doctoral thesis

【背景】インターロイキン(IL)-33は, 宿主防御, 神経損傷, 炎症などに重要なIL-33/ST2シグナル経路を誘導する. 一方, IL-33のデコイ受容体である可溶性ST2(sST2)は, IL-33/ST2シグナル経路を抑制する. sST2は種々の神経疾患患者の血清中で増加するが, 低酸素性虚血性脳症(Hypoxic-ischemic encephalopathy; HIE)では知られていない. 【目的】本研究の目的は, HIEにおける血清中のIL-33, 及びsST2濃度を測定し, HIE重症度と神経学的予後との関連性を検討することである. 【対象と方法】2017年1月から2022年4月の期間に, 山口大学医学部附属病院総合周産期母子医療センターに入院した, 在胎期間36週以上, かつ出生体重1,800g以上の新生児を対象とし, HIE群23名, 対照群16名を本研究に登録した. HIEの重症度はSarnat分類により軽症, 中等症, 重症に分類し, 生後6時間以内, 及び1-2, 3, 7日目の血清IL-33及びsST2濃度を測定した. プロトン磁気共鳴スペクトロスコピーによりHIE群の基底核におけるlactate/N-acetylaspartate(Lac/NAA)比を算出し, 退院後の神経学的後遺症の有無を追跡調査した. 【結果】血清中IL-33濃度は各群で差を認めなかった. 一方, 中等症及び重症HIE群の血清sST2濃度は, 対照群に比し著明に高値で, HIE重症度と相関して高値であった. 血清中sST2濃度はLac/NAA比と有意な正の相関を示し(相関係数=0.527, P=0.024), 神経学的後遺症を来したHIE児では予後良好の児に比し, sST2濃度及びLac/NAA比が有意に高かった(それぞれP=0.020, <0.001).【結論】血清sST2濃度はHIEの重症度および神経学的予後予測に有用である可能性が示唆された.

【背景】結腸直腸癌（colorectal cancer:CRC）の予後については、腫瘍の特性だけでなく、宿主の免疫反応も重要な因子となる。我々は宿主の免疫反応として全身および腫瘍微小環境（tumor microenvironment: TME）の炎症性サイトカイン発現に注目し、これらを評価することにより、免疫抑制状態と患者の予後との関係を検討した。 【方法】 切除可能CRC患者209名において、術前に採取した血清サンプルを用いてサイトカイン濃度（IL-1β、IL-6、IL-8、TNF-α）を電気化学発光法により測定し、予後との関連を検討した。また切除切片における腫瘍組織でのサイトカイン発現を腫瘍細胞、間質細胞に分けて免疫組織化学的に評価した。さらに、切除したCRC患者10例において、新鮮な切除切片から抽出した腫瘍浸潤細胞を用いたマスサイトメトリーによるシングルセル解析を行った。 【結果】 無再発生存期間において、血清IL-1β、IL-8、TNF-α濃度の高低では有意な関係を認めなかったが、血清IL-6高値群で有意に予後不良であった。また血中IL-6濃度上昇は腫瘍組織中の間質細胞におけるIL-6高発現と関連していた。シングルセル解析の結果、腫瘍浸潤免疫細胞のうちIL-6+細胞は主に骨髄球系細胞で構成され、リンパ球系細胞ではIL-6発現をほとんど認めなかった。またIL-6高発現群では、CD33+HLADR-骨髄由来抑制細胞（myeloid-derived suppressor cell: MDSC）およびCD4+FOXP3highCD45RA-エフェクター型抑制性T細胞(effector regulatory T cell: eTregの割合がIL-6低発現群に比べ有意に高かった。さらに、MDSCにおけるIL-10+細胞の割合、eTregにおけるIL-10+細胞またはCTLA-4+細胞の割合は、IL-6高発現群で有意に高かった。 【結論】血清IL-6濃度の上昇は間質細胞のIL-6発現と関連し、予後不良であった。腫瘍浸潤免疫細胞におけるIL-6高発現は、TMEにおけるMDSCやeTreg等の免疫抑制性細胞の蓄積と関連し、その機能性マーカーの上昇も認めた。これらIL-6を介した抑制性免疫機構がCRC患者の予後不良の一因となっている可能性がある。

アミノグリコシド系抗菌薬は有害反応として聴覚障害をきたすことがあり、蝸牛基底回転の外有毛細胞が障害されやすいことが知られている。本研究では、ネオマイシンの有毛細胞障害に対するアスタキサンチンナノ製剤の保護効果を検討した。ネオマイシンを加えたCBA/Nマウスの卵形嚢培養に対し、培養液にアスタキサンチンナノ製剤を投与した群では有毛細胞の減少および酸化ストレスが有意に抑制された。さらに、アスタキサンチンナノ製剤の鼓室内投与を行い音響曝露前後の聴性脳幹反応（ABR）閾値の変化、有毛細胞減少率を評価した。アスタキサンチン投与群では音響曝露後のABR閾値変化、有毛細胞減少率が抑制される傾向が見られた。血液内耳関門の存在により、鼓室内投与に適した薬剤は限られるが、アスタキサンチンナノ製剤の形態は正円窓膜を浸透する可能性があり、内耳障害抑制の効果を有する可能性が示唆された。

