDAR蛋白がEDAR、DARADD、TRAF6からなる正しい蛋白複合体を形成できないことを示唆しており、EDAR遺伝子変異に起因するHEDの鍵となっている可能性があるが、本現象の病的意義を解明するためには今後のさらなる検討を要する。 本研究で得られた結果に基づき、各変異を機能喪失の度合いで評価した。R358QとT403Mを「重度」、p.I388Tを「中等度」、p.G382Sを「軽度」とした。各変異を報告した文献に提示されていた表現型と比較検討した結果、EDAR遺伝子変異の機能喪失の程度がHEDの重症度と相関している可能性が示唆された。

間葉系幹細胞（MSC）は、再生医療において有用な細胞である。MSCの培養工程の初期に、組織から抽出した単核球を培養容器に播種し、MSCの単離を行う。単離工程において、単核球の播種密度を最適化することで、単離後のMSCの収量や分化能が向上することが報告されている。しかし、単核球の播種密度がMSCの品質に影響する機構は明らかになっていない。 我々はその機構を明らかにする為、ヒト骨髄単核球を様々な播種密度（4.0×104、1.25×105、2.5×105、6.3×105、1.25×106cells/cm2）で培養し、単核球の播種密度とMSCのコロニー形成過程との関係を解析した。播種密度が高い条件（6.3×105、1.25×106cells/cm2）では、培養容器に接着したMSC同士の距離が小さく、互いの増殖スペースが制限される為、早期に高密度に達した。一方、低い播種密度（4.0×104、1.25×105 cells/cm2）では、MSCは単一細胞由来のコロニーを形成し、コロニーの直径や密度は様々な形態を示した。コロニーを継時観察したところ、高い増殖能力を持つMSCは、培養時間の経過に伴い、全体におけるその割合が増加した。それに対して、増殖能力が低いMSCは肥大化し、老化が進行した。老化細胞は剥離処理の時間を調整することで除去できることを見出した。単核球の播種密度が低い条件で、上記機構により、増殖能が高いMSCの純度が向上することが明らかになった。この機構をもとに単離工程のパラメータを最適化し、得られたMSCの増殖能と分化能を評価した。単核球の播種密度が高い条件（1.25×106cells/cm2）と比較し、最適化した条件（播種密度：1.25×105cells/cm2）から得られたMSCは、高い増殖能を示し、骨以外の脂肪や軟骨への分化能が有意に上昇した。 本研究により、高品質なMSCの純度を向上させる方法として、（1）単一細胞由来のコロニーを形成させる為に単核球の播種密度を最適化すること、（2）高い増殖能を持つMSCの割合を増やす為に培養期間を調整すること、（3）老化細胞を除去できる剥離処理の時間を設定することを提案する。

The objective of Chapter 1 of the present study was to evaluate the effect of heat-killed Lactobacillus sakei HS-1 (HK-LS HS-1) on the health and fecal bacteriological change of suckling Japanese Black calves as a supplement in milk replacers. To this end, they were randomly assigned to an HK-LS HS-1 supplement or a control without HK-LS HS-1 group in milk replacers. HK-LS HS-1 was administered from separation day to 3 weeks. Blood and fecal samples were examined. The result is glucose and vitamin A levels on day 7 were significantly higher in the supplement group than in the control group. No significant differences were observed in haptoglobin or serum amyloid A between the groups. The number of Escherichia coli in feces was lower in the control group than in the supplement group on day 21. No difference was observed in the number of Bifidobacteria, but that of lactic acid bacteria was significantly higher in the supplement group on day 21. The number of medications administered was significantly lower in the supplement group than in the control group during the experimental period. The results indicated that HK-LS HS-1 is potentially beneficial for improving intestinal microbes and reducing the number of medical treatments. In the second study, we evaluated the effects of supplementing cattle feed with difructose anhydride III (DFA III) by measuring urinary sterigmatocystin (STC) concentrations using 20 Japanese Black cattle aged 9–10 months from one herd. DFA III was supplemented for 2 weeks for 10 animals, and non-treated animals served as controls. STC concentration in the dietary feed was 0.06 mg kg−1(mixture of roughage and concentrate) at the beginning of the study (Day 0). The urine STC concentration was measured using liquid chromatography with tandem mass spectrometry 1 d prior to DFA 2 III administration, 9 and 14 d thereafter, and 9 d following supplementation cessation, concomitant with the measurement of serum amyloid A (SAA). The number of heifers in which STC was detected in the urine was low in the DFA III group compared to that in the control group on Day 9. After 9 d following supplementation cessation (Day 23), STC concentrations were significantly lower (P = 0.032) in the DFA III group than in the control group, although there was no difference in the number of heifers in which urinary STC was detected or in SAA concentrations between the two groups. Our findings demonstrate the effect of DFA III on reducing the urinary concentration of STC in Japanese Black cattle.

本論文は，申請者が山口大学大学院東アジア研究科在学中において進めてきた，遠隔教育支援環境における授業と学習活動の支援に関する研究をまとめたものである． 人口減少社会の到来により，特に地方において，適正規模での学校運営が難しい小規模の学校が増加傾向にある．小規模校では，きめ細やかな指導等が行える一方で，人間関係が固定化しがちであったり，多様な意見や考え方に触れることが少なくなったりなど，教育上の課題を抱えている．その解決策の１つとして，複数校の児童生徒が1つの学校に集まって合同授業が実施されているが，時間的・金銭的な問題などから実施回数が制限されるケースが多い．この合同授業を補う形で，日進月歩の勢いで進歩しているICT技術を活用した遠隔合同授業の取組みが期待され，教育実践が進められてきている． 一方，小学校や中学校だけでなく，大学などの高等教育機関においてもICTを活用した遠隔教育は進められてきている．さらに，新型コロナウィルス感染症のパンデミックを契機に，小学校・中学校・高校や高等教育機関だけでなく，塾など様々な場でオンライン授業あるいはオンライン学習といった遠隔教育の取り組みも行われてきている． しかしながら，オンライン授業や遠隔合同授業を実践する上では，環境整備面や授業実施面などで課題が存在する．特に，授業実施時における教員の作業負荷が非常に高いことが問題であり，オンライン授業や遠隔合同授業の特性や授業プロセスを踏まえた仕組みの構築や機能の開発が必要となる．さらに，オンライン授業や遠隔合同授業における学習者個人あるいはグループによる学習活動や，教員による教授活動や学習者グループの活動支援のプロセスを分析した上で，その状況に応じた支援手法を検討し，学習支援機能やその機能と連動した授業支援機能を遠隔教育支援ツール・アプリとして提供することが必要となる． そこで本研究では，高校・大学などにおけるオンライン授業や小学校・中学校の小規模校における遠隔合同授業といった遠隔教育を対象にして，校種やICT活用能力に応じた機能や画面に切り替えることができる設計思想を持った，課題解決的な学習が展開される際の学習者の学習活動と教員の教授活動を支援するための遠隔教育支援環境を設計して開発することを目的にする． 本論文では，本研究の目的を達成させるために，以下に示す5つの調査・研究・開発の成果がまとめられている． (1) 遠隔教育の支援環境に関する研究動向の調査 (2) 遠隔教育を支援する環境・機能についての整理と必要機能の提案 (3) 「クラス（学級）」と「個」をつなぐ遠隔教育支援環境「つながる授業アプリ」の開発と評価 (4) 「比べる」協働的思考活動支援機能の開発と評価 (5) グループ学習活動・対話状況確認支援機能とグループ活動状況管理機能の開発と評価 (1)では，遠隔教育に関する研究動向を調査した．これらの整理・分類などを踏まえ，遠隔教育における学習環境，学習支援機能，授業支援機能の研究に対する本研究の位置づけを行った． (2)では，遠隔教育をICTで支援する環境について説明した．さらに，2つのつながり（「クラス（学級）」と「個」）を保障する遠隔教育システムと協働学習支援システムに必要な機能を整理した． (3)では，遠隔教育システムと協働学習支援システムの両方の機能を併せ持つ遠隔教育支援環境「つながる授業アプリ」を設計・開発した．さらに，「つながる授業アプリ」のの有用性を評価し，基本機能を完成させた． (4)では，遠隔合同授業における小学生を対象として，より深い学びを実感できることを目的として，グループ活動時の思考に必要なプロセスを整理し，最も基本的な思考活動の1つである「比較思考」を協働的に進めることができる支援を「つながる授業アプリ」の拡張機能として実装した．「比べる」協働的思考活動支援機能は，「比べる」思考手順を１スライドずつ学習者間の協働的な思考手順として提示して，思考活動プロセスをガイドする仕組みである．さらに，開発した支援機能についてその有用性を評価した． (5)では，大学の遠隔教育を対象にして，グループ学習活動における学習者の対話内容の確認や振り返り，教員による見とりを支援する目的として，対話音声データ（バーバル情報）とノート記述データ（ノンバーバル情報）を活用した活動状況・対話状況の可視化機能を「つながる授業アプリ」の拡張機能として実装した．さらに，教員の見とり支援としてグループ活動状況管理機能も拡張機能として実装し，これらの機能の有用性を評価した． 以上により，オンライン授業が展開でき，学習者の個別学習やグループ学習の活動支援や教員の見とり支援が可能な「つながる授業アプリ」の開発・授業実践を通して，遠隔教育において必要な学習環境の要素機能，学習支援・授業支援の機能を確認することができた．

