Pandemic influenza virus A(H1N1)pdm09 infection occurred in healthy children and young adults, but asthmatic patients presented more rapid progression of respiratory distress and plastic bronchitis. To investigate the pathogenesis of worsening respiratory symptoms after A(H1N1)pdm09 infection, we focused on matrix metalloproteinase‐9 (MMP‐9) and tissue inhibitor of metalloproteinases‐1 (TIMP‐1). MMP‐9 and TIMP‐1 levels in bronchoalveolar lavage fluid and serum from mice with and without asthma were evaluated after A(H1N1)pdm09 or seasonal A(H1N1) infection. MMP‐9 levels were more elevated in Asthma/A(H1N1)pdm09‐infected mice than in non‐Asthma/A(H1N1)pdm09‐infected mice on both 3 and 7 days post‐infection. Immunohistochemical findings in this pneumonia model showed that MMP‐9 and TIMP‐1 positive cells were observed in blood vessels and bronchus of lung tissue in severe pathological findings of pneumonia with asthma. Microscopically, shedding cells and secretions were conspicuous in the trachea on days 3 and 7 postinfection, in the A(H1N1)pdm09‐infected mice with asthma. Our results suggest that MMP‐9 and TIMP‐1 expressions are related to severe pneumonia in the A(H1N1)pdm09 infection with asthma, leading to cause epithelial cell shedding.

背景：がん幹細胞 (CSC) は、発がん、再発、転移、治療抵抗性に重要な役割を果たすと考えられている。我々は、化学療法抵抗性と転移能を有するがん幹細胞様スフィア細胞 (CSLC) の誘導に成功した。CSLC に対する標的治療の開発を可能にするため、CSLC のこの表現型の原因となる遺伝子を同定した。 方法：ヒト肝がん細胞株SK-HEP-1 を用い、独自のスフィア誘導培地を用いてCSLCを誘導し、HuH-7 細胞を非スフィア形成細胞として同条件で使用した。RNA シーケンシングを行った後、定量的 RT-PCR とウェスタンブロッティングで検証した。ノックダウン (KD) 実験はCRISPR-Cas9 によるゲノム編集により行い、レスキュー実験は発現プラスミドベクターを用いて行った。細胞の化学療法抵抗性と肝転移は、MTS アッセイと細胞の重度免疫不全マウスへの脾臓注入後の解析で評価した。培地中のエクソソームの定量は、EL ISA 法を用いて行った。 結果： RAB3B は、RNA シーケンシングによりCSLC と予後不良の肝細胞がん (HCC)の両方で発現が増加している遺伝子として同定された。RAB3B-KD 細胞は、スフィア形成、化学療法抵抗性、転移能などのCSLC 表現型の変化を示し、これらはRAB3Bの相補化によって回復された。CSLC ではエクソソーム分泌の増加が観察されたが、RAB3B-KD 細胞では観察されなかった。また、RAB3B の発現は、ABCG2、APOE、LEPR、LXN、TSPAN13 の発現と相関していた。 結論：RAB3B のアップレギュレーションは、CSLC の化学療法抵抗性と転移能に重要な役割を担っている可能性がある。

Some textbooks of formal languages and automata theory implicitly state the structural equality of the binary n-dimensional de Bruijn graph and the state diagram of minimum state deterministic finite automaton which accepts regular language (0+1)*1(0+1)^{n-1}. Although the isomorphism in binary case is relatively easy to prove, it is desirable to rigorously prove such an isomorphism in general k-ary case. To achieve this purpose, the author introduces a new computational model, called "colored finite automata (CFA)," and give a certain characterization of the general k-ary de Bruijn graphs by regular languages. The second purpose of this study is to investigate the potential of this automaton with multi-colored accepting states. By the way, when CFA is nondeterministic (NCFA), it is desirable that the colors of accepting states are unmixed (i.e., there are no inputs that are accepted with differently colored accepting states) in order to pursuit the accurate identification. Thus, the author proposes the three decision problems (Unmixedness Verification problem, Unmixedness Partitioning problem, and Unmixedness Extension problem) concerning unmixedness and show that UV, UP, and UE problems are shown to be NLOG-complete, P, and NP-complete, respectively. The author also illustrates the applications of colored finite automata, e.g. to existing regular expression engines and model checking tools for the purpose of improvement of their efficiency and conveniency. Next, the author introduces "colored pushdown automaton (CPDA)" which is an ordinary pushdown automaton with colored accepting states. It is shown that while the computational complexity of the above-mentioned UV, UP, and UE problems of CPDA are all undecidable, restriction of CPDA are all undecidable, restriction of CPDAs to unambiguous ones simplifies some problems of them to the permanently true problems. In this way, the concept of colored accepting states can be applied to a wide range of automata that have a set of accepting states and expected to be useful in a wide range of theoretical and practical field of automata applications in the future.