Generalized pustular psoriasis (GPP) is a rare form of psoriasis, which is characterized by sudden onset of repeated erythema and pustule formation with generalized inflammation. Recent advances in molecular genetics have led to the identification of several genes associated with GPP, including IL36RN, CARD14, AP1S3, SERPINA3, and MPO. Of these, only limited cases of GPP have been reported to carry mutations in the AP1S3, SERPINA3, or MPO to date. In the present study, we investigated a Japanese patient with GPP and found a bi-allelic missense mutation c.1769G>T (p.Arg590Leu) in the MPO gene. Structural analysis predicted that the mutant MPO protein would abolish its ability to bind with heme protein. In vitro studies using cultured cells revealed that the mutant MPO was stably expressed, but completely lost its myeloperoxidase activity. Immunohistochemistry (IHC) using an anti-MPO antibody showed markedly-reduced expression of MPO protein in the patient’s skin, suggesting that the mutation would lead to an instability of the MPO protein in vivo. Finally, IHC with an anti-citrullinated Histone H3 antibody demonstrated a sparse formation of neutrophil extracellular traps within a Kogoj's spongiform pustule of the patient’s skin. Collectively, we conclude that the c.1769G>T (p.Arg590Leu) in the MPO is a complete loss-of-function mutation associated with GPP in the patient. Our data further underscore critical roles of the MPO gene in the pathogenesis of GPP.

Objective: Anastomotic leakage is a common and severe complication of esophageal reconstruction. Accordingly, there is a clinical need for novel methods to prevent it. We developed multilayered, growth factor-secreting fibroblast sheets that promote wound healing and angiogenesis. The present study aimed to assess the utility of allogenic multilayered fibroblast sheets in preventing esophageal anastomotic leakage in a rat model of esophageal reconstruction. Methods: Allogenic multilayered fibroblast sheets prepared from oral mucosal tissues were implanted at esophageal anastomotic sites. Results: The allogenic multilayered fibroblast sheet group had significantly higher burst pressure and collagen deposition compared to a control group five days postoperatively. The expression levels of collagen type I and III mRNAs around esophageal suture sites were higher in the allogenic multilayered fibroblast sheet group compared to the control group on postoperative days 0, 3, and 5. There was a trend toward lower anastomotic leakage and lower abscess scores in the allogenic multilayered fibroblast sheet group compared to the control group; however, these differences did not reach statistical significance. Allogenic multilayered fibroblast sheets completely disappeared at ten days after implantation. Further, no inflammation was observed at suture sites with implanted allogenic multilayered fibroblast sheets at five days after surgery. Conclusion: Allogenic multilayered fibroblast sheets may represent a promising method of preventing esophageal anastomotic leakage.

Many mail filtering methods have been proposed, but they have not yet achieved perfect filtering. One of the reasons for this is the influence of modified words created by spammers to slip through the mail filtering, in which words are modified by insert symbols, spaces, HTML tags, etc. For example,“ price$ for be$t drug$! ”，“ priceC I A L I S ”， “ <font>se</font>xu<font>al</font> ”, etc. These are frequently replaced with new strings by changing the combination of symbols ,HTML tags etc. Mail filtering is a technique that captures trends in words in training mails (mails received in the past) and applies these trends to words in test mails (newly received emails). Some of the above modified words appear in both training and test mails, i.e., words that could be used as features of spam mail by using them unprocessed, while others appear only in test mails, i.e., words that have not been learned and require special processing (e.g., removal of symbols, search for similar words, etc.) for their use. However, existing methods do not make these distinctions and treat them in the same way. Therefore, in order to bring the filtering performance of the existing methods closer to perfect filtering, we developed a method in which the above modified words are separated into words that appear in both training and test mails and words that appear only in test mails, and each of these words is used for mail filtering. In this study, we treat the above modified words as ”strange words”. Typical examples of such strange words include, in addition to the above, new words included in ham mails, proper nouns used in close relationships, and abbreviations. The results of this study are as follows (1) In order to compare the filtering performance between strange words and other words, filtering experiments were conducted using existing methods with strange words, nouns, verbs, and adjectives. The results showed that the filtering performance of the strange words was the best. This means that strange words have a significant impact on the filtering performance, and we expect to improve the filtering performance of existing methods by developing a new method to utilize strange words. (2) In order to examine the breakdown of strange words, we counted the number of words that appeared in both training and test mails, and the number of words that appeared only in test mails. The results were compared with those obtained for nouns, verbs and adjectives. We found that there are a significant number of strange words that appear in both training and test mails, but only in one of the groups, i.e., ham or spam mail. Words with this appearance pattern are most useful for mail filtering. On the other hand, we found that there are many strange words that appear only in test mails, i.e., words that cannot be learned. We expect to improve the filtering performance by separating these strange words and developing a new method to use each of them. (3) For the use of strange words, we developed (A) a method for using words that appear in both training and test mails, and (B) a method for using words that appear only in test mails, respectively. (A) To examine the breakdown of strange words that appear in both training and test mails, we divided them into two categories: words that appear only in ham and spam mails, i.e., words with patterns that improve filtering performance, and words that do not, and examined their frequency of occurrence. The results showed that the words with appearance patterns that improve filtering performance tend to appear more frequently than those without such patterns. This means that by using words with a certain number of occurrences in filtering, it is possible to use more words that improve filtering performance. We developed a method to do this and conducted experiments with different threshold values to find the optimal value, and confirmed that setting the threshold around 7 improves filtering performance. (B) We compared the number of strange words that appear only in the test mails between ham and spam mails, and found that the number tends to be higher in spam mail than in ham mail. In order to utilize this difference for filtering, we proposed a method to set a uniform spam probability for strange words that appear only in the test mails, and attempted to find the optimal spam probability. As a result, setting the spam probability to 0.7 improved the filtering accuracy from 98.2% to 98.9%. By using (A) and (B) above together, both words that appear in both training and test mails and words that appear only in test mails can be used for mail filtering to increase accuracy. Mail filtering has been improved and its performance has reached its limit. In order to further improve accuracy, i.e., to approach perfect filtering, a new perspective is needed, and this paper provides one such perspective: the use of strange words. This paper is organized as follows. In Chapter 1, we review the background of mail filtering methods, discuss how spammers use strange words to slip through such filters. The purpose and structure of this paper are then presented. In Chapter 2, we will discuss related research on examples of filtering methods that have been proposed so far are given. In Chapter 3, we describe the mail datasets, word handling, and strange words used in the this paper. This is followed by an explanation of the ROC curve, which is the measure used to evaluate the filtering performance, and explanation of scatter plots and box-and-whisker plots. In Chapter 4, we compare the filtering performance between strange words and other words, and show that strange words have a significant impact on the filtering performance. Furthermore, based on the results of a breakdown of the number of strange words, we discuss the possibility of improving filtering performance by separating words that appear in both training and test mails from those that appear only in the test mails. We will work on this in the next chapters and report the results. In Chapter 5, we develop a method to use (A) above, i.e., strange words that appear in both training and test mails. From the results of counting the number of words used in the subject and body of each email, we show that the number tends to be smaller for words that degrade the filtering performance. Based on these results, we propose a method that sets a threshold for the number of words used in the subject and body of mails, and uses only those words that exceed the threshold for classification. Experiments are conducted to find the optimal value by varying the threshold, and the effect of this method on performance is reported. In Chapter 6, we develop a method to use (B) above, i.e., strange words that appear only in the test mails. We compare the number of types of these words in ham and spam mails, and show that the number tends to be larger in spam mails, and that this feature can be used as a bias for detecting spam mails. In this paper, we deal with experiments using bsfilter and develop a method to set spam probabilities uniformly for strange words that appear only in the test mails. After searching for the optimal spam probability, we report that a spam probability of 0.7 greatly improves the filtering performance. In Chapter 7, we describes the processing flow combining the methods developed in Chapter 5 and Chapter 6. The paper is then summarized, including future prospects.