Access to clean water has been crucial global problem, especially with climate change, increasing population, and industrial activities. As one of Malaysia’s leading economic activities, the Oil and Gas Industry generates a massive amount of wastewater called Produced Water (PW). Dissolved organics in produced water, such as organic acids and phenolic compounds, are concerning due to the possibility that they can be toxic, non-biodegradable, and have bioaccumulation properties. Conventional treatments such as adsorption, incineration, and biological treatment seem to have difficulties treating these dilute but toxic components in an economical and environmentally friendly manner. Regulations on wastewater management has also been stricter around the globe. Therefore, there is a need on a new water treatment method to treat the diluted organics in a large volume of wastewater. Membrane technology has been of interest in the water treatment technologies’ industrial and research scenes. It offers simpler configuration and maintenance. However, the application is limited by the reduction of performance over time due to fouling phenomena. Photocatalyst offer an effective method to decompose organics in an environmentally friendly manner. This study researched on photocatalytic removal of diluted organic in water and potential of biofouling reduction by deposition of AgTiO2 coating on membrane surface. In Chapter 1: Introduction, the research background and purpose of this research were discussed. At the end of this chapter, the thesis framework was shown. In Chapter 2: Preparation and characterization of TiO2 and AgTiO2 coatings; the method to prepare AgTiO2 coatings on membrane support were explained. The prepared membranes were characterized with XPS, SEM, TEM, and ICP analysis to understand the prepared coatings. Results shows that the concentration of silver deposited on the membrane can be control by the concentration of silver in the precursor (silver acetate solution) used during the photochemical deposition step. Via XPS, it was found that the state of silver prepared through this method is oxide state. In Chapter 3: Removal of dissolved organic pollutants in water by photooxidation, the photocatalytic performance of prepared membranes was studied. Decomposition of diluted formic acid was performed under UV-light, and the concentration was evaluated using UVspectrophotometer. AgTiO2 membranes show better photocatalytic activity then TiO2 membrane. The concentration of silver on the membrane was found to influence its photocatalytic performance. In relation to PW application which commonly contain high salts, influence of salt types; NaCl, MgSO4, MgCl2 and K2SO4 and concentration were studied. All salts were found to inhibit the membranes’ photocatalytic performance. In Chapter 4: Antibacterial activity of AgTiO2 membranes, the antibacterial activity towards E. coli by prepared AgTiO2 membranes were investigated. Silver dissolution from membrane was found to be significantly increased in the presence of NaCl as compared to only water. Comparing membrane with lower Ag deposition, and around 20 times higher deposition, the silver dissolution from these membranes reached almost the same value after some time. However, as the amount of silver deposited on the membrane was higher, the antibacterial performance show around four times higher than the lower silver membrane. Based on other tests performed, it was concluded that there are potential of contribution from the silver oxide deposited on the membrane surface on the antibacterial activity of the AgTiO2 membranes In Chapter 5: Antibacterial activity in filtration system, prepared AgTiO2 membrane was used to filter E. coli suspension in water. E. coli growth was found to be inhibited by short contact with silver on the coated membrane. Finally, Chapter 6: Conclusion, the thesis was summarized, and future works were proposed.

Nutrient pollution is one of our most pervasive, expensive, and challenging environmental problems, according to the United States Environmental Protection Agency (EPA). Phosphorus is one of the nutrients that are essential for the growth of living organisms. However, excessive amounts of nutrients released into the environment by human activities can harm ecosystems and impact human health. In surface waters, phosphorus can contribute to an overgrowth of algae called algal "blooms" that can sicken or kill wildlife and endanger aquatic habitats. Algal blooms consume dissolved oxygen in the water, leaving little or no oxygen for fish and other aquatic organisms. Algal blooms can harm aquatic plants by blocking the sunlight they need to grow. Some algae produce toxins and encourage the growth of bacteria that can make people sick who are swimming or drinking water or eating contaminated fish or shellfish. Phosphorus is often a major limiting nutrient freshwater system. Consequently, many of the wastewater treatment plant discharged into freshwater systems such as lakes, ponds, and rivers have phosphorus discharge limits. In an attempt to prevent harmful environmental effects of excess phosphorus, several techniques have been designed to remove phosphorus from wastewater. These techniques range from adsorption and precipitation to enhanced biological phosphorus removal and constructed wetlands. Biological phosphorus removal (BPR) was first used at a few water resource recovery facilities in the late 1960s. A common element in EBPR implementation is the presence of an anaerobic tank (no nitrate and oxygen) before the aeration tank. In the next aerobic phase, these bacteria can accumulate large amounts of polyphosphate in their cells and phosphorus removal is said to be increased. The group of microorganisms that are largely responsible for P removal are known as the phosphorus accumulating organisms (PAOs). One of the options to remove phosphorus is to utilize bacteria from nature, besides being easy to obtain and inexpensive. The application of bacteria from sediment and seawater was able to reduce phosphorus in wastewater. In this study, for screening salt-tolerant phosphorus accumulating organisms (PAOs) and investigating the P release and uptake of the organisms in saline wastewater. The samples used were sediment and seawater from Yamaguchi Bay, Yamaguchi, Japan. Sediment and seawater added 150 mL of artificial saline wastewater with media (anaerobic media). The samples were then cultured and given feed media every three hours day at 25 °C and shaken at 140 rpm. The hydraulic retention time of the cultivation was 16 h and 8 h under anaerobic and aerobic conditions, respectively. 10 sponges made of polyurethane with dimensions of 2 cm were put in Erlenmeyer flasks and was used as a bio-carrier surface for microorganisms to adhere to. Water was passed over the sponge surface to acclimatize the microorganisms growing outside the sponge as well as within its pores, ensuring sufficient growth surface. The cultivation duration was 112 days. Batch experiments were conducted over 98 days in solutions with a salinity of 3.5% and P concentrations of 1, 5, 10, and 20 mg-P/L. The P-uptake ability of microorganisms increased by increasing P concentration from 1 to 20 mg-P/L. A high P removal percentage with an average of 85% was obtained at 10 mg-P/L after day 56. The uptake and release of P were observed in saline wastewater, signifying that salt-tolerant PAOs could grow in the saline solution. Bacterial screening by isolation and sequence analysis using 16S rRNA demonstrated that two cultivated strains, TR1 and MA3, had high similarity with Bacillus sp. and Thioclava sp. EIOx9, respectively. The colony morphology analysis showed that the colonies of TR1 were rod-shaped, milky-colored, round, shiny-viscous, smooth with a defined margin, while colonies of MA3 were cream-colored with smooth surfaces and raised aspect. The TR1 was gram-stain-positive with approximately 6-10 μm long and 1.2 μm wide cells, and MA3 was gram-stain-negative with about 0.9 μm long and 0.5 μm wide cells. The results demonstrated the involvement of Bacillus sp., and Thioclava sp. in the release and uptake of P, owing to their ability to grow in saline wastewater. Furthermore, Bacillus sp. (TR1) and Thioclava sp. (MA3) were assessed for their abiotic adaptability and phosphorus removal efficiency in saline wastewater. The effects of abiotic factors such as carbon source, pH, temperature, and salinity on bacterial growth were examined through a series of batch experiments. Both bacteria used carbon sources such as glucose, sucrose, and CH3COONa for their growth. The pH study indicated that Bacillus sp. (TR1) preferred the pH range of 6 8 and Thioclava sp. (MA3) preferred the pH range of 6-9. Bacillus sp. favorably multiplied in the temperature range of 25- 40 °C, while 25 35 °C was preferred by Thioclava sp. Salinity range of 0% 10% was favorable for TR1, with optimum growth observed at 3.5% 5%, and Thioclava sp. (MA3) preferred the salinity range of 1% 10% with optimal growth at 4%, but was absent in non-saline water. Bacillus sp. and bacterial combination (TR1 and MA3) showed similar values for phosphorus removal efficiency (100%) at 1.0 mg-P/L total P compared to Thioclava sp. (38.2%). The initial phosphorus concentration of 2.5 mg-P / L showed a slightly higher 72.35% P removal efficiency compared to the individual strains. However, phosphorus removal did not increase, but showed a downward trend with increasing at initial phosphorus. The combination possibly built a synergistic activity between the individual strains to remove phosphorus. The results demonstrated that when used individually, Bacillus sp. showed a reasonably high phosphorus removal ability than Thioclava sp., and exhibited good synergy when used in combination to remove phosphorus from saline wastewater.

Innovation is the practical implementation of ideas that result in the introduction of new goods or services or the improvement in them (Schumpter, 1983). Innovation is closely related to invention as innovation is more on involving the practical implementation of a new or improved invention to make a meaningful impact in a market or society (Schumpter, 1939). On the other hand, innovative design is a process of identifying, pinpointing, and understanding the needs of the user or audience (Shaulis, 2021). Previously, Dixon (1966) defined innovative design as any design that is: new or different, or elegant or uses new ideas, or is an improvement over its peers. Once the market need has been identified, a solution can then be designed. In our proposed innovative design method, we introduced and investigated a method that is able to be applied in designing an intergrated system that could be a valuable solution to the society. This method starts with directly observe activities of things and real people in real trouble in the real field. Then, we think about the value of "I wish there were such things as…", visualize the story, draw a clear sketch to accomplish the story concretely. Next, we solidify the functions and specifications while investigating needs and competition. Then, we create a prototype that able to show and test your ideas, demonstrate to the people who need it, let them experience it, and gain feedback. Lastly, we evaluate the value of product design and development and plan methods for implementing it as an organization, and plan ways to improve and expand globally. All of the steps in this method are important for innovative design, however, in this research this time we focused on co-designing value, big idea, and considering as integrated steps for identifying latent needs of the consumers. It is because identifying needs is an important part in the product development process. Latent needs are those that many consumers recognize as important in a final product but unable to articulate in advance (Ulrich, 2015). The latent needs addressed in this study was focusing on identifying consumer requirements in product development in the innovative design method. The challenge in identifying latent needs is finding the method to elicit from consumers the needs which are not addressed by any inventors yet in the present market but would delight the consumers if delivered tomorrow. The purpose of this study is to propose and verify the method in the elicitation of latent needs from consumer needs by introducing a working prototype to the consumers, interviewing, and analyzing responses from the consumers. This research was conducted during the year the start of the COVID-19 pandemic. As the pandemic spread, most countries were forced to go into lockdown or declare an emergency state. The school was closed and business organizations needed to switch to working from home to prevent the spread. The parents were unable to work from home efficiently as they were worried their children will involve in dangerous incidents if the children were left by themselves. Based on this situation, this study was conducted in finding the latent needs of the parents, childcare workers, and children in order to assist them in going through their problems during this COVID-19 pandemic. The working prototype was used as material to prepare presentation slides for the consumers' interviews. The first presentation slides were focused on the background problems and ideas for the solutions while the second presentation slides provided consumers with a prototype and story of the product that was believed would be one of the solutions to the problems. Interviews were conducted after both slide presentations. Consumers' responses were obtained and interpreted into consumers' needs in terms of product functions. In the first study, consumers' interpreted needs from Problem-based interviews and Prototype and Story-based interviews were compared. Based on the results, latent needs interpreted from interviewees' responses and the categories of the needs obtained from the Prototype-based interviews are more than from the Problem-based interview. The latent needs that we were able to obtain from this research were for example, “The device is able to detect small changes in a child while watching he/she sleeping” which could lead into the prevention of unwanted incident such as sudden infant death syndrome (SIDS). This supports our assumption that showing working prototype-based materials with story descriptions can be effective in uncovering potential latent needs. In the second study, it is assumed that experience, empathy, and knowledge of working prototype is essential elements in product development, therefore, new additional guidelines which are “to write a statement with empathy”, “to write a statement as a designer”, and “to write a statement as someone with experience” were proposed during consumers' needs interpretation to see whether these new guidelines will influence the process of identifying latent needs of consumers. From the result, it is concluded that the number of interpreted needs increased when we applied the new proposed guideline. Although the number is small, the needs might not be interpreted if the new guidelines were not considered. We were also able to obtain a few important latent needs when we applied these new guidelines. A latent need collected from applying the guideline "to write a statement as someone with experience" is “The device is not for teaching love and humanity but for monitoring by watching facial expression, posture, and vital signals such as temperature and heart rate”. We could conclude that including these guidelines upon interpreting raw data from the consumers’ interviews might lead into discovering important and critical latent needs of the consumers. In the third study, a quantitative evaluation method for identifying latent needs was introduced. The consumers' interpreted needs were rated according to a basis of rating from the three perspectives of importance, latent-ness, and technological feasibility. The Degree of Latent Needs (DLN) was calculated by multiplying these three metrics. Based on the result for the average and variance of DLN mean value for each evaluator which is sufficiently small, it indicates that the basis of rating for three metrics of the DLN is effective. The results also indicate that the 20 highest DLN points of the interpreted needs contain attractive features in terms of design. However, we had gotten some pushback on the average of each interpreted need and its variance which indicates opposing opinions among evaluators. As it is possible that attractive needs are hidden and may lead to the discovery of latent needs through individual pinpoint interviews, the interviews with the minority evaluators were conducted. The interview results indicate that the latent needs with low DLN rates but valuable might be able to be discovered by conducting follow-up interviews such as “The device is able to recognize items (food or not) that a child wants to put in the mouth”. From the results in all three studies, we could conclude that a number of important latent needs are able to be elicited from consumers’ needs by applying the proposed method. In our fourth study, a decision-making method based on the patent analysis between the conceptual design stage and the prototyping stage in the innovative design method was introduced. Conducting a patent strategy was assumed to support how to select the right concept precisely. In this study, by conducting a patent search in this stage by the designer who understood best the product functions and working principles, a supporting method was introduced to assist the designer in their decision-making process. Based on the result, the method was able to observe whether there are dominating companies or not for our concept design. If there is a dominating company, the possibility of not being able to produce our concept becomes bigger. This method may be applied as an indicator to support decisionmaking in the concept design stage in the innovative design method, whether to proceed with the concept design or not and to reduce the possibility of product failure in the future. From the results of all the studies, we could conclude that these above methods may be applied as assistive tools to support designers’ understanding of consumers’ requirements and selecting the right concept design.