Political connections are considered a valuable resource in not only high-corruption countries but also low-corruption countries. This dissertation investigates the relationship between political connections and Sharia compliance, aiming to deepen understanding of the nature of political connections, as Sharia compliance prohibits engaging in corruption. Specifically, I analyze whether: 1) political connections and Sharia compliance affect merger and acquisition (M&A) performance; 2) connections to politicians affect the market response to firms' inclusion in or exclusion from the Indonesia Sharia Stock Index (ISSI); 3) political connections and Sharia compliance have been valuable during the COVID-19 pandemic; and 4) political connections affect firms' environmental performance. This dissertation comprises six chapters. Chapters 1 and 6 respectively introduce and conclude this dissertation. The remaining four chapters (i.e., Chapters 2-5) are essays on the empirical relationship between political connections and Sharia compliance. In Chapter 2, I study the impact of political connections and Sharia compliance on M&A performance, focusing on M&A deals in Indonesia during 2010-2016. I find that while political connections can improve market reactions to M&A announcements, Sharia compliance has a positive but insignificant impact on M&A performance. I further find that there is a substitution relationship between Sharia compliance and political connections: Sharia -compliant firms with political connections have poorer M&A performance than non-Sharia-compliant firms with political connections. Chapter 3 examines market responses to firms' addition to and removal from the ISSI and how political connections influence those market responses. I employ two kinds of analysis. First, using the event-study methodology, I measure abnormal returns surrounding the announcement of each firm's addition to or removal from the ISSI. Second, to more precisely identify the relationship between political connections and Sharia compliance, I use a pooled regression analysis. The results show that neither addition to nor removal from the ISSI produces abnormal returns for Indonesian firms, indicating that investors are little concerned with ISSI reconstitutions. Furthermore, political connections increase firms' value before inclusion in the ISSI, but the benefits of these connections are lost after their addition to the index. Chapter 4 offers novel evidence by investigating the value of political connections and Sharia compliance during the COVID-19 pandemic. I use the event-study methodology to measure the stock market reaction to the COVID-19 pandemic, and conduct a pooled regression analysis to more precisely identify the value of political connections and Sharia compliance during the pandemic. I find that a stock market anomaly occurred during the COVID-19 pandemic in Indonesia, in that the pandemic had a positive impact on the stock market. I also find that there was value for firms in being Sharia-compliant during the pandemic when the government announced tax incentives for firms. In Chapter 5, I investigate the relationship between political connections and the environmental performance of Sharia -compliant firms. I use a unique sample of firms covered by Indonesia's Program for Pollution Control Evaluation and Rating during 2013-2019. I find that political connections are less (more) valuable for Sharia-compliant firms (non-Sharia-compliant firms) m enhancing their environmental performance.