Steel truss bridges, which are one of the bridge structures applicable to long spans, are widely used as marine bridges connecting mainland and remote island. Since such steel truss bridges are built on the sea, they are exposed to severe corrosive environment due to the influence of airborne salt. In addition, there are many parts where it is not easy to inspect to detect abnormalities, so it is more difficult to eliminate the risk of member damage in such steel truss bridges than in general bridges. On the other hand, once the marine bridges are built, they become an indispensable facility for the life of the island. Therefore, if there are no other traffic routes to access an island, the sustainability of the marine bridge is an important issue that is directly linked to the sustainability of the remote island life. When member damage occurs in a steel truss bridge, it depends on redundancy, which means the margin for the load-bearing capacity and load-bearing function, whether the damage develops into chain damage or remains limited damage. Bridges with redundancy could be restored by repairing even if the member damages occurred, because they did not develop into chain damages. In some cases, vehicles could pass through with traffic restrictions. Although redundancy is an important performance for maintaining life on remote islands that have no alternative traffic routes, there are few studies on evaluation and improvement methods of redundancy for long steel truss bridges used for marine bridges. The purpose of this study is to propose a method for improving the redundancy of long steel truss bridges, and three research subjects are set to achieve this purpose. The first study subject is the investigation of the effect of truss joint modeling on redundancy evaluation, and is the subject to appropriately evaluate the redundancy of steel truss bridges. The second is also the subject related to the redundancy evaluation of steel truss bridges, and is the development of dynamic response calculation method that considers the vibration characteristics of steel truss bridges, which are vibration systems with multiple degrees of freedom. The third study subject is a proposal for methods to improve the redundancy of long steel truss bridges. This paper consists of five chapters. Chapter 1 is an introduction, and describes the background of the research, the setting of the purpose and research subjects, and the previous studies. Chapter 2 describes the study on the modeling of the truss joint. In the analysis of healthy steel truss bridges with no member damage, the sectional forces can be calculated appropriately even with analysis modeling in which frame elements of truss members are rigidly connected at the truss joints, simply. On the other hand, in the redundancy analysis of steel truss bridges with member damages, it is shown that it is necessary to consider the shape of the gusset plates at the truss joints in analysis modeling. Chapter 3 describes the study on the calculation method of dynamic response caused by damage of truss members. There are cases where the dynamic response due to member damage is calculated in the same way as a single-degree-of-freedom vibration system. However, this study develops a dynamic response calculation method considering the vibration characteristics of long steel truss bridges by using the eigenvector of steel truss bridges with member damage. A method is proposed to set the magnitude of the eigenvector using balance equation of the work given to the steel truss bridge by the sectional force unloaded from damaged member and the strain energy stored in the steel truss bridge. In addition, a method is proposed to calculate the dynamic response by setting the range of vibration modes using the sum of effective mass ratio, and selecting the eigenvector that has the greatest effect on the dynamic response for each member. It is shown that the proposed calculation method gives redundancy evaluation closer to time-history-response analysis than the method that calculates the dynamic response in the same way as a single-degree-offreedom vibration system. Chapter 4 describes the study on redundancy improvement for a long steel truss bridge. A combination of the countermeasure against members that trigger chain damage and the countermeasure against members with insufficient load-bearing capacity is planned. Analysis clarifies that the X bracing, which is a reinforcing structure in X shape, is an efficient reinforcement that works against multiple member damage cases as the countermeasure against the trigger member of member chain damage. Also, the load-bearing capacity is verified by a loading test of specimens with reinforced structures. Since the subject bridge has 18 truss panels where X bracing can be installed, the placement patterns were examined by the optimization method. It is clarified that the weight of reinforcing material can be reduced by installing X-braces only at four truss panels in the alternating areas, rather than installing at all 18 truss panels. Chapter 5 describes the summary of this study and future developments.