自動車事故に際しシートベルトに沿って生じる帯状の皮下出血斑はシートベルト兆候（seat belt sign：SBS）と呼ばれている。特に腹部SBS が上前腸骨棘（anterior superior iliac spine：ASIS）よりも上方に位置する場合、腹部臓器損傷の危険性が高い。本研究の目的は、腹部SBS 位置に関連するシートベルト腹部部分（ラップベルト）の位置に影響を与える因子について解析することである。本研究は、健康な成人100名（男性50名、女性50名）の身体所見と、カーシート座位時のラップベルト位置との関係を前向きに検討したものである。身体所見は、年齢、身長、Body Mass Index（BMI）、腹囲を測定した。それぞれ平均年齢37.9歳、平均身長164.9cm、平均BMI 23.9kg/m2、平均腹囲83.4cmであった。X線学的所見は、腰椎前弯（lumbar lordosis：LL）、仙骨傾斜（sacral slope：SS）を測定し、ラップベルト位置は運転席側のラップベルトの中央とASIS相当の位置に鉛テープでマーキングすることで計測した。側面X線撮影を行い、ASISから中央マーカーまでの水平距離（X値）、垂直距離（Z値）を計測した。ラップベルト角度は、2つのマーカーの上端を結ぶ直線と水平線とのなす角度を計測することで求めた。これらの身体所見とX線学的所見との関係を統計学的に解析した。X値とZ値は体重（X値r = 0.73、Z値r = 0.56）、BMI（X値r = 0.77、Z値r = 0.56）、腹囲（X値r = 0.74、Z値r = 0.52）と正の相関があり、ラップベルト角度は体重（r = -0.33）、BMI（r = -0.35）、腹囲（r = -0.37）と負の相関があった。これらの結果からは、BMIの高い乗員ではラップベルトがASISより高い位置にあるため、シートベルト損傷を引き起こす可能性が高い。この解析は、より安全なシートベルトの開発に役立つと思われる。

背景：The Union for International Cancer Control（UICC）の tumour、node、metastasis（TNM）分類によって大腸がんの治癒あるいは再発の可能性を予測出来るため、治療方針の決定に役立っている。しかしこの分類法単独による予後の予測は不十分である。そのため予後予測因子として新たなバイオマーカーの同定が望まれている。過去に我々は、根治切除を行ったステージI、II、およびIIIの結腸直腸がん（CRC）患者において免疫組織化学によって同定されたCD4およびforkhead box P3（FOXP3）陽性T細胞密度の組み合わせの無再発生存期間（RFS）・全生存期間に対する有用性を報告した。本研究は、統計的パターン認識の手法である離散ベイズ識別則を応用することにより、T3/T4a ステージIIのCRC患者の再発を予測するマーカーの最適な組み合わせを抽出した。 方法：T3/T4aステージII患者の137症例の切除標本を用いて、12の臨床病理学的および免疫的因子を再発の予後予測因子の候補として解析を行った。 結果：比較的に予後良好とされている T3/T4aステージ II 症例で、CD4・FOXP3陽性T細胞両方の腫瘍浸潤度に最も強い影響があることが示唆された。CD4・FOXP3陽性T細胞両方の腫瘍浸潤度が低い症例群のRFSが明らかに不良であった。 結論：CD4とFOXP3陽性T細胞を組み合わせた腫瘍浸潤度がCRCの予後予測因子となり得ることが示唆され、従来のステージ分類では不十分な患者の層別化も可能になるという新規の知見が明らかになった。補助化学療法は、CD4・FOXP3陽性T細胞両方の腫瘍浸潤度が低い患者に対して考慮されるべきであることが示唆された。

小胞体ストレスの増加は、動脈硬化において血管平滑筋培養細胞（VSMCs）の形質転換（分化→脱分化）と強く関連している。小胞体のCa2+貯蔵量減少は、VSMCs における小胞体ストレスの増加の主要な原因の一つである。リアノジン受容体（RyR）は筋小胞体膜上に存在する主要なCa2+放出チャネルである。正常細胞の安静状態ではカルモジュリン（CaM）はRyR と結合し、RyR を閉鎖した状態で安定化させている。CaM とRyR の結合が減弱すると、RyR から異常なCa2+漏出が起こり、Ca2+貯蔵量が減少し、小胞体ストレスが増加する原因となり得る。そこで我々は、マウスのVSMCs を用いてRyR に結合しているCa（CaM-RyR）が小胞体ストレスにより引き起こされるVSMCs の形質転換に重要な役割を果たしているか否か、また、CaM-RyR の結合親和性を高める作用を有するダントロレン（DAN）がVSMCs の形質転換に影響を与えるか否かを評価した。 小胞体ストレスによりCaM がRyR から解離し、核内へ移行することにより、MEF2 とKLF5 の核内での発現量が増加し、このMEF2-KLF5 のシグナル伝達経路が活性化することでVSMCs が形質転換(分化→脱分化)し、増殖能や遊走能を有するようになり動脈硬化巣の形成や不安定化につながるという新たな知見を得ることができた。さらにCaM-RyR の結合親和性を高めるDAN は、RyR チャネルを安定化させ、異常なCa2+漏出を抑制し、小胞体内のCa2+貯蔵量を保持することで小胞体ストレスの増加を抑制し、さらにCaM の核内への移行を制御することで、MEF2-KLF5 経路の活性化を抑制し、その結果としてVSMCs の形質転換を抑制することが示され、動脈硬化巣の進展化、不安定化に対する全く新しい治療戦略となり得る可能性があることが示唆された。

The development and implementation of industrial policy are essential in shaping a country’s economic landscape. It promotes industrialization, which, in turn, generates employment opportunities, enhances productivity, and diversifies the economy. the present dissertation studies the subject of industrial policy, with a particular emphasis on resource allocation and computable general equilibrium in Ghana. Chapter 2 delves into the concept of industrial policy and its implementation in Africa in general, and Ghana, in particular. First, we examine Ghana’s past experience with industrial policy implementation and the reasons for its inability to attain the desired outcomes. Subsequently, in response to the call for a return to industrial policy, we argue in favor of a renewed implementation of industrial policy in Ghana. We posit that the likelihood of success is significantly higher with the benefit of better institutions. Chapter 3 examines the subjects of firm-level productivity, productivity distribution, and resource allocation. In the first instance, we decompose labor productivity in Ghana and draw the conclusion that within-sector resource allocation primarily drives productivity growth, with structural change playing a limited role. Next, we analyze the gross allocative effect, finding evidence that resources are migrating toward sectors of lower productivity. Finally, we also examine productivity distribution through the lens of the power law distribution, establishing that firms involved in international trade exhibit higher levels of aggregation. Thus, allocating resources to such firms leads to greater productivity, thereby minimizing resource misallocation. Chapter 4 presents a dynamic recursive computable general equilibrium for Ghana, employing a Social Account Matrix (SAM) with 2015 as the benchmark year, and we conclude this chapter with a brief analysis of SAM. Chapter 5 examines several possible simulation scenarios. We build our simulations around two industrial policy strategies: labor-intensive and capital-intensive, furthermore our simulation is informed by Ghana’s industrial policy plan. We analyze various policies such as efficiency improvement, trade protection, free trade, and taxation policy. We conclude that capital-intensive industrialization would work better under a free trade policy. Moreover, we discovered that the cost of protecting labor-intensive industries is less than the cost of safeguarding capital-intensive industries. We conclude the dissertation with a discussion of the implications of our findings. we aim to provide a comprehensive discussion of the implications of our findings, as well as acknowledge the limitations of our study and propose potential avenues for further research. By doing so, we hope to contribute to the existing body of knowledge in our field and inspire future researchers to expand upon our work.