The role of optimization can be found in almost all aspects of human life. Optimization is common in but not limited to the fields of engineering, economics, design, and planning. Although the optimization problems to be solved change, the optimization goal never changes. That is to find effective solutions efficiently. In modern optimization studies, the metaheuristic algorithm has been one of the most interesting methods, considering the demands of a reasonable computational time. Many metaheuristic algorithms have been introduced. However, based on the number of tentative solutions used in the search process, metaheuristic algorithms can be categorized into (1) population-based or (2) single-trajectory-based algorithms. The searching with singletrajectory-based metaheuristic algorithms manipulates and modifies a single solution point in every iteration. In contrast, the population-based metaheuristic algorithms combine a set of solution points to create new solutions in every iteration. A metaheuristic algorithm usually consists of two components, i.e., exploration and exploitation. Exploration means searching for solutions in the global space. In contrast, exploitation means searching for a solution by focusing on a small area or an area near an already known solution. The single-trajectory-based metaheuristic algorithm is exploitation-oriented. On the other side, the population-based metaheuristic algorithm is exploration-oriented because of searching by many points distributed on all search spaces. Balance settings between exploration and exploitation are needed to produce good solutions. In fact, most population-based algorithms will encounter decreasing in exploration and become too exploitation-oriented as the iteration increase. Any metaheuristic algorithm applies parameters to control the behavior. However, the parameters usually do not provide a good intuition of the rate of exploration and exploitation. Hence, reaching a balance between them is hard to predict just by the algorithm parameters. This dissertation proposes a conceptual design combining the spy algorithm and B-VNS. The spy algorithm is a population-based metaheuristic algorithm that mimics the strategy of a group of spies, the spy ring. The spy algorithm is a new concept with the main idea to ensure the benefit of exploration and exploitation, and cooperative and non-cooperative searches always exist. This goal is implemented by utilizing three kinds of dedicated search operators and regulating them in a fixed portion. The occurrences of exploration and exploitation are controlled by algorithm parameters. Thus, the spy algorithm parameters provide good before-running intuition to easier reach the balance between exploration and exploitation. The spy algorithm is first designed to be used in the continuous optimization model. The spy algorithm was compared to the genetic algorithm, improved harmony search, and particle swarm optimization on a set of non-convex functions by aiming at accuracy, the ability to detect many global optimum points, and computation time. The Kruskal-Wallis tests, followed by Games—Howell post hoc comparison tests, were conducted using a. for the comparison. The statistical analysis results show that the spy algorithm outperformed the other algorithms by providing the best accuracy and detecting more global optimum points within less computation time. Furthermore, those results indicate that the spy algorithm is more robust and faster than other algorithms tested. On the other hand, the B-VNS algorithm is a modification of the variable neighborhood search (VNS) algorithm. The benefit of VNS comes from its thorough search while avoiding the local optimum trap by moving to the neighboring point called shaking. The local search after shaking is another benefit of VNS that makes VNS a prominent algorithm. However, the thorough search has the drawback of long computation time. This dissertation introduces a modified neighborhood structure to reduce the computation times. The main idea is to apply the binomial distribution to create the neighboring point. As a result, the neighborhood distance has a random pattern. However, it follows a binomial distribution instead of a strictly monotonic increase like in VNS. The B-VNS is a modification of VNS and is classified as a single solution-based algorithm. The B-VNS is intended to solve combinatorial optimization problems, particularly the quadratic unconstrained binary optimization (QUBO) problems categorized as NP-hard problems. The B-VNS and VNS algorithms were tested on standard QUBO problems from Glover and Beasley, on standard max-cut problems from Helmberg-Rendl, and those proposed by Burer, Monteiro, and Zhang. Finally, Mann-Whitney tests were conducted using a. to compare the performance of the two algorithms statistically. It was shown that the B-VNS and VNS algorithms are able to provide good solutions, but the B-VNS algorithm runs substantially faster. Furthermore, the B-VNS algorithm performed better in all of the max-cut problems regardless of problem size and in QUBO problems with sizes less than The spy algorithms and B-VNS have different designs in the process and the domain of the solved problems. However, considering the benefit of the spy algorithm and B-VNS, their combination has the potential to provide good results. Conceptually, the spy algorithm can be seen as the first step of B-VNS. Conversely, B-VNS can be considered an additional refinement for the spy algorithm.