キメラ抗原受容体(CAR)-T細胞療法は造血器腫瘍において優れた抗腫瘍効果を示している一方、固形がんにおいては臨床応用がまだなされていない。臨床応用におけるハードルとなっている理由の一つとして、固形がんでは適切なCAR ターゲットが欠如しているということにある。GM2 は糖鎖にシアル酸を有するスフィンゴ糖脂質の一群であるガングリオシドの一つであり、様々なタイプの固形がんにおいて過剰発現している。本研究では、我々が以前に開発したインターロイキン7(IL-7)/ケモカインリガンド19(CCL19)産生型ヒトCAR-T細胞システムを用いて、GM2が固形腫瘍に対するCART細胞療法のターゲットとなりうるかという点についてヒト肺小細胞がん異種移植マウスモデルを用いて探究した。IL-7/CCL19 産生型抗GM2 CAR-T細胞治療を行ったところ、GM2 陽性腫瘍の完全な退縮が観察され、腫瘍内部への豊富なT 細胞浸潤や長期のメモリー応答形成も観察されたが、有害事象は認めなかった。加えて、臨床においてCAR-T 細胞使用時に問題となるサイトカイン放出症候群や神経毒性をコントロールする方法として、ガンシクロビル(GCV)投与によりアポトーシスが誘導される自殺システムである単純ヘルペスウイルス-チミジンキナーゼ(HSV-TK)をCAR-T細胞に遺伝子導入した。HSV-TK発現IL-7/CCL19産生型抗GM2 CAR-T細胞は、in vivoにおいてGCV投与により効果的に除去された。以上より、我々の研究はIL-7/CCL19産生型ヒト抗GM2CAR-T細胞のGM2陽性固形がん治療への臨床応用における安全性を実証し、その有望な治療効果を明らかにした。

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.

Since social infrastructure, which was intensively developed during the high economic growth period,will deteriorate all at once in the future, maintenance and management of facilities will be an issue in thefuture. Currently, facility inspection records are based on paperbased forms, and are not premised onautomatic processing by computer. The authors have developed the “Smart Chosa” and realized a databaseof facility inspections and a GIS System. The Smart Chosa was able to record the location of the inspectionphoto on a two dimensional map, but because it was necessary to approach the deformed part when takingthe inspection photo, it was not possible to grasp the position, direction, and size of the entire facility.Therefore, we applied 3D GIS to Smart Chosa for sabo dams, created a 3D model from photographs takenon site, and conducted research to manage inspection results on the 3D model. This study summarizes the results of research on management of inspection photographs on a 3D GISin order to improve the efficiency of management of inspection photographs of sabo facilities. This thesisconsists of 6 chapters, and the main content of each chapter is as follow. [Chapter 1: Introduction] In this chapter, the current status and issues of the maintenance and management of socialinfrastructure in Japan were summarized. Utilization of 3D models for maintenance and management ofcivil engineering facilities, high-precision positioning used for alignment of 3D models, efficientinspection of concrete structures, and existing research trends on iPhone LiDAR applications wereorganized. On that basis, the purpose and points of focus of this research were organized, and the structureand outline of this paper were described. [Chapter 2: Comparison of 3D models and examination of application to sabo facilities maintenance management system] In this chapter, three types of models, a BIM/CIM model, a 3D point cloud model, and a 3D surfacemodel, are compared and examined as 3D models to be applied to the maintenance management system.The problem setting in this chapter is the selection of a 3D model to be used in this system. The constraintis that the Sabo dams including the existing dam can be modeled in 3D. At present, there are few BIM/CIMmodels for sabo dams, so when applying to existing sabo dams, we believe that a 3D surface model thatcan be created by SfM/MVS technology from photographs taken by UAVs will be useful. The 3D modelin the research was a 3D surface model. Knowledge about the 3D model that can be applied to the sabo facility maintenance managementsystem and knowledge for utilizing the 3D model in the 3D GIS were obtained. [Chapter 3: Performance evaluation of RTK receiver used for Sabo facility investigation support system] In this chapter, a survey of high-precision positioning technology necessary for positioning 3D modelsof sabo dams and inspection photos on a 3D GIS, and evaluation of positioning performance in sabo damsand surrounding forests are conducted. The problem setting in this chapter is whether or not locationinformation can be acquired during surveys of Sabo facilities, and accuracy verification. The constraintis that real-time high-precision positioning is required using inexpensive and small devices inenvironments that are unfavorable for satellite positioning (such as sabo dams and forests). It was confirmed that the multi-band receiver whose performance was evaluated has a horizontalvariation of 22 mm (2DRMS) even in a poor environment where about 70% of the sky directly below thesabo dam is covered. It was confirmed that the method can be applied to aligning 3D models ofphotographs. [Chapter 4: Investigation of image synthesis for creation of sabo dams inspection image] In this chapter, as a basic examination of 3D model creation, the image synthesis method is organized.The problem setting in this chapter is normalization and image combination necessary for synthesizinginspection photographs (2D). The constraint is that the inspection photography equipment is a smartphonefor field survey. In the feature point detection method for image synthesis, we compared two types offeature amounts, SIFT feature amount and AKAZE feature amount, and confirmed the accuracy byexperiments. In addition, RANSAC was used as an outlier removal method. By combining these methods,we performed image synthesis using multiple photographs of the concrete surface of the sabo dam. [Chapter 5: 3D model creation by SfM/MVS and application to 3D GIS] The problem setting in this chapter is a superimposed display of a 3D Sabo dam model and inspectionphotographs. The constraint is that the equipment that can be used to create a 3D model of inspectionphotographs is limited to equipment (compact and lightweight) that can be brought by local workers. Inthis chapter, we first present an overview of the "smart chosa" system that expands the scope of applicationfrom 2D to 3D in this research. After that, we investigated SfM/MVS processing to create a 3D surfacemodel. By creating a 3D model of the sabo dam and a 3D model of inspection photos by SfM/MVSprocessing, and importing them into a 3D GIS, we succeeded in superimposing the sabo dam andinspection photos on a 3D map. In addition, we examined a method of creating a 3D surface model using the iPhone LiDARapplication that can perform 3D measurement using the LiDAR function installed from iPhone12Pro. Wecompared the 3D model created with the iPhone LiDAR app and the 3D model created using MetaShape,a software that implements SfM/MVS processing, and confirmed the image resolution and positionalaccuracy for use as inspection photographs. In order to incorporate the created 3D model into 3D GISsoftware, we examined a method for matching the orientation and position, and actually superimposedthe 3D model of the sabo dam and the 3D model of the inspection photograph on the 3D GIS. I haveconfirmed that it is possible. [Chapter 6: Summary] In this chapter, a summary of the results obtained in Chapters 2 to 5 and future issues were discussed. The result of this research is a visualization method that makes it easy for people other than fieldinvestigators to understand the situation of the site by importing 3D surface models acquired by variousmethods into 3D GIS. 3D surface models include SfM models created from photos taken with aUAV/smartphone, SfM models created from photos taken with a handheld RTK rover, and 3D modelscreated with the iPhone LiDAR app. By using this method, it is possible to grasp the deformation position and deformation direction of thesabo dam in 3D space, and by superimposing the photographs of each inspection, it is possible to graspthe change over time.