Fiber-reinforced polymer (FRP) rods fabricated from unidirectional fibers and a polymer matrix strengthen effectively reinforced concrete (RC) members. The pultrusion is a production method of FRP rod. The FRP rods show various advantages, such as light and no-corrosion. Most FRP rods have higher tensile strength than standard steel bars. Therefore, the FRP rods can be used as an alternative reinforcement of steel bars in RC structures. In addition, FRP rods can be applied in near-surface mounted (NSM) systems for strengthening existing concrete structures. The tensile properties of FRP rods in adhesively bonded anchorages are expected to be studied in detail. Numerous experimental studies were conducted on FRP rods made of glass, carbon, aramid, or basalt fibers. The previous studies have reported that the tensile properties of FRP rods are affected by the shear-lag effect. However, these studies referred to the tensile failure, the shear-lag effect of FRP rods as a phenomenon without a mechanical explanation. Moreover, the effects of mechanical properties of fibers, matrix, fiber-matrix interface on FRP rod properties have not been investigated in detail. To quantify factors affecting the tensile properties of FRP rods, this study performed a numerical investigation on aramid FRP rods to assess the shear-lag effect, tensile load-capacity, and tensile strength. In addition, the effects of fiber, matrix, and fiber-matrix interface on the behavior of FRP material in three dimensions were demonstrated by micro-models. Firstly, two representative volume element (RVE) models of fibers and matrix were proposed to predict engineering constants and strengths of the FRP material in three dimensions. Based on the predicted strength, the criteria were designed. Then, the main simulation, including the FRP rod, the filling material, and the steel tube, was carried out to analyze FRP rods under the variation of interfacial conditions between materials, including full-bonding strength and partiallybonding strength models. In the partially-bonding strength model, the interfaces between materials were simulated as cohesive zone models with the variation of bond strengths and fracture energy release rate. A technique called submodeling was applied to enhance the simulation results. The submodel was cut from the main simulation model and only applied to simulate FRP rods with finer meshes. The study proposed a procedure for calculating the stress distribution in any cross-section of an FRP rod. The simulation results agreed well with the previous experimental study. The findings clearly indicated the position of the failure section in which the tensile stress distribution is unequal. The load-capacity, failure modes, shear-lag effect were predicted based on the maximum stress criterion. The results revealed that the FRP material strengths enforce the failure in two modes associated with the transverse and longitudinal directions of FRP rods. In addition, diameter is a significant factor that increases the shear-lag effect and reduces the tensile strength of the FRP rods. The numerical simulation provided a new method to predict the load-capacity of FRP rods. The study consists of 6 chapters. Outline of the chapter was presented as follows: Chapter 1 introduces about kinds of FRP rods and their application in civil engineering. The chapter shows the research objects, the gaps in composite studies, and the scopes of the present research. Chapter 2 summrizes the review of previous studies related to the theoretical studies of the composite materials. The chapter reveales the gap of theory. In addition, the study compares the advantages and disadvantages of previous studies and proposes methods and models for the present study. Chapter 3 presents the simulations of the representative volume element (RVE) models to determine the mechanical properties and strengths of composite materials. The study investigates the effects of the fiber properties and fiber-matrix interface on composite mechanical properties in detail. The RVE-1 model was employed to predict engineering constants of the FRP material. The RVE-2 was applied to predict the tensile and shear strengths in three dimensions. Chapter 4 shows the numerical simulations of the FRP rod tensile tests with various cases of the materials in Chapter 3. The models are built in two cases of the interface between the FRP rod and filling material: full-bonding and partially-bonding strengths. In the case of the full-bonding strength, three models are built with three hypotheses of FRP rod material. Three models, A, B, and C, were proposed to demonstrate the effect of fiber properties on FRP properties. Model A was built based on the hypothesis that the FRP rod is made of transversely isotropic fibers. Model B was made to simulate with an FRP rod of isotropic fibers. Model C assumes the FRP rod as an isotropic material. In the case of the partially-bonding strength, the study models various interface cases between the FRP rod and the filling materials to investigate the bonding effects. The proposed models were applied to simulate FRP rods from D3 to D8 to analyze the diameter effect. In Chapter 5, the difference between the proposed models was discussed to show the advantages and disadvantages of each model. Firstly, the study compared models (A, B, and C) to highlight the effect of fiber properties on FRP rods. Secondly, the study compared the partially-bonding strength and full-bonding strength models to investigate the bonding effects on the tensile properties of FRP rods. Moreover, the chapter illustrates the existence of the shear-lag effect and demonstrates the diameter effect on tensile strength in FRP rods. Chapter 6 summarizes the novel findings and research significance of the study. In addition, recommendations for future works were also presented.

角膜の剛性を表す生体力学的指標の一つとして角膜ヒステリシス（CH）があり，CHは眼圧（IOP）や中心角膜厚（CCT）などの影響を受け，眼球全体の剛性も反映することから，CH低下が緑内障の進行リスクとして注目される。一方で，硝子体は眼球を外力から保護する緩衝材としての作用があり，眼球の剛性に影響するが，硝子体とCHとの関連は明らかになっていない。本研究では硝子体切除がCHに与える影響を評価することを目的に，白内障単独手術と白内障手術併施硝子体切除術の術後早期のCHを比較した。白内障手術（PEA+IOL）を施行した18例20眼（PEA+IOL群），黄斑上膜あるいは黄斑円孔に対してPEA+IOL併施の経毛様体扁平部硝子体切除術（PPV）を施行した27例28眼（PPV triple群）を対象とした。術前，術後2週，術後3か月のCH，IOP，CCTおよびCHとCCTの相関関係について後ろ向きに検討した。CHは術前，術後2週，術後3か月で，PEA+IOL群において11.1±1.1mmHg，10.4±1.1 mmHg，11.0±1.0 mmHgであり，PPV triple群において11.0±1.4mmHg，9.8±1.4 mmHg，10.6±1.6 mmHgであった。CHはPEA+IOL群で術前後に有意差は認めなかったが，PPV triple群の術後2週で有意に低下していた。IOPおよびCCTは両群とも術前後に有意な変化は認めなかった。PEA+IOL群の全時点とPPV triple群の術前にはCHとCCTの正の相関関係を認めたが，PPV triple群の術後には相関関係を認めなかった。以上より，PPV triple手術ではIOPやCCT以外による要因で術後にCHが低下することが示され，硝子体切除が眼球の剛性変化をもたらしCH低下に寄与した可能性が考えられた。CHの低下は，外力や眼圧の影響を受けやすい眼球構造であると言えることから，PPV 術後のCH評価は眼圧管理の指標や緑内障の発症，進行リスクを反映する可能性がある。

Aldosterone is a steroid hormone synthesized in the adrenal cortex and is part of the renin-angiotensin-aldosterone system (RAAS). It accelerates renal sodium retention and elimination of potassium through its action on the mineralocorticoid receptor (MR), and has a major role in regulating body fluid volume and blood pressure. Excessive secretion of aldosterone and activation of the MR cause cardiovascular inflammation, fibrosis and remodeling, and tubulointerstitial fibrosis and glomerular injury in the kidney. There are several reports on plasma aldosterone concentration (PAC) in healthy, chronic kidney disease (CKD), systemic hypertension, and chronic heart failure in cats and dogs. Measurement of urinary aldosterone/creatinine ratio has also been reported in cats and dogs. However, it has been suggested that measuring aldosterone in feline urine using the available methodology has limited or no utility in investigating feline hypertension associated with CKD. It may be important to evaluate PAC in cats and dogs with CKD associated with the activation of RAAS. On the other hand, angiotensin-converting enzyme inhibitors (ACEIs) and angiotensin receptor blockers suppress the RAAS during hypertensive, renal, and cardiac diseases in cats. The Randomized Aldactone Evaluation Study in humans showed that the aldosterone antagonist, spironolactone, reduced the mortality of patients with chronic heart failure who received ACEI and loop diuretics. Spironolactone also reportedly reduced the mortality rate in cats with congestive heart failure secondary to cardiomyopathy. Another selective aldosterone antagonist, eplerenone, not only antagonizes MR but also blocks the nongenomic effects of aldosterone in vascular tissues not susceptible to spironolactone. These effects of eplerenone may be more effective than spironolactone in treating hypertension due to vasoconstriction. Although eplerenone reduces mortality and hospitalization in human patients with chronic heart failure, there are no available reports on eplerenone’s use in feline practice. Since elevated PAC is a risk factor for kidney injury in humans, and MR antagonists are beneficial in rodent models of CKD and human patients, it was hypothesized that if an elevated PAC is detectable in the early stages of the disease in cats, the use of eplerenone may prolong lifespan. However, the relationship between PAC and the survival time in cats and dogs with CKD has not been investigated. Therefore, this study aimed to investigate PAC in cats and dogs with CKD, and evaluate the influence of high PAC on the survival time of CKD animals and the effect of treatment with eplerenone in CKD cats with high PAC. In chapter 1, PAC in cats with CKD was investigated retrospectively, and the survival time of cats with high PAC was evaluated. Furthermore, the effect of treatment with eplerenone on survival time in CKD cats with high PAC was examined prospectively. The eplerenone study was conducted including both cats with CKD only and CKD cats complicated cardiac disease or systemic hypertension. The PAC was measured retrospectively in blood samples obtained from 156 client-owned cats that visited a veterinary hospital. The cats were designated into 2 groups: clinically healthy (n = 101) and CKD (n = 55). The PAC was measured by solid-phase radioimmunoassay. Median (minimum–maximum) PAC in healthy cats was 97 (10–416) pg/mL and the upper limit (95th percentile) was 243 pg/mL. In the CKD group, PAC [126 (10–981) pg/mL] was significantly higher than in the clinically healthy group. In the CKD group as classified by the International Renal Interest Society (IRIS) stage, the PACs were higher in IRIS stage 2 than in the healthy group. Similarly, PACs in IRIS stage 3 and 4 cats were higher than in the healthy group. In cats with CKD, the survival time of those with high PAC (n = 16) (> 243 pg/mL) was significantly shorter than that of those (n = 39) with normal PAC. In cats with high PAC and CKD, eplerenone administration (2.5 to 5 mg/kg body weight; n = 8) prolonged significantly the survival compared to cats not receiving eplerenone (n = 18). These results indicated that PAC could be a prognostic marker of CKD in cats and that eplerenone may prolong the survival in cats with CKD and high PAC complicated with cardiac disease or hypertension. In chapter 2, PAC in dogs with CKD was investigated retrospectively, and the survival time of CKD dogs with high PAC was evaluated. PAC was measured in blood samples obtained from 145 client-owned dogs. The dogs were divided into two groups: clinically healthy (n = 106) and CKD (n = 39). In clinically healthy group, median (minimum–maximum) PAC was 56 (10–250) pg/mL, and the upper limit (95th percentile) was 182 pg/mL. PAC (median 69 pg/mL; range 10–553 pg/mL) in CKD group was significantly higher than in the healthy group. In the CKD group as classified by IRIS stage, PAC (median 97 pg/mL) in IRIS stage 2 and 3 was significantly higher than in the healthy group. A significant positive correlation between PAC and IRIS stage was observed in CKD dogs, suggesting that the lower survival rate in high PAC group may be related to severity of CKD. In dogs with CKD, the survival time of those with high PAC (n = 10) (> 182 pg/mL) was significantly shorter than that of those with normal PAC (n = 24). These results suggested that high PAC might indicate shorter survival time in dogs with CKD. In conclusion, this study revealed that both cats and dogs with CKD had significantly higher PAC than clinically healthy animals. In CKD, the survival time of cats and dogs with high PAC was significantly shorter than those with normal PAC. The use of eplerenone also significantly prolonged the survival of cats with high PAC in CKD complicated with cardiac disease or hypertension. This study proposes PAC as a prognostic marker of cats or dogs with CKD. Eplerenone may be useful in prolonging cats’ survival with high PAC in CKD complicated with cardiac disease or hypertension. However, further study on PAC level in CKD progression and treatment response in a larger population may be required. This study provided new information on the relationship between PAC and the survival of cats or dogs with CKD, and the effect of eplerenone treatment for the survival time of cats with high PAC and CKD.