Rapid population growth and economic progress over the past decades have triggered a sharp increase in the global demand for fossil fuels thereby resulting in an energy crisis. The problem may be alleviated by upgrading and producing gaseous energy, but one of the major challenges associated with gaseous energy is to separate it effectively from other less desirable gases. Thus, energy-saving and high-efficiency separation technology is needed. In the past three decades, gas separation membranes, including polymeric membranes and inorganic membranes, have attracted much attention due to their advantages in terms of energy efficiency, operational simplicity, cost competitiveness, and small footprint. Although polymeric membranes have been utilized in practical gas separation, their separation performance is not sufficient for widespread practical application. Carbon molecular sieve (CMS) membranes, one of the inorganic porous membranes, can be prepared by pyrolyzing polymeric precursors.Their pore structures provide molecular sieving ability and possess good thermal and chemical resistance. Especially, the separation properties of the CMS membranes for a variety of gas pairs have exceeded the upper bound of polymeric membranes. These characteristics have made them attractive candidates for gas separation. The pore structures, separation properties, and transport mechanism of the CMS membranes depend critically on the type of the polymeric precursors, pyrolysis conditions and pre- and post-treatments. Thus, in this thesis, I prepared CMS membranes derived from different polymeric precursors and investigated the effect of pyrolysis conditions and post-treatment on the gas permeation properties. In Chapter 2, toluene vapor addition was performed for the first time during the pyrolysis process to prepare highly selective CMS membranes. Adding toluene vapor in the pyrolysis process was a simple method to improve the selectivity compared with the traditional chemical vapor deposition post-treatment technique. Additionally, the use of toluene can avoid the high transportation costs of gaseous hydrocarbons. The results indicated that toluene vapor addition increased selectivities of the H2-related gas pairs compared with CMS membranes without toluene vapor addition. This could not be realized simply by increasing the pyrolysis temperature without toluene vapor addition. The CMS membrane with toluene vapor addition also showed higher permeance with a moderate selectivity compared with the CMS membrane with gaseous hydrocarbon addition reported in the literature. Furthermore, the gas permeance and selectivity could be readily controlled by adjusting the pyrolysis temperature and duration of the addition. The optimal preparation conditions of the CMS membrane with toluene vapor addition depend on the targeted gas pair to be separated. This study indicated that adding liquid hydrocarbon vapor in the pyrolysis process can be a simple and effective method for preparing highly selective CMS membranes. In Chapter 3, the mechanism of achieving high selectivity for the CMS membranes prepared by adding toluene vapor was further investigated. The physical and chemical properties of CMS membrane with adding toluene vapor were characterized using some sophisticated characterization techniques. It was found that toluene vapor addition formed carbon deposition on the outer surface region of the CMS membrane, which agreed with the previous report. The gas adsorption experiment suggested that toluene vapor addition also resulted in the loss and the narrowing of ultramicroporosity. Additionally, I preliminary analyzed the mechanism of achieving high selectivity for the CMS membrane with toluene vapor addition. In Chapter 4, a novel porous carbon fiber (PCF) was investigated to prepare supported CMS membranes derived from wood tar solution, as the development of wood tar-derived CMS membranes has been limited by the availability of porous supports in recent years. Moreover, the CMS membranes supported on commercially available porous ceramic tubes were also prepared under the same conditions for comparison purposes. The PCF consisted of interconnected pore structures, which provide additional paths and channels for gas transport, whereas the porous structure of the ceramic support consisted of voids between the alumina particles. It was found that for both supports, 70 wt% wood tar solution was the optimal solution for preparation of CMS membranes. The PCF-supported CMS membranes exhibited higher gas permeance and selectivity than the NA3-supported membranes. Furthermore, a series of PCF-supported CMS membranes from 70 wt% wood tar solution were prepared at different pyrolysis temperatures, the membrane pyrolyzed at 600 °C exhibited the highest H2 selectivity. This study demonstrated that PCF can be used for supported CMS membranes derived from wood tar solution. Additionally, PCF is also a promising support for the supported CMS membranes derived from other polymeric precursors. Finally, Chapter 5 summarized the main contents of this thesis.