Japan's declining birthrate and aging population are difficult to resolve in the short term, and the working-age population, which refers to the population aged 15 to 65, continues to decline. On the other hand, mental illness patients are increasing in the working age population. The medical side, which is responsible for treatment, is required to improve efficiency by reforming the way doctors work. Therefore, expectations are placed on psychotherapy performed at home for the purpose of effective treatment of the working-age population. In this study, neurofeedback, which is one of the psychological therapies, was taken up, and applied equipment development and verification were carried out for its implementation at home. In this study, as a preclinical stage, measures were verified based on measurements of general cooperators. Neurofeedback (NFB) is considered to be one of psychotherapy using electroencephalogram signals, and is a psychotherapy that visualizes one's own electroencephalogram and self-controls the visualized electroencephalogram. It is attracting attention because it is a non-drug therapy and provides neuromodulation. NFB is being investigated for many clinical applications. The target diseases are diverse, including chronic pain, ADHD, depression, and mood disorders. However, we believe that there are four tasks to ensure the effectiveness of this therapy. Task 1 is overcoming the difficulty of installing electroencephalogram electrodes. NFB is considered to be a therapy that affects the plasticity of the cranial nerves, and is a therapy that actively promotes the development of neural networks, and is expected to be highly effective if the training frequency is high. It is necessary to be able to perform it at home, and it is required that electroencephalogram electrodes can be easily installed. Therefore, we made a prototype of an electroencephalogram headset with bipolar gel electrodes, and as a result of trial verification with children, we were able to confirm the electroencephalogram signals of 30 people aged 5 to 20 years old. Analysis of the recorded electroencephalogram revealed an age-dependent left-brain tendency in β waves, etc., confirming consistency with previous findings. Task 2 is determining the brain wave derivation part of the NFB training target. For electroencephalography, lead electrodes are usually placed in the scalp, but it is difficult to place the electrodes in the scalp by yourself. There is a need to consider forehead derivation for easy EEG electrode placement at home. There are regional differences in EEG waveforms within the forehead, and it is necessary to select the most appropriate extraction site. For NFB, we explored the optimal site based on the correlation with the top of the head, which is usually the electroencephalogram derivation position. Next, we performed an EEG network analysis at the time of NFB using the EEG derived from the top of the head and the EEG derived from the optimal forehead region, and analyzed the difference in the brain network during NFB due to the difference in the derivation region. For the second task, we explored the optimal site for deriving brain waves from the forehead, and proved that NFB from the brain waves derived from this site works on the same network as NFB from the brain waves derived from the top of the head. Task 3 is a method of selecting an electroencephalogram frequency band to be derived and self-controlled in NFB therapy (training target electroencephalogram frequency band). In previous studies, the EEG frequencies targeted for NFB therapy are diverse and not standardized. Even for the same disease, various electroencephalogram frequency bands are selected and NFB is performed. A personalized frequency band decision is made according to the patient's pathology and condition. In order to make the frequency band determination method more logical, we thought it necessary to determine the electroencephalogram frequency for therapy from the comparison of the basic rhythms of healthy subjects and patients. In this study, we created an electroencephalogram basic rhythm evaluation program and collected electroencephalogram basic rhythm data from randomly selected subjects. The electroencephalogram basic rhythm evaluation program consists of 7 stages. Eyes open stage, Eyes closed stage, 0Back stage, Rest1 stage, 2Back stage, Rest2 stage, Healing Picture stage. Changes in brain waves occur due to external stimuli such as eye opening and eye closing, concentration, and relaxation. An electroencephalogram basic rhythm evaluation program was created considering multiple stimuli that affect electroencephalogram dynamics. The usefulness of this program was confirmed as a preliminary examination of the dominant fluctuation region and network analysis by topographic analysis during the execution of the electroencephalogram basic rhythm evaluation program. EEG basic rhythm brain standard program electroencephalogram measurements were carried out for 89 subjects recruited from the general public, and a database was created. Using the forehead optimally measured parts (left and right) obtained in Task 2 as electroencephalogram derivation parts, a significant difference test was performed for each electroencephalogram frequency band Power value and content rate of each stage of the electroencephalogram basic rhythm evaluation program. The α Power value increased 2.52 times when the eyes were closed, and the θ Power value increased 1.67 times during 2 Back compared to 0 Back. We examined the possibility of clinical application by analyzing the correlation between the score of the questionnaire used in clinical diagnosis and the electroencephalogram component. The questionnaires used were mainly CSI (CENTRAL SENSITIZATION INVENTORY), and POMS2 (Profile of Mood States 2). Task 4 is NFB scoring. Continuation of psychotherapy requires a score as a reward to be visualized. We compared the two scores, the time ratio score and the amplitude ratio score, analyzed the correlation between the questionnaire used in task 3 and the two scores, and examined the optimal score. In results, the frequency band that correlates better with psychological activity during NFB was suggested SMR. Some of the psychological scales included the data probably above the general average level, which might have provided hypotheses at the preclinical stage. 4 tasks were conducted to demonstrate the technical requirements and effectiveness evaluation for the practical application of cognitive psychological training NFB, which is expected to be used with high frequency at home for children to working-age adults. The technological requirements and effectiveness evaluation for the practical application of NFB are presented. This research attempted four tasks and realized the possibility of frequent NFB training at home for patients from children to productive age. As a preclinical stage, it was a study within a range that can be resolved as a stage of policy verification based on the general participant study. In the future, the efficacy of this study will be further evaluated by comparing it with clinical data in the area of mental illness such as depression and developmental disorders, including chronic pain. In the future, the effectiveness of this study will be further evaluated by comparison with clinical data in the area of chronic pain and other mental illnesses such as depression and developmental disorders.