膵β細胞量の進行性の減少が糖尿病の発症・進展に関与する。糖尿病病態では、インスリン作用不足による高血糖に対する代償的なインスリン産生の増加や脂質異常のためにβ細胞において小胞体ストレスが亢進する。一方、セリン・スレオニンキナーゼGsk-3 はPI3/Akt により抑制され、Gsk-3 の活性制御障害がβ細胞不全と小胞体ストレス関連β細胞死に関連する。しかし、Gsk-3 を介した細胞障害の分子機構は十分に解明されていない。本研究では、Gsk-3 を介する小胞体ストレス応答調節と膵β細胞アポトーシス誘導の関連について検討を行った。方法：マウス単離膵ランゲルハンス氏島(ラ氏島)およびマウス膵β細胞株で薬理学的に小胞体ストレスを誘導した。Ins2 遺伝子に変異を持ち、変異インスリンの蓄積により小胞体ストレスが亢進しβ細胞死を来すAkita mouse 由来の膵β細胞株を遺伝的小胞体ストレスのモデルとして用いた。低分子化合物、あるいは酵素不活性型Gsk-3βの導入によりGsk-3 活性を阻害した。結果：小胞体ストレス下ではGsk-3 を抑制するAkt の活性が減弱しGsk-3 が活性化した。Gsk-3 はATF4 のSer214 リン酸化を介してATF4 とSCF-βTrCP の結合とそれに続くATF4 のユビキチン化、蛋白分解を促進する。したがって、Gsk-3 の抑制によりATF4 の分解速度が低下し、ATF4 蛋白質量が増加することが明らかになった。このとき、β細胞のアポトーシスが減弱することを確認した。この抗アポトーシス効果はdominant negative-ATF4 導入あるいはATF4 ノックダウンにより有意に減弱した。Gsk-3 抑制によるアポトーシス抑制のメカニズムについて、ATF4 の転写標的であるGADD34 および4E-BP1 の発現増強を介した全般的な蛋白翻訳の動態との関連が考えられた。さらに、Akita mouse 由来の膵β細胞株でもGsk-3 活性阻害によるアポトーシス抑制が確認され、これにはATF4 の発現増強と全般的な蛋白翻訳抑制が関連した。以上の結果より、インスリンシグナルとストレス応答のクロストークが明らかになり、Gsk-3/ATF4 経路が糖尿病におけるβ細胞保護の治療標的となる可能性が示唆された。

背景：糖尿病患者における聴覚障害の有病率は有意に高く、その予防法の開発が望まれている。 目的：本研究では、糖尿病マウスに対するエイコサペンタエン酸（EPA）投与による早期難聴の予防効果を検討した。 方法：糖尿病モデルとしてTSOD（Tsumura, Suzuki, Obese Diabetes）マウスを、コントロールとしてTSNO（Tsumura, Suzuki, Non Obesity）マウスを使用した。TSNO群とTSOD（EPA-）群（ひまわり油投与）、TSOD（EPA＋）群（EPA投与）の3 群に分けた。聴性脳幹反応（ABR）を測定し、蝸牛を組織学的に評価した。 結果：TSOD（EPA＋）群はTSOD（EPA-）群に比べ、閾値の上昇が小さい傾向を認めた。TSOD（EPA+）群では、生後11 ヶ月から14 ヶ月にかけて、4kHzでのABR 閾値がTSOD（EPA-）群よりも有意に低かった。TSOD（EPA-）群では、血管条の毛細血管内腔の狭小化と蝸牛軸における血管壁の肥厚が観察された。 結論：TSOD マウスに対するEPA 投与による蝸牛血管の動脈硬化の抑制は、加齢に伴う早期難聴を抑制することが示唆された。

Fertility decreases during aging in human and bovine females, but the exact pathophysiological mechanisms in the oviducts and uteri are not clarified yet. Anti- Müllerian hormone (AMH) is a glycoprotein that belongs to the transforming growth factor -β superfamily. Plasma AMH concentration can predict the fertility of adult female goats, ewes, cows, and women via unknown physiological mechanisms. This thesis study attempted to clarify whether AMH, and the main receptor for AMH, AMH receptor type 2 (AMHR2) have important roles for the age-related infertility. In first, I investigated whether the primary receptor for AMH, AMHR2, is expressed in bovine oviducts and endometria. Reverse transcription polymerase chain reaction (RTPCR) detected expression of AMHR2 mRNA in oviductal and endometrial specimens. Western blotting and immunohistochemistry were performed to analyse AMHR2 protein expression using anti-bovine AMHR2 antibody. Immunohistochemistry revealed robust AMHR2 expression in the tunica mucosa of the ampulla and isthmus, as well as in the glandular and luminal epithelium of the endometrium. The number of AMHR2-positive fibroblasts increased, suggesting the presence of fibrosis in the oviducts and uteri of old cows. AMHR2 mRNA (measured RT-qPCR) and AMHR2 protein expression in these layers did not significantly differ among oestrous phases in adult Japanese Black (JB) cows (P>0.1). In addition, AMHR2 mRNA and protein expression in these layers did not differ Among old Holsteins (mean (±SEM age 91.9±6.4 months ) and young (26.6±0.8 months) and old (98.8±10.2 months) JB cows. Therefore, AMHR2 is expressed in bovine oviducts and endometria. Other important hormones for endocrinological regulation have paracrine and autocrine roles. Therefore, in the next study, I investigated whether bovine oviducts and endometria produce AMH. RT-PCR and western blotting detected AMH expression in oviductal and endometrial specimens. Immunohistochemistry revealed robust AMH expression in the ampulla and isthmus epithelia, and the glandular and luminal endometrial epithelia (caruncular endometria). The number of AMH-positive fibroblasts increased, suggesting the presence of fibrosis in the oviducts and uteri of old cows. AMH mRNA and protein expression in these layers did not significantly differ among estrous phases in adult JB heifers (p > .1). Furthermore, the expression in these layers also did not differ among Holstein cows (93.8 ± 5.8 months old), JB heifers (25.5 ± 0.4 months old), and JB cows (97.9 ± 7.9 months old). We also compared AMH concentrations in the oviduct and uterine horn fluids among the three groups (measured by immunoassays). Interestingly, the AMH concentration in the oviduct fluid, but not in the uterine horn fluid, of Holstein cows was lower than those in JB heifers and cows (p < .05). Therefore, bovine oviducts and endometria express AMH and likely secrete it into the oviduct and uterine fluids. Collagen, the most abundant extra-cellular matrix in oviducts and uteri, performs critical roles in pregnancies. I hypothesised that the locations and amounts of both denatured collagen and the collagen-specific molecular chaperone 47-kDa heat shock protein (HSP47) in the oviducts and uteri of old cows are different compared with those of young heifers because of repeated pregnancies. Since detecting damaged collagen in tissues is challenging, we developed a new method that uses a denatured collagen detection reagent. Then, we compared damaged collagen in the oviducts and uteri between postpubertal growing nulliparous heifers (22.1 ± 1.0 months old) and old multiparous cows (143.1 ± 15.6 months old). Further, I evaluated the relationship between denatured collagen and HSP47 by combining this method with fluorescence immunohistochemistry. Picro sirius red staining showed collagen in almost all parts of the oviducts and uteri. Expectedly, damaged collagen was increased in the oviducts and uteri of old cows. However, damaged collagen and HSP47 were not located in the same area in old cows. The number of HSP47- positive fibroblasts increased, suggesting the presence of fibrosis in the oviducts and uteri of old cows. These organs of old cows showed higher HSP47 protein amounts than those of heifers. However, the uteri, but not oviducts, of old cows had lower HSP47 mRNA amounts than those of heifers. These findings revealed the specific location and amounts of denatured collagen and HSP47 in the oviducts and uteri of old cows compared with those of heifers. Therefore, I discovered the AMH, AMHR2 expression in several important layers of oviducts and uteri, and I discovered the increased AMH, AMHR2, and HSP47 in the fibroblasts after aging. However, still role of AMH, AMHR2 in oviducts and uteri were not clarified yet. Therefore, in the next study, I hypothesized that AMH stimulate HSP47 expression in fibroblast and epithelium. I cultured uterine fibroblasts and epithelial cells obtained from heifers. Then, I treated the cells with recombinant with increasing concentrations (0, 1, 10, or 100 ng mL-1) of AMH. HSP47 expression was measured by western blotting. AMH stimulated (P<0.05) HSP47 expression in epithelial cells but not in fibroblasts. Therefore, these findings suggested the role of AMH to cause the abnormal high HSP47 expression in the oviducts and uteri of old cows. In conclusion, this thesis discovered the AMH and AMHR2 in bovine oviducts and uteri, which have important roles for collagen synthesis via HSP47.