It is important to anticipate problems such as a large amount of spring water that occurs during shafts construction and maintenance problems such as concentration of lining cracks after shafts construction in advance, and to carry out construction in a rational manner. Crack tensor from the rock mass information (cracks, strength) obtained by the construction of the shafts of the Horonobe Underground Research Laboratory of the JAEA, aiming at the selection of support and the implementation of spring water countermeasures. Based on the theory, the research results were summarized with the aim of evaluating the water hydraulic conductivity of the rock mass during shafts construction and the deteriorated condition of the lining after shafts construction, and establishing a simple prediction method for these. Chapter 1 summarizes the current state of rock mass geological observation and lining maintenance in the shafts, the water permeability coefficient of the rock mass, and the past domestic and overseas studies on the deterioration state of the lining, and the hydraulic conductivity of the rock mass using the rock mass information. The purpose of this study was clarified with the task of evaluating the deterioration state of the lining and establishing these simple prediction methods. In Chapter 2, crack tensor theory and stereology (statistical geometry) are used using information (length, direction, opening width) of rock cracks during shafts construction. By applying the concept, the three-dimensional permeable tensor was estimated accurately. Then, when the hydraulic conductivity obtained from the three-dimensional hydraulic tensor and the hydraulic conductivity based on the result of the in-situ permeability test using the deep borehole near the shafts were compared, it was clarified that they were in good agreement. A high correlation was obtained between the crack frequency obtained by dividing the total length of the cracks obtained in the shafts construction by the evcavation surface area and the hydraulic conductivity obtained from the three-dimensional hydraulic tensor. Therefore, we proposed a method to easily predict the hydraulic conductivity of rock from the frequency of cracks. The obtained prediction formula targets the depth at which three cross sections orthogonal to each other can be obtained on the rock crack observation surface, but the prediction formula was also obtained at a depth where three cross sections orthogonal to each other cannot be obtained. Comparing the hydraulic conductivity of the rock mass and the hydraulic conductivity based on the in-situ permeability test results, it was clarified that they are in good agreement. In Chapter 3, the orientation dependence of the earth pressure is recognized in the underground environment of the Horonobe Underground Research Center, and it is the major principal stress direction in the shafts of the underground Laboratory. Cracks have occurred on the lining wall surface in the east-west direction. However, the lining cracks in the shafts at that point may depend not only on the orientation dependence of the ground pressure, but also on the rock cracks on the back surface of the lining, the ground cover and the rock strength. If a prediction formula for predicting lining deterioration can be created in consideration, it will be useful information for countermeasures during construction, and based on the crack tensor calculated based on the information on cracks, from the rock crack tensor and rock strength. I proposed a method to estimate the deterioration condition of the lining. The estimated value of the crack tensor regarding the deterioration of the lining using the obtained prediction formula and the measured value calculated from the information of the lining crack, assuming that the margin of error due to the relative error is 0.1, the estimated value and the measured value are well one. Im adei t clear that Ia md oing it. In Chapter 4, aiming at selection of rational support for shafts extension of underground Research Laboratory planned in the future and implementation of spring water countermeasures. Ip roposed ac onstruction management system that applies the crack tensor prediction method for rock hydraulic conductivity during shafts construction and deterioration of lining after shafts construction. In the future, in order to select more rational support and implement measures against spring water, the rock mass information (cracks and rock mass strength) acquired during shafts construction will be obtained using the prediction formulas established in Chapters 2 and 3. By predicting the hydraulic conductivity of the rock and the crack tensor related to the deterioration of the lining, and reflecting it in the measures against spring water in the rock mass and the measures against the deformation of the lining, it can be expected to contribute to the reduction of maintenance costs. Chapter 5 summarizes the research results in each chapter and raises future issues for conclusion.

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。

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.

転写因子CCAAT/enhancer-bringing protein beta（C/EBPβ）は、IGF-binding protein-1（IGFBP-1）やprolactin（ERL）遺伝子のプロモーターおよびエンハンサー領域において、転写活性マーカーであるHistone-H3 lysine-27 アセチル化(H3K27ac)を誘導するパイオニア因子であり、ヒト子宮内膜間質細胞（ESC）の脱落膜化に貢献することを我々はこれまでに報告している。パイオニア因子の一部はヒストンアセチルトランスフェラーゼ（HAT）活性を有するコファクターと複合体を形成することで機能する。我々は、C/EBPβと共役するHATタンパクとしてp300を同定しているが、それ以外のコファクターについては不明である。Peroxisome proliferator-activated receptor gamma comma coactivator 1-α（PGC-1α）はH3K27acを制御することが知られている転写共役因子である。PGC-1αはESCでも発現しているが、脱落膜化におけるPGC-1αの機能は不明である。そこで、PGC-1αがC/EBPβの転写共役因子として働き、脱落膜化過程においてH3K27ac誘導に関与しているのではないかと考え検討した。脱落膜化を誘導するためにESCをcAMP存在上で培養した。cAMPによるIGFBP-1およびPRLの発現上昇はPGC-1αのノックダウンにより抑制された。また、cAMPはIGFBP-1およびPRLのプロモーターとエンハンサー領域に存在するC/EBPβ結合部位へのPGC-1αとp300のリクルートを増加させた。さらに、PGC-1αをノックダウンするとC/EBPβとp300に結合、およびH3K27acレベルが低下したことから、PGC-1αはこれらの領域でC/EBPβおよびp300とヒストン修飾複合体を形成することでH3K27ac誘導に関与していることが示された。さらにPGC-1αの発現制御機構を調べるために、C/EBPβを上流因子として着目した。PGC-1αのエンハンサー領域へのC/EBPβの結合はcAMPで増加した。また、これらのエンハンサーをゲノム編集により欠失させた細胞ではPGC-1αの発現が減少したことから、C/EBPβは我々が見出したエンハンサー領域に結合することでPGC-1αの発現を上昇させると考えられた。以上より、PGC-1αはC/EBPβの新規エンハンサーへの結合によって発現誘導されること、また、C/EBPβとp300と共にヒストン修飾複合体を形成して、IGFBP-1およびPRLのプロモーターとエンハンサーにエピゲノム変化を引き起こすことで脱落膜化に貢献していることがわかった。