Open source software (OSS) are adopted as embedded systems, server usage, and so on because of quick delivery, cost reduction, and standardization of systems. Therefore, OSS is often used not only for the personal use but also for the commercial use. Many OSS have been developed under the peculiar development style known as the bazaar method. According to this method, many faults are detected and fixed by developers around the world, and the fixed result will be reflected in the next release. Also, many OSS are developed and managed by using the fault big data recorded on the bug tracking systems. Then, many OSS are developed and maintained by several developers with many OSS users. According to the results of the 2022 Open Source Security and Risk Analysis (OSSRA), OSS is an essential part of proprietary software, e.g., the source code containing OSS is 97%, all source code using OSS is 78%. On the other hand, OSS has issues from various perspectives. Therefore, OSS users need to decide on whether they should use OSS with consideration of each issue. In addition, the managers of open source projects need to manage their projects appropriately because OSS has a large impact on software around the world. This thesis focuses on the following three issues among many ones. We examine a method for OSS users and open source project managers to evaluate the stability of open source projects. 1. Selection evaluation and licensing: Methods for OSS users to make selections from the many OSS available situation, 2. Vulnerability support: Predicted fault fix priority for the reported OSS, 3. Maintenance and quality assurance: Prediction of appropriate OSS version upgrade timing, considering the development effort required after OSS upgrade by OSS users. In “1. Selection evaluation and licensing,” we attempt to derive the OSS-oriented EVM by applying the earned value management (EVM) to several open source projects. The EVM is one of the project management methodologies for measuring the project performance and progress. In order to derive the OSS-oriented EVM, we apply the stochastic models based on software reliability growth model (SRGM) considering the uncertainty for the development environment in open source projects. We also improve the method of deriving effort in open source projects. In case of applying the existing method of deriving effort in open source projects, it is not possible to derive some indices in the OSS-oriented EVM. Thus, we resolve this issue. The derived OSSoriented EVM helps OSS users and open source project managers to evaluate the stability of their current projects. It is an important to use the decision-making tool regarding their decisions and projects of OSS. From a different perspective, we also evaluate the stability of the project in terms of the speed of fault fixing by predicting the time transition of fixing the OSS faults reported in the future. 2. In “Vulnerability support”, in terms of open source project managers, we create metrics to detect faults with a high fix priority and predicted a long time for fixing. In addition, we try to improve the detection accuracy of the proposed metrics by learning not only the specific version but also the bug report data of the past version by using the random forest considering the characteristic similarities of bugs fix among different versions. This allows the project managers to identify the faults that should be prioritized for fixing when a large number of faults are reported, and facilitates project operations. In “3. Maintenance and quality assurance”, as an optimum maintenance problem, we predict the appropriate OSS version-up timing considering the maintenance effort required by OSS users after upgrading the OSS. It is dangerous in terms of the vulnerability to continue using the specified version of OSS ignoring the End of Life. Therefore, we should upgrade the version periodically. However, the maintenance cost increase with the version upgrade frequently. Then, we find the optimum maintenance time by minimizing the total expected software maintenance effort in terms of OSS users. In particular, we attempt to reflect the progress of open source projects by using the OSS-oriented EVM in deriving the optimal maintenance time. In conclusion, we found that there is the applicability as the stability evaluation of open source projects from three perspectives. Particularly, the OSS-oriented EVM discussed in “1. Selection evaluation and licensing” can contribute to the visualization of maintenance effort in open source projects. The proposed method will potentially contribute to the development of OSS in the future.