Not only taste but also flavor has a significant impact on the "deliciousness" of food as perceived by people. Flavor characteristics of food have a significant impact on its palatability and influence the qualitative value of the food itself. In this study, the molecular mechanisms of biosynthesis of flavor compounds derived from amino acids in soybean (Glycine max) and fatty acids in mushrooms were elucidated. In Chapter 1, I identified the characteristic aromatic properties of soybean due to sulfur-containing amino acids. Several soybean germplasms, such as Nishiyamahitashi 98-5 (NH) among local varieties in Nagano Prefecture, have an intense seaweed-like flavor after cooking because of their high seed S-methylmethionine (SMM) content. In this study, I compared the amounts of amino acids in the phloem sap, leaves, pods, and seeds between NH and the common soybean cultivar Fukuyutaka. This revealed a comparably higher SMM content alongside a higher free L-methionine (L-Met) content in NH seeds, suggesting that the SMM-hyperaccumulation phenotype of NH soybean was related to L-Met metabolism in seeds. To investigate the molecular mechanism behind SMM hyperaccumulation, I examined the phenotype-associated gene locus in NH plants. Analyses of the quantitative trait loci in segregated offspring of the cross between NH and the common soybean cultivar Williams 82 indicated that one locus on chromosome 10 explains 71.4% of SMM hyperaccumulation. Subsequent fine-mapping revealed that a transposon of about 6 kbp insertion into the intron of a gene, Glyma.10g172700, is associated with the SMM-hyperaccumulation phenotype. The Glyma.10g172700-encoded recombinant protein showed Met-γ-lyase (MGL) activity in vitro, and the transposon-insertion mutation in NH efficiently suppressed Glyma.10g172700 expression in developing seeds. Exogenous administration of L-Met to sections of developing soybean seeds resulted in transient increases in L-Met levels, followed by continuous increases in SMM concentrations, which was likely caused by L-Met methyltransferase activity in the seeds. Accordingly, I propose that the SMM-hyperaccumulation phenotype is caused by suppressed MGL expression in developing soybean seeds, resulting in transient accumulation of L-Met, which is converted into SMM to avoid the harmful effects caused by excess free L-Met. In Chapter 2, I studied the biosynthesis mechanism of 1-octen-3-ol, the main volatile component of mushrooms. 1-Octen-3-ol is a volatile oxylipin found ubiquitously in Basidiomycota and Ascomycota. As 1-octen-3-ol attracts mosquitoes and flies, its involvement in emitter–receiver ecological communication has been proposed. Although the biosynthetic pathway to form 1-octen-3-ol from linoleic acid through linoleic acid 10(S)-hydroperoxide has been proposed in mushrooms, the enzymes involved in this pathway have not been identified. I determined that the Coprinopsis cinerea dioxygenase 1 and 2 (CcDOX1 and CcDOX2) genes in the mushroom C. cinerea contains an N-terminal cyclooxygenase-like heme peroxidase domain and a C-terminal cytochrome P450-related domain. Through analysis of products formed from linoleic acid by the recombinant CcDOX1 and CcDOX2 proteins, I found that CcDOX1 preferentially catalyzes to form the 10(S)-hydroperoxide of linoleic acid (10S-HPODE), meanwhile CcDOX2 form the 8-hydroperoxide of linoleic acid. Moreover, disruption of Ccdox1 in C. cinerea (ΔCcdox1) mycelia suppressed 1-octen-3-ol synthesis. Administration of the 10S-HPODE to the microsome fraction prepared from mycelia resulted in the efficient production of 1-octen-3-ol. Together, these results indicate that CcDOX1 is essential for the biosynthesis of 1-octen-3-ol as the oxygenase that forms 10S-HPODE, followed by the cleavage enzyme. I studied physiological and ecological significance of 1-octen-3-ol of mushroom. ΔCcdox1 was less attractive to fruit fly larvae, while the feeding behavior of fungus gnats on ΔCcdox1 mycelia showed little difference from that on the mycelia of the wild-type strain. The proliferation of fungivorous nematodes on ΔCcdox1 mycelia was similar to or slightly worse than that on wild-type mycelia. Thus, 1-octen-3-ol seems to be an attractive compound for some animals that interact with mushrooms.

中央構造線(MTL; Median Tectonic Line)は、西南日本を東西に横断する延長約1000kmの断層である。愛媛県西条市付近には、MTLは三波川変成帯と和泉層群を境する構造線としての低角度な断層帯(MTLTB; MTL inactive terrane boundary)と、この断層の北側に並走する活断層としての高角度な断層帯(MTLAFZ; MTL active fault zone)がある。地表でのMTLAFZの傾斜角度を明らかにするために、川上断層を横断する延長約10m、深さ約2mのトレンチ調査を行った。また、地表部で約10mの間隔で並走する両断層の地下での接合関係と断層面の傾斜角度を明らかにするために、断層を横断する80-330mの6本のボーリング掘削を実施した。更に、より広範囲の断層構造や地盤の物性を把握するために延長1200mの反射法地震探査と延長500mの高密度電気探査を実施した。採取した断層試料を用いて断層岩の化学分析、変形構造記載、カルサイトの双晶密度の測定、断層の変形フェーズの解析を行い、低角度横ずれ断層のメカニズムや断層活動史を明らかにした。 トレンチ調査、ボーリング調査、高密度電気探査により、地表部で北方へ約70゜の角度で傾斜する川上断層が、地下で北方へ30゜の角度で傾斜するMTLTBに収れんすることが示唆され、地下のMTLTBは活断層であることが分かった。MTLTBの上盤に分布する小断層の卓越した和泉層群の比抵抗値は、主破砕帯の割れ目の少ない安山岩ブロックと推定される高比抵抗部を除き、断層下盤に分布する堅硬な三波川変成岩類の比抵抗値よりも低い値を示した。また、断層に沿って深部流体が上昇していると推定される低比抵抗帯が確認された。反射法地震探査では、MTLTBに相当する北方へ約30゜の角度で傾斜する明瞭な反射面が確認され、より深部まで断層が延長することが分かった。主破砕帯を構成する蛇紋岩中の鉱物のEPMA分析結果によると、マントル起源のマグネシオクロマイトを含むことが分かった。既往の深部地震探査の結果は、MTLの深部延長が下部地殻まで達しいることを示しているが、これにより、MTLTBの延長がマントルまで達し、蛇紋岩が断層変位とダイアピルによって表層部まで上昇してきたことが示唆された。MTLTBは断層面の傾斜角度が低角度であり、本来は横ずれ断層として動きにくいと考えられる。MTLTBの断層ガウジや主破砕帯に大量の層状珪酸塩鉱物が存在することや断層沿いの深部流体の存在は、断層のせん断強度を低下させる要因となり、低角度の断層でも横ずれ運動が可能になったと考えられる。カルサイトの双晶密度から求めたMTLTBを横断する歪み分布は断層から直線的で緩やかに低下する傾向を示し、断層のせん断強度が低下していることを示唆する。 変形フェーズの解析では、MTLTBとMTLAFZの幾何学的な特徴やそれぞれの断層と地層との接合関係、断層の変位センス等の構造地質学的特徴、古応力場の解析等に基づいて変形フェーズを古いほうからD1～D4の4つに定義した。D1フェーズはNNE-SSW圧縮の応力場の変形であり始新世中期(47 -46 Ma) 頃に断層の上盤が西方へ変位した左横ずれセンスの運動、D2フェーズはE-W伸張の応力場の変形であり中新世中期(15 -14 Ma) 頃に断層の上盤が北方へ変位した正断層センスの運動、D3フェーズはNNW-SSE圧縮の応力場の変形であり中新世中期から鮮新世後期(14-3Ma) 頃に断層上盤が南方へ変位した逆断層運動、D4フェーズはWNW-ESE圧縮の応力場の変形であり鮮新世後期から更新世前期(3-1 Ma) 以降に断層上盤が東方へ変位した右横ずれ運動である。 西南日本を横断する中央構造線沿いには多くの都市が分布しており、MTLの傾斜角度等の幾何学的な情報は、地震災害分布や地震の規模等を予測する上で重要パラメータになると考えられる。また、MTLAFZは地下数km以内の浅い深度でMTLTBに収れんすると考えられ、従来、非活動的な地質断層として考えられていたMTLTBが、将来、活断層として変位する可能性があることを示唆している。

　本研究では、小児期（幼児期、学童期、青年期）の生活習慣、遺伝要因と肥満との関連を調査し、肥満予防のための公衆衛生的アプローチを検討した。食物繊維摂取が学童期の子どもの肥満および血圧、血中脂質パラメーターに及ぼす影響を明らかにすること（調査1：10～11歳の横断研究）、FT0遺伝子多型が幼児期から青年期の体格変化に及ぼす影響を明らかにすること（調査2：3歳、10歳、13歳に渡る縦断研究）、さらに、学童期から青年期にかけて身体活動がFT0遺伝子多型と体格変化に及ぼす影響を明らかにすること（調査3：13歳、18歳に渡る縦断研究）、を本研究の目的とした。 　調査1では、学童期の食物繊維摂取は肥満およびコレステロール高値のリスク低減に有効であることが示唆された。 　調査2では、幼児期から学童期にかけてFT0遺伝子の影響が強く表れ、中学生の年代になるとこの影響は逆に減弱し、部活動等が始まるこの時期の身体活動の影響が関連している可能性があることが推測された。 　調査3では、青年期で、身体活動量が多いほどBMIの増加を弱め、FT0遺伝子多型の影響を減弱させた。 　本論文の構成は5章からなる。 第1章では、緒言として、研究の背景と本研究の目的を述べる。 第2章では、食物繊維摂取が学童期の子どもの肥満および血圧、血中脂質パラメーターに及ぼす影響を検討した。 第3章では、FT0遺伝子多型が幼児期から青年期の体格変化に及ぼす影響を検討した。 第4章では、身体活動がFT0遺伝子多型と体格変化に及ぼす影響について検討した。 第5章では、結言として、本研究のまとめと今後の研究の方向性、今後の展望を述べた。

背景：肝癌は肝内転移を生じやすく、その予後は不良である。がん幹細胞様細胞（CSLCs）とは、幹細胞性、腫瘍形成能、治療抵抗性に加え、転移能亢進といった特徴を有している。近年では、がんの転移形成や制御において、宿主の免疫機構が重要な働きを担っていると考えられており、今回、肝癌細胞株から誘導したCSLCsの免疫逃避能について検討を行った。 方法：Sphere誘導培地を用いて、Sphere細胞の形態で肝癌細胞株からCSLCsを誘導した。免疫逃避に関わる遺伝子およびタンパク発現を、RNAシーケンス、フローサイトメトリー、ELISA法を用いて解析し、親株とSphere細胞とで比較した。親株とSphere細胞それぞれの、NK細胞に対する感受性について、クロム放出試験を用いて比較検討した。BALB/cヌードマウスを用いた異種移植実験にて、親株とSphereの腫瘍形成能を比較した。 結果：肝癌細胞株SK-HEP-1から誘導したSphere細胞（SK-sphere）では、親株と比較し、細胞膜上のPD-L1、PD-L2、CEACAM1の発現が亢進し、ULBP1、MICA/MICBの発現は低下していた。また、SK-sphereの培地中では、可溶型MICAの濃度が上昇していた。SK-sphereにおけるHLA class I発現低下は見られなかった。肝癌細胞株SK-HEP-1およびHLEから誘導したSphere細胞を用いて、NK細胞に対する感受性をそれぞれの親株と比較したところ、どちらのSphere細胞においてもNK細胞による細胞障害性がより低下していた。NK細胞を保持するヌードマウスにおいて、親株の移植と比較してSK-sphereを移植した際により大きな腫瘍を形成した。 結論：肝癌細胞株から誘導したCSLCsはNK細胞を介した免疫系からの逃避能が亢進している事が示唆された。

