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Abstract Purpose: The purpose of this study was to investigate the effects of two different postures (sitting and standing) and three different ambient temperatures (10℃, 20℃, and 30℃) on heart rate variability (HRV) among healthy young adults. Methods: Twelve young adult volunteers (males 6, females 6) were recruited. Following acclimatization to any the room temperature (10℃, 20℃ or 30℃), 5-min measurements of HRV were conducted in sitting and standing postures of the subjects. Results: Compared to the sitting posture, measurements obtained in the standing posture revealed a significant decrease in high-frequency power/HF, root mean square of successive differences between RR intervals, standard deviation of Poincaré plot perpendicular to the line-of-identity or SD1 and SD1/standard deviation of Poincaré plot along the line-of-identity or SD2, and a significant increase in lowfrequency power/LF and LF/HF under all experimental conditions (p<0.05 to 0.005). Majority of HRV parameters showed significant differences while the values obtained under 10℃ were compared with 20℃ and 30℃ conditions, respectively (p<0.05 to 0.001). Conclusions: Our findings suggest the predominance of sympathetic tone in the standing compared with sitting posture. Furthermore, colder conditions caused a predominance of the parasympathetic activity in both sitting and standing postures, and such effects of ambient temperature on the sympathovagal balance were stronger in the latter posture.

Abstract Background: Recently, the importance of nutritional management in pressure ulcer control has been pointed out. In this study, we analyzed the relationship between nutritional indicators and the presence or absence of pressure ulcers in order to reveal the importance of nutritional management in pressure ulcer control. Method: We investigated 407 inpatients for blood tests, height, weight, BMI, the Ohura-Hotta (OH) scale, nutrition method, living independence, and the presence or absence of pressure ulcers. Results: In the comparison of patients with and without pressure ulcer, significant differences were found in gender, nutrition method, serum total protein, serum albumin, hemoglobin concentration, and the OH scales. Multiple logistic regression analysis showed that gender, intravenous nutrition, serum albumin levels, and the OH scale were associated with the presence or absence of pressure ulcers. The results suggest that not only the OH scale, but the nutritional support was also important in the prediction of the pressure ulcer. It was suggested that shifting from intravenous feeding to tube feeding or oral feeding is important. Conclusion: we found that pressure ulcer was related to gender, intravenous nutrition, serum albumin level and the OH scale. The importance of nutritional management for pressure ulcer prevention was confirmed.

Abstract Objectives: This study examined the short-term outcomes of endovascular therapy (EVT) with the GORE VIABAHN VBX (VBX) which is a balloon-expandable covered stent in patients with aortoiliac occlusive disease (AIOD) and acute limb ischemia (ALI). Methods: This was a single-center retrospective review of patients with AIOD and ALI who underwent EVT with the VBX between December 2018 and January 2021. Primary, assisted, and secondary patency rates were assessed, along with 30-day mortality rates. Results: Twenty-two patients underwent EVT with the VBX; 18 patients had AIOD, whereas 4 had ALI. The incidence of TransAtlantic Intersociety Classification II D lesions was 50% (n=9). Technical success was 100%. One patient with unilateral common iliac artery stenosis developed an intraoperative dissection of the distal edge of the VBX, which required an additional self-expanding VIABAHN stent on the distal edge of the VBX. The primary patency rate at 24 months was 100%. No patients required additional target vessel treatment at a median follow-up of 10 months (range, 1-27). EVT was also successful in the four patients with ALI, who had no complications of the target vessel. Conclusion: The present study demonstrated that EVT with the VBX is effective for AIOD and ALI.

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。

【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。

右心室 (RV) の機能障害とそれに関連する不整脈は、肺動脈性肺高血圧症 (PAH) の予後の重要な決定要因として認識されている。今回心臓リアノジン受容体 (RyR2) の安定剤であるダントロレン (DAN) による右室心筋への直接的な薬理学的介入が、モノクロタリン (MCT) 誘発性PAHラットモデルにおいてRV機能障害および不整脈に対する保護効果を有するかどうかを調査することを目的とした。方法として雄の8週齢のSprague-Dawleyラットに、PAHの誘発のためにMCTを腹腔内投与した。カテコールアミンによる心室頻拍 (VT) の誘発も、単離された心筋細胞におけるRyR2を介したCa^{2+}放出特性に関連して評価された。RVの形態と機能に対する慢性的な圧力過負荷の独立した影響を評価するためには、肺動脈縮窄モデルも確立した。結果、MCT誘発PHAラットモデルでは、RV肥大、拡張、および機能低下が観察され、MCT誘発2か月後の生存率は0%であった。対照的に、慢性DAN治療はこれらすべてのRVパラメータを改善し、生存率を80%に増加させた。慢性的なDAN療法はまた、RyR2からのカルモジュリンの解離を防ぎ、それによってMCTによって誘発された肥大したRV心筋細胞におけるCa^{2+}スパークと自発的なCa^{2+}トランジェストを抑制した。エピネフリンは、MCT誘発性PAHのラットの50%以上でVTを誘発したが、慢性DAN治療によってVTの完全に抑制した。以上よりDANによるRyR2の安定化は、PAHに関連するRV機能障害および致命的な不整脈の発症に対する新しい治療薬としての可能性を秘めている。