Hyperspectral (HS) imaging can capture the detailed spectral signature of each spatial location of a scene and leads to better understanding of different material characteristics than traditional imaging systems. However, existing HS sensors can only provide low spatial resolution images at a video rate in practice. Thus reconstructing high-resolution HS (HR-HS) image via fusing a low-resolution HS (LR-HS) image and a high-resolution RGB (HR-RGB) image with image processing and machine learning technique, called as hyperspectral image super resolution (HSI SR), has attracted a lot of attention. Existing methods for HSI SR are mainly categorized into two research directions: mathematical model based method and deep learning based method. Mathematical model based methods generally formulate the degradation procedure of the observed LR-HS and HR-RGB images with a mathematical model and employ an optimization strategy for solving. Due to the ill-posed essence of the fusion problem, most works leverage the hand-crafted prior to model the underlying structure of the latent HR-HS image, and pursue a more robust solution of the HR-HS image. Recently, deep learning-based approaches have evolved for HS image reconstruction, and current efforts mainly concentrated on designing more complicated and deeper network architectures to pursue better performance. Although impressive reconstruction results can be achieved compared with the mathematical model based methods, the existing deep learning methods have the following three limitations. 1) They are usually implemented in a fully supervised manner, and require a large-scale external dataset including the degraded observations: the LR-HS/HR-RGB images and their corresponding HR-HS ground-truth image, which are difficult to be collected especially in the HSI SR task. 2) They aim to learn a common model from training triplets, and are undoubtedly insufficient to model abundant image priors for various HR-HS images with rich contents, where the spatial structures and spectral characteristics have considerable difference. 3) They generally assume that the spatial and spectral degradation procedures for capturing the LR-HS and HR-RGB images are fixed and known, and then synthesize the training triplets to learn the reconstruction model, which would produce very poor recovering performance for the observations with different degradation procedures. To overcome the above limitations, our research focuses on proposing the unsupervised learning-based framework for HSI SR to learn the specific prior of an under-studying scene without any external dataset. To deal with the observed images captured under different degradation procedures, we further automatically learn the spatial blurring kernel and the camera spectral response function (CSF) related to the specific observations, and incorporate them with the above unsupervised framework to build a high-generalized blind unsupervised HSI SR paradigm. Moreover, Motivated by the fact that the cross-scale pattern recurrence in the natural images may frequently exist, we synthesize the pseudo training triplets from the degraded versions of the LR-HS and HR-RGB observations and themself, and conduct supervised and unsupervised internal learning to obtain a specific model for the HSI SR, dubbed as generalized internal learning. Overall, the main contributions of this dissertation are three-fold and summarized as follows: 1. A deep unsupervised fusion-learning framework for HSI SR is proposed. Inspired by the insights that the convolution neural networks themself possess large amounts of image low-level statistics (priors) and can more easy to generate the image with regular spatial structure and spectral pattern than noisy data, this study proposes an unsupervised framework to automatically generating the target HS image with the LR-HS and HR-RGB observations only without any external training database. Specifically, we explore two paradigms for the HS image generation: 1) learn the HR-HS target using a randomly sampled noise as the input of the generative network from data generation view; 2) reconstructing the target using the fused context of the LR-HS and HR-RGB observations as the input of the generative network from a self-supervised learning view. Both paradigms can automatically model the specific priors of the under-studying scene by optimizing the parameters of the generative network instead of the raw HR-HS target. Concretely, we employ an encoder-decoder architecture to configure our generative network, and generate the target HR-HS image from the noise or the fused context input. We assume that the spatial and spectral degradation procedures for the under-studying LR-HS and HR-RGB observation are known, and then can produce the approximated version of the observations by degrading the generated HR-HS image, which can intuitively used to obtain the reconstruction errors of the observation as the loss function for network training. Our unsupervised learning framework can not only model the specific prior of the under-studying scene to reconstruct a plausible HR-HS estimation without any external dataset but also be easy to be adapted to the observations captured under various imaging conditions, which can be naively realized by changing the degradation operations in our framework. 2. A novel blind learning method for unsupervised HSI SR is proposed. As described in the above deep unsupervised framework for HSI SR that the spatial and spectral degradation procedures are required to be known. However, different optical designs of the HS imaging devices and the RGB camera would cause various degradation processes such as the spatial blurring kernels for capturing LRHS images and the camera spectral response functions (CSF) in the RGB sensors, and it is difficult to get the detailed knowledge for general users. Moreover, the concrete computation in the degradation procedures would be further distorted under various imaging conditions. Then, in real applications, it is hard to have the known degradation knowledge for each under-studying scene. To handle the above issue, this study exploits a novel parallel blind unsupervised approach by automatically and jointly learning the degradation parameters and the generative network. Specifically, according to the unknown components, we propose three ways to solve different problems: 1) a spatial-blind method to automatically learn the spatial blurring kernel in the capture of the LR-HS observation with the known CSF of the RGB sensor; 2) a spectral-blind method to automatically learn the CSF transformation matrix in the capture of the HR-RGB observation with known burring kernel in the HS imaging device; 3) a complete-blind method to simultaneously learn both spatial blurring kernel and CSF matrix. Based on our previously proposed unsupervised framework, we particularly design the special convolution layers for parallelly realizing the spatial and spectral degradation procedures, where the layer parameters are treated as the weights of the blurring kernel and the CSF matrix for being automatically learned. The spatial degradation procedure is implemented by a depthwise convolution layer, where the kernels for different spectral channel are imposed as the same and the stride parameter is set as the expanding scale factor, while the spectral degradation procedure is achieved with a pointwise convolution layer with the output channel 3 to produce the approximated HR-RGB image. With the learnable implementation of the degradation procedure, we construct an end-toend framework to jointly learn the specific prior of the target HR-HS images and the degradation knowledge, and build a high-generalized HSI SR system. Moreover, the proposed framework can be unified for realizing different versions of blind HSI SR by fixing the parameters of the implemented convolution as the known blurring kernel or the CSF, and is highly adapted to arbitrary observation for HSI SR. 3. A generalized internal learning method for HSI SR is proposed. Motivated by the fact that natural images have strong internal data repetition and the crossscale internal recurrence, we further synthesize labeled training triplets using the LR-HS and HR-RGB observation only, and incorporate them with the un-labeled observation as the training data to conduct both supervised and unsupervised learning for constructing a more robust image-specific CNN model of the under-studying HR-HS data. Specifically, we downsample the observed LR-HS and HR-RGB image to their son versions, and produce the training triplets with the LR-HS/HR-RGB sons and the LR-HS observation, where the relation among them would be same as among the LR-HS/HR-RGB observations and the HR-HS target despite of the difference in resolutions. With the synthesized training samples, it is possible to train a image-specific CNN model to achieve the HR-HS target with the observation as input, dubbed as internal learning. However, the synthesized labeled training samples usually have small amounts especially for a large spatial expanding factor, and the further down-sampling on the LR-HS observation would bring severe spectral mixing of the surrounding pixels causing the deviation of the spectral mixing levels at the training phase and test phase. Therefore, these limitations possibly degrade the super-resolved performance with the naive internal learning. To mitigate the above limitations, we incorporate the naive internal learning with our selfsupervised learning method for unsupervised HSI SR, and present a generalized internal learning method to achieve more robust HR-HS image reconstruction.

To investigate whether dantrolene (DAN), cardiac ryanodine receptor (RyR2) stabilizer, improves impaired diastolic function in an early pressure-overloaded hypertrophied heart, pressure-overload hypertrophy was induced by transverse aortic constriction (TAC) in mice. Wild-type (WT) mice were divided into four groups: sham-operated mice (Sham), sham-operated mice treated with DAN (DAN+Sham), TAC mice (TAC), and TAC mice treated with DAN (DAN+TAC). The mice were then followed up for 2 weeks. Left ventricular (LV) hypertrophy was induced in TAC, but not DAN+TAC mice, 2 weeks after TAC. There were no differences in LV fractional shortening among the four groups. Catheter tip micromanometer showed that the time constant of LV pressure decay, an index of diastolic function, was significantly prolonged in TAC but not in DAN+TAC mice. Diastolic function was significantly impaired in TAC, but not in DAN+TAC mice as determined by cell shortening and Ca2+ transients. An increase in diastolic Ca2+ leakage and a decrease in calmodulin (CaM) binding affinity to RyR2 were observed in TAC mice, while diastolic Ca2+ leakage improved in DAN+TAC mice. Thus, DAN prevented the progression of hypertrophy and improved the impairment of LV relaxation by inhibiting diastolic Ca2+ leakage through RyR2 and the dissociation of CaM from RyR2.

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。

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。

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。

Chromatography is considered as a key operation in the downstream process (DSP) of biopharmaceuticals, including proteins. Therapeutic proteins such as monoclonal antibodies (mAbs) with high economic values in the global market require immediate innovation in the purification step to adapt to the increased throughput from upstream. Authorities have also initiated changes toward a more modernized pharmaceutical manufacturing platform which is agile and flexible without extensive oversight. Instead of the conventional batch operation and empirical models, the design and application of in silico modeling and simulation for integrated multi-column processes to improve their performance in capture chromatography steps have been explored in the dissertation. Due to the fact that mechanistic models can reveal adsorption and mass transfer behaviors better in the chromatography compared to statistical models, mechanistic frameworks were applied in the study. Ion-exchange and protein A chromatography, the main categories of therapeutic protein chromatography were examined. With an example of oligonucleotides, the mass transfer phenomenon of biomolecules in different types of ion-exchange resins was explored by mechanistic models. The results demonstrate the effectiveness of modeling approaches to understand the chromatography process of biopharmaceuticals. By focusing on the DSP of mAbs, multi-column continuous chromatography was examined with IgG samples. The study covered the repeating batch to 4-column settings in the continuous periodic counter-current (PCC) chromatography, with development in modeling and simulation tools for process quantification and evaluation. Process performances including productivity, capacity utilization, and buffer consumption were investigated by simulations with the aim to increase productivities and lower buffer consumptions, which are the main bottleneck in the current DSP. The critical operation parameter, breakthrough percent (BT%) for column switching in PCC processes, requires the information from binding capacity, mass transfer, and non-loading operations. To obtain the optimal BT% under synchronized conditions, numerical solvers developed from mechanistic models were employed. It was found that over 20% improvement in buffer consumption and resin utilization can be observed in PCC processes while the same productivity as batch operation is maintained. Furthermore, regressive relations were developed for predictions of process performances and BT% based on the findings from PCC simulations. With high coherence in R2 over 0.95, the linear regression function can act as an accelerated method in the PCC process design. Finally, a new strategy of linear flow-velocity gradient (LFG) in the loading step was explored as a supplement to increase process efficiency. The method controls the total column capacity and the loaded amount as functions of time. Based on the relationship between the dynamic binding capacity and residence time, the gradient time of LFG was obtained. The optimal flow velocities and time gradients were examined by scanning through the range of applicable residence times. A case study of the 4-column PCC process is presented. By integrating a linear decreasing flow gradient in the PCC loading operation, the productivity has 1.4 times enhancement along with a 13% reduction in the cost of resin per amount of processed mAbs compared to constant flows. Undoubtedly, the next generation of DSP platform technology is directed toward continuous and integrated systems. Regarding the advantages in process performances and regulation perspectives, continuous manufacturing can advance development and manufacturing while assuring the product quality. The evolution in modeling and simulation enables faster development of in silico process prediction and evaluation. With the support from models, process design and optimization in chromatography can rise to the challenge.

Phosphorus is an indispensable nutrient to sustain the daily life of all living things on Earth. However, the over-enrichment of the aquatic ecosystem with phosphorus leads to eutrophication, which is still a global environmental problem. More stringent regulations have been put in place for the limit of phosphorus discharge to address this problem and resulted in the removal of phosphorus removal becomes exceptionally crucial. Furthermore, phosphorus deposits are a non-renewable resource and forecasted to deplete until 2170, given the current usage and global population growth. Thus, the removal of phosphorus coupled with the recovery and reuse of phosphorus offer the best strategies to meet the future phosphorus demand. Accordingly, adsorption represents a fascinating separation