近年、遺伝子改変T細胞を用いた養子免疫療法の臨床応用が進められているが、未だ治療成績は満足のいくものではなく、更なる治療の進歩が求められている。我々はこれまで、自律性にIL7とCCL19を発現するCAR-T細胞（7×19 CAT-R細胞）が優れた抗腫瘍活性を発揮することを示した。本研究ではIL7及びCCL19がTCR-T細胞の抗腫瘍活性を増強させる可能性を検証するため、腫瘍抗原であるP1Aを特異的に認識するTCR-T細胞（P1A-T細胞）にIL7及びCCL19を分泌させる遺伝子改変を加えた7×19 P1A-T細胞を作製した。P1Aを有する細胞株であるP815を皮下接種したマウスに対する養子免疫療法において、7×19 P1A細胞はIL7及びCCL19を分泌しないP1A-T細胞（Conv. P1A-T細胞）と比較し優れた抗腫瘍効果を示した。腫瘍拒絶マウスにおいては、Conv. P1A-T細胞を使用したマウスと比較し優れた免疫記憶が形成されていた。また抗PD-1抗体を用いた複合免疫療法を行うことで、7×19 P1A-T細胞の有する抗腫瘍効果は更に増強された。CRISPR/ Cas9システムを用いPdcd1をknockdownした7×19 P1A-T細胞は対照7×19 P1A-T細胞と比較し強い抗腫瘍効果を示したが、抗PD-1抗体を併用することで更なる抗腫瘍効果の増強を認めたことから、7×19 P1A-T細胞の発揮する抗腫瘍効果には7×19 P1A-T細胞そのものばかりでなくマウス内在性のT細胞が関与していることが分かった。また、7×19 P1A-T細胞においても抗PD-1抗体との併用により抗腫瘍効果が増強することを確認した。本研究における検証により、7×19 TCR-T細胞及び7×19 CAR-T細胞を用いた免疫療法が抗悪性腫瘍療法の発展に寄与できる可能性が示された。

現在の日本は少子高齢化・医療高度化を背景に、要介護者数の増加、医療費増加、人材不足、医療格差などの医療課題に直面している。近年、人工知能（ArtificialintelligenceAI）技術やシステム医学に基づいたデータ駆動型医療の登場によって、これら課題を解決できる可能性が広がった。筆者自身は呼吸器診療に携わる立場でもあることから、呼吸器診療を支援する以下3つの医療AI技術を開発した。1つ目の技術では、副作用ビッグデータ（JapaneseAdverseDrugEventReportJADER）に基づきベイズ推定を行うことで、AUC0.93の精度で副作用の原因薬を推定できた。呼吸器領域では薬剤性肺障害など重篤な副作用があるが、本技術の臨床応用によって、副作用による健康被害の最小化と副作用管理の効率化が期待できる。2つ目の技術では、喘息患者の臨床データ（年齢、BMI、血中好酸球数、呼気NO値、増悪回数）をもとに教師あり機械学習を行うことで、喘息患者の気流閉塞の急速進行1秒量低下）をAUC0.85の精度で予測できた。実用化によって、早期の治療介入が必要な喘息患者を同定でき、重症化を防ぐための先制治療につながる。3つ目の技術では、喘息質問票（AsthmaControlQuestionnaire-5:ACQ-5）のデータを用いて教師なし機械学習を行うことで、症状から喘息病態である気流閉塞、2型気道炎症、増悪リスクを推定できた。従来、治療選択のため病態評価には専門的検査が必要だが、本技術ではACQ-5に含まれる喘息症状の評価のみから、個々の喘息の病態に応じた治療選択（個別化治療）につなげることができる。本技術は、発展途上国、過疎地域、プライマリケアの現場など、医療環境が不十分な地域で、適切な喘息治療薬の選択を支援できる。ひいては、医療格差の是正につながる可能性が期待できる。本研究で開発したAI技術の実用化によって、臨床現場における副作用管理、先制医療、個別化治療を支援する。これによって、健康寿命の延伸、医療費抑制を目指す。同時に、開発したAI技術によって専門医療の一部を補完することで、医療従事者の業務負担軽減と、医療格差の是正均てん化につながることが期待できる。

抗がん剤の副作用である口腔粘膜炎の改善にはエレンタール^(R)は有効であることが報告されている。5-フルオロウラシル (5-FU) の副作用には口腔粘膜炎がある。そこでエレンタール^(R)の中で口腔粘膜炎の改善に最も有用な成分の特定を試みた。マウス（対照群を除く）に5-FUを4日間腹腔内注射し、生理食塩水（対照群、5-FU群）、デキストリン（デキストリン群）、アミノ酸（17AA群）、エレンタール^(R)（エレンタール^(R)群）のいずれかを7日間投与した。

本研究の目的は、健常人において、血漿中の尿酸値（UA）と分枝鎖アミノ酸および芳香族アミノ酸（BCAAおよびAAA）の濃度の関連を明らかにすることである。 合計2,804人の健常人について、血漿UAレベルに応じて3つのグループに分類した。その3グループ間における、BCAAとAAAの濃度の関連を、分散分析（ANOVA）と共分散分析（ANCOVA）により解析した。 この研究で対象としたすべてのBCAAとAAAの濃度は、トリプトファンを除き、全てがUA濃度の各レベルに応じて、漸進的に有意に増加した（P<0.001）。また、全体として、個々のBCAAおよびAAAについて、最低のカテゴリーの差にくらべ、高いカテゴリーの差の方が、より大きかった。本研究は、血漿中のBCAAおよびAAAと、UAのレベルについて、潜在的に、密接な関係が存在していることを示唆した。今後、それらの間の因果関係および相互作用を解明する研究が必要である。

方法： 本研究は圧縮センシングを用いた自由呼吸下での多相ダイナミックEOB-MRIを撮像された96人の患者を対象とした。多相ダイナミック撮像として自由呼吸下で11秒毎に1相を5分間、脂肪抑制T1強調画像を撮像し、単純1相と造影28相を撮像した。造影剤投与後20分後に肝細胞相を撮像し、30相目とした。ROI: region of intensityを肝右葉に2つ、左葉に1つを脈管を避けながら可能な限り大きく設定した。3つのROIの信号強度の平均値をそれぞれの時相の信号強度とした。 以下の増強効果のパラメータについて評価した。 CER (contrast enhancement ratio) CERy-x: (SIy -SIx)/SIx (x相目からy相目)とし、 CER4-pre：動脈相 CER7-5：門脈相から動脈相にかけて CER7-pre：門脈相 CER28-pre：5分早期肝細胞相 CER28-7：門脈相から5分早期肝細胞相 CERHBP-pre：20分肝細胞相 GRL (gradient of regression line)：回帰曲線の傾き GRLy-x: x相目からy相目とし、 GRL4-2：動脈相での傾き GRL7-4 ：門脈相での傾き GRL 7-2 ：動脈相、門脈相にかけての傾き GRL 28-7：門脈相から早期肝細胞相での傾き 先行研究によると、肝実質の線維化の重症度(F0-F2 vs F3-F4)に基づいてCERHBP-preでcut off値を0.703として肝細胞相での増強効果がinsufficient HBP enhancement groupとsufficient HBP enhancement groupに分けれるとしている。CERHBP-pre<0.703またはCERHBP-pre>0.703により患者を2群に分け、CERy-x、GRLy-xについて検討した。 上記に加えて年齢、性別、総ビリルビン、プロトロンビン時間、アルブミン、eGFRについても、この2群間で比較した(ウィルコクソンの順位和検定)。 肝細胞相の増強効果に対する影響の大きさを調べるためにノンパラメトリック検定も用いられた(スピアマンの順位相関係数)。 結果： 動脈相(CER4-pre、GRL4-2)に関する結果として、これらはsufficient HBP enhancement groupがinsufficient HBP enhancement groupの間に有意差を認めなかった。 動脈門脈相(1相目～7相目)に関する結果としては、CER7-preはsufficient HBP enhancement groupがinsufficient HBP enhancement groupより有意に高い値となった(0.55 vs 0.44, p<0.001)。CER4-pre、GRL4-2、Gradient7-4、Gradient7-2では2群間に有意差は見られなかった。 5分後の早期肝細胞相(1相目～28相目)に関する結果としては、CER28-pre、CER28-7、GRL28-7においてsufficient HBP enhancement groupがinsufficient HBP enhancement groupより有意に高い値となった(0.64 vs 0.47, 0.10 vs 0.03, 1.27 vs 0.27、すべてp<0.001)。 血液データ(総ビリルビン、プロトロビン時間、アルブミン、eGFR)においても2群間で有意差が認められた(p=0.004-0.049)。CER7-pre、CER28-pre、CER28-7、GRL28-7の各パラメータは血液データのパラメータよりも相関係数が高かった。 CER28-preが最も相関係数が高かった(0.838)。 考察： 動脈相では2群間(sufficient HBP enhancement groupとinsufficient HBP enhancement group)に有意差を認めず、門脈相のパラメータでは有意差が見られた。機序としては推測になってしまうが、肝の線維化の進行に伴い門脈血流は低下しやすいが、動脈血流は比較的保たれることが原因であろうか。 また、肝細胞相の信号強度は、血液データなどによって得られた肝機能を示す数値と相関することはこれまでにも報告されてきた。肝機能が良好であるほど、肝細胞がEOBを取り込みやすいためとされる。 本研究では腎機能と肝細胞相の信号強度とにも相関が見られたが、腎機能が低い症例の方がより肝排泄の割合が増えるからと思われた。 本研究のように、ダイナミック撮像中に得られるパラメータが肝細胞相における肝実質の信号強度と相関することを報告した研究はこれまでになかった。 CER7-pre、CER28-pre、CER28-7、GRL28-7の各パラメータは、いずれも血液データのパラメータよりも相関係数が高かった。 本研究のようにダイナミック撮像中に得られたパラメータを用いれば、血液データよりも高い精度で肝実質の信号強度を予測することができる。 これにより、肝細胞相の撮像タイミングを症例により短縮できることが期待される。 結語： 圧縮センシングを用いた自由呼吸下でのEOB-MRIのダイナミック撮像おいて、肝実質の信号強度の変化を連続データとして捉えることにより得られたパラメータは、肝細胞相での肝実質の信号強度と強い相関を示す。これにより、肝細胞相の撮像タイミングを症例により短縮できることが期待される。

ダントロレン(DAN)はRyR2のN末端ドメインLeu601-Cys620に直接結合し、RyR2の4量体構造を安定化させることにより、RyR2からの拡張期Ca2+漏出を防ぐ。以前我々は、RyR2へのCaM高親和性KIマウス(V3599K)を用いて、横行大動脈縮窄(TAC)による圧過負荷誘発性心肥大マウスモデルにおいてRyR2からのCaM解離を抑制することで、Ca2+漏出を防ぎ、左室リモデリングを抑制することを報告した。そこで本研究では、横行大動脈縮窄（TAC）による圧負荷誘発性心肥大マウスモデルにおいてダントロレンの慢性投与がCaMとRyR2の結合親和性を遺伝的に強化した場合と同様の機序で左室リモデリングを抑制するかを調べた。横行大動脈縮窄（TAC）による圧負荷誘発性心肥大マウスモデルを作成した。野生型マウスを、Sham群、TAC群、TAC-DAN群(ダントロレン20mg/kg/day腹腔内投与)の3群に割り付けた。ShamまたはTAC手術から8週後の生存率、心機能および組織評価、単離心筋細胞を用いたCa2+ハンドリング、RyR2-CaM結合性の評価を行った。TAC-DAN群はTAC群と比較し、TAC手術から8週後の生存率は良好であった(TAC群 49% vs TAC-DAN群83%)。また、心エコーと心筋組織においては、TAC群で認めた左室リモデリングは、TAC-DAN群で抑制された。TAC手術から8週後の単離心筋細胞ではTAC群で拡張期Ca2+スパーク頻度の増加およびRyR2-CaM結合親和性の低下を認めたが、TAC-DAN群ではそれが抑制された。我々の研究はダントロレンの慢性投与によりRyR2を安定化させ、RyR2からのCaM解離を抑制することで、RyR2からの拡張期Ca2+漏出を防ぎ、左室リモデリングが抑制され、予後が改善することを示した。