We previously discovered that SPC/Fyn/Rho-kinase (ROK) pathway mediates the Ca^{2+}-sensitization of coronary arterial smooth muscle (CASM) contraction leading to vasospasm, a major cause of sudden death. Lately, we have been trying to find and develop more natural edible compounds which can treat and/or prevent the SPC-induced abnormal CASM contraction, and finally the first to discover that tangeretin (5,6,7,8,4'-prentamethoxyflavone), a natural compound extracted from citrus plants, can inhibit the SPC-induced CASM contraction both in the pretreatment and posttreatment. In porcine CASM tissues, tangeretin showed remarkable inhibitory effects on the SPC-induced contraction with modest inhibitory effects on the high K^+-depolarization-induced Ca^{2+}-dependent contraction, both in pretreatment and posttreatment at the optimal concentrations; Regarding the mechanisms, tangeretin markedly abolished the SPC-induced cell contraction through inhibiting the SPC-induced activation and translocation of Fyn and ROK from the cytoplasm to the cell membrane in cultured CASM cells, resulting in the reduction of phosphorylation of myosin light chain. Taken together, these findings indicate that tangeretin, upon pre- or post- treatment, inhibits the SPC-induced CASM contraction through suppressing the Fyn/ROK signaling pathway, thereby suggesting that tangeretin can be a potential candidate for the treatment and/or prevention of vasospasm.

Huntingtin-associated protein 1 (HAP1) is a neural huntingtin interactor and being considered as a core molecule of stigmoid body (STB). Brain or spinal cord regions with abundant STB/HAP1 expression are usually spared from neurodegeneration, whereas the regions with little STB/HAP1 expression are always neurodegenerative targets. The enteric nervous system (ENS) can act as a potential portal for pathogenesis of neurodegenerative disorders. To date, the expression of HAP1 and its neurochemical characterization have never been examined there. In the current study, we determined the expression and immunohistochemical phenotypes of HAP1 in ENS of adult rodents using Western blotting and light/fluorescence microscopy. HAP1 immunoreactivity was strongly expressed in both myenteric and submucosal plexuses of ENS. STBs were observed in the cytoplasm of most of the HAP1-immunoractive (ir) cells in ENS. In myenteric plexus, a large number of calretinin, calbindin, NOS, VIP, ChAT, SP, somatostatin, and TH-ir neurons showed HAP1 immunoreactivity. In contrast, most of the CGRP-ir neurons were devoid of HAP1-immunoreactivity. In submucosal plexus, almost all the cholinergic secretomotor neurons containing ChAT/ CGRP/ somatostatin/ calretinin, non-cholinergic secretomotor neurons containing VIP/TH/calretinin and vasodilator neurons containing VIP/calretinin express HAP1. Our current study is the first to clarify that HAP1 is highly expressed in excitatory motor neurons, inhibitory motor neurons, and interneurons but almost absent in sensory neurons in myenteric plexus. While, HAP1 is expressed in all neuronal subgroups of Meissner’s plexuses. These suggest that due to lack of putative STB/HAP1 protectivity, the sensory neurons (Dogiel type II) might be more vulnerable to neurodegeneration than STB/HAP1-expressing Dogiel type I neurons in myenteric plexus and secretomotor/vasodilator in Meissner’s plexuses. Our current results may reflect the involvement of HAP1 in modulation of excitatory and inhibitory motor neuron functions in myenteric plexus and the secretomotor and vasodilator functions of submucosal neurons. It will be of great interest to elucidate the physiological or pathological roles of HAP1 in ENS. Our current results might lay a basic foundation for future studies that seek to clarify the physiological/pathological effects of STB/HAP1 in the ENS.