背景：顕微鏡的多発血管炎 (MPA) は全身臓器が障害される疾患である。しかしながら、MPA患者における心臓超音波検査（心エコー図検査）における指標の特徴については不明な点が多い。 目的：本研究は、単施設後ろ向き研究で、ステロイド療法の新規導入または再導入後2週間以内に心エコーを行ったMPA患者15名を対象とし、その心エコー所見の特徴について検討することを研究の目的とした。30人の年齢・性別をマッチさせた心疾患のないコントロール群と比較検討した。 方法と結果：左室径、左室駆出率、拡張早期僧帽弁輪最大移動速度 (e’) に2群間で有意差は認めなかった。一方、MPA群では左房径、左房容積係数が有意に高く、左室流入血流速波形 (TMF) の早期拡張期充満速度(E波)と肺静脈流入拡張期血流速度 (D波) の高さ、三尖弁における収縮期右室-右房最大圧較差も有意に高く、TMFのE波の減速時間 (DCT) は短縮していた。血清CRPはTMFのE波高、E/A比およびDCTと相関が見られた。今回の研究では、MPA患者におけるe’の有意な低下がみられなかったことから、左室弛緩能の低下よりはむしろ左室スティフネスの上昇によって左室拡張機能低下が生じ、結果として左房拡大が生じている可能性が示唆された。 結語：急性期MPA患者では左室拡張機能低下によると考えられる左房拡大を生じていた。MPA患者、特に強い炎症反応を伴う患者では、心機能評価を行うことが重要であることが示唆された。

Sleep is an essential physiological process for the human body. People spend about one-third of their lives sleeping. Both sleep duration and sleep quality are important to human health. Sleep quality describes how restful and restorative the sleep process is. Over 80 sleep disorders are known to affect sleep quality. Among them, sleep-related breathing disorder (SRBD) is the second factor. Sleep-related breathing disorders are sleep disorders in which breathing abnormalities occur during sleep. Abnormal snoring and respiratory arrest or abnormally low breathing during sleep reduce oxygen levels in the blood, increasing the risk of depression, cardiovascular disease, stroke and even death. Therefore, monitoring and analysis of respiration during sleep is gaining increasing importance in healthcare. Polysomnography (PSG) is considered the gold standard for diagnosing sleep disorders, but PSG is usually performed in an unfamiliar sleep laboratory under the supervision of a medical technician and is often worn with many sensors that interfere with sleep. It is often the case. This research group is developing a breathing sound measurement system that constantly monitors the quality of sleep in general home environment. This system can easily measure breath sounds during sleep all night with high accuracy without disturbing sleep. The purpose of this research is to develop a technique to classify patterns of breathing sounds and to analyze the quality of breathing in order to more accurately analyze the state of sleep from breath sound information. There are various patterns of sleep breath sounds, such as normal breath sounds and snoring, and abnormal breath sounds and snoring. To develop a method to classify these patterns, to develop an algorithm to calculate ventilation from breath sounds, to estimate the sleep apnea index (AHI), and to assess the quality of breathing during sleep. try. Specifically, the temporal feature waveform (TCW) is calculated after partly removing the noise of the breathing sounds of sleep with a band-pass filter. Based on the time feature waveform, a respiratory signal effective for analysis is extracted from low-level signals and phase-divided into a respiratory phase and an apnea or low signal. Mel-frequency cepstrum coefficients (MFCC) are then obtained for the respiratory phases, and an agglomerative hierarchical clustering (AHC) algorithm is applied to distinguish between normal/abnormal breathing, normal/abnormal snoring, and normal/abnormal breathing. , tossing and turning, etc., which are less relevant to breathing. The categorized breathing patterns are analyzed every 30 seconds and the relative tidal volume of the breath is calculated. In addition to verifying the effectiveness and accuracy of the technology and analysis method proposed in this study, a method of estimating the apnea syndrome index (AHI) and converting the ventilation volume into high, medium, and low levels, We propose a method to evaluate the quality of breathing in a patient and verify its effectiveness. This paper consists of six chapters, including an introduction and conclusion. Chapter 1 introduces the background and overview of this research. Chapter 2 describes a signal-processing technique for analyzing breath sounds during sleep and a method for classifying breathing patterns. Breathing sound data during sleep often includes disturbed breathing due to bruxism or body movement, ambient environmental noise, etc. In this chapter, the Time Characteristic Waveform (TCW) and the Characteristic Moment Waveform (CMW) are calculated for respiratory sound signals that have undergone preprocessing, such as filtering noise to preprocess the respiratory sounds, and the segmentation of inspiration and expiration is performed. The Mel-Frequency Cepstrum Coefficients (MFCC) are obtained for each respiratory cycle and applied as a feature vector to the Agglomerative Hierarchical Clustering (AHC) algorithm. This method is used to classify ordinary respiratory signals (normal and abnormal breathing, normal and abnormal snoring) from signals less relevant to respiration, such as tossing and turning and environmental noise. In Chapter 3, using the technology described in Chapter 2, breathing sound data during sleep are classified into apnea, hypopnea, normal breathing, abnormal breathing, normal snoring, and abnormal breathing for each 30-second frame. In addition, we describe a method for classifying events such as no snoring and rolling over and determining the respiratory state. In Chapter 4, we propose a method for estimating the apnea-hypopnea Apnea-Hypopnea Index (AHI) for classified abnormal breath sounds and low-level breath sound signals, compare it with the diagnostic results of PSG, and examine its validity. And verify usefulness. Chapter 5 describes a method for estimating ventilation volume from breath sounds. Because normal breath sounds are correlated with ventilation, this study used a quantitative approach to calculate normal breathing and normal snoring and a qualitative method to calculate apnea/hypopnea and abnormal breath sounds. We will propose and compare it with the diagnosis result of PSG and verify its validity. In Chapter 6, as an application development, an example of applying the breathing sound classification method proposed in this study to heart sound analysis is presented. Finally, we will explain the construction of a data collection distribution system for sharing auscultation data collected at different facilities and hospitals using blockchain technology. Chapter 7 presents the conclusions and prospects of this study.

【目的】代謝状態の朝晩の変化を理解することは、代謝障害を管理するためには重要である。我々は、インスリン分泌とインスリン感受性の観点からこの朝晩の変化を解析し、さらに概日リズムとの関連性を含めて検討することを目的とした。 【方法】非糖尿病成人男性14人と10人に対し、それぞれ75ｇ経口ブドウ糖負荷試験(OGTT)と高インスリン正常血糖（HE）クランプ実験を行った。各被験者に対し、午前8時と午後8時にOGTTまたはHEクランプを1回ずつ行った。ただし試験時間の順序は無作為とした。各試験直前に毛根を採取し、毛包における時計遺伝子発現量をリアルタイムPCR法で解析した。また、マウスの肝臓と筋肉でもAKTリン酸化をウエスタンブロット法により解析した。 【結果】OGTTの結果から、耐糖能は午前8時の方が良好であることがわかった。これは、OGTTにおける負荷後60分までのインスリン早期分泌とHEクランプで説明される骨格筋インスリン感受性の違いに起因すると考えられた。一方、OGTTにおける肝臓のインスリン抵抗性指数によって推定される肝インスリン感受性は、午後8時の方が良好であった。60分までのインスリン分泌量と肝臓のインスリン抵抗性指数の朝晩の差は、Per2のｍRNAの相対的発現量と有意に相関していた。ΔGIR（20時の値－8時の値）は、Δ非エステル化脂肪酸（NEFA）と有意に相関したが、時計遺伝子発現との相関は認められなかった。ΔNEFAは、E4bp4のmRNA発現量およびΔコルチゾールと有意に相関していた。マウスでは、ヒトの研究から予想されるように、AKTリン酸化は活動時間の初めに肝臓で減少し、筋肉で増加していた。 【結語】各組織の糖代謝は朝と晩で大きく異なっており、脂質代謝、時計遺伝子およびコルチゾールの影響を受けていることがわかった。この関連性についてのより深い知見が代謝障害の新たな改善法の開発に寄与する可能性がある。

ミエリン関連糖蛋白 (myelin-associated glycoprotein: MAG) は，神経組織の髄鞘に局在する膜貫通糖蛋白であり，MAGに対するIgM型自己抗体 (MAG抗体) を有する患者では脱髄性末梢神経障害をきたす．末梢神経には血液神経関門 (blood-nerve barrier: BNB) があるために，通常は免疫グロブリンなどの大きな分子が神経内に侵入することはできない．しかし，MAGニューロパチー患者から採取した腓腹神経内有髄神経線維の髄鞘にはMAG抗体が沈着しており，MAG抗体はBNBを通過していることが想定される．MAG抗体のBNB通過機序を明らかにすることを目的に，MAGニューロパチー患者血清，ヒトBNB構成内皮細胞株と周皮細胞株，MAGニューロパチー患者から採取した腓腹神経検体を用いて解析を行った．MAGニューロパチー患者血清を作用させた内皮細胞に対して，全RNAトランスクリプトーム解析と免疫染色を用いたハイコンテントイメージング解析を行い，NF-κBの活性化とTNF-αの発現増加を確認した．次に内皮細胞と周皮細胞を共培養したBNB in vitroモデルで透過性を解析した． MAGニューロパチー患者血清を作用させた結果，10 kDaデキストランやIgGの透過性を変化させることなく，IgMやMAG抗体の透過性を亢進させ，TNF-αの中和抗体を添加することでIgMやMAG抗体の透過性は抑制された．電子顕微鏡による観察ではMAGニューロパチー患者の腓腹神経内でBNBを構成する微小血管の密着結合は保たれており，内皮細胞内に多数の小胞が確認された．MAGニューロパチー患者では，MAG抗体はBNB構成内皮細胞の自己分泌TNF-αの増加を介して内皮細胞質内をトランスサイトーシスの機序により通過していることが示唆された．TNF-α阻害薬は既存の薬剤であり，本研究結果から，TNF-α阻害薬によるMAGニューロパチー患者への新たな治療戦略が期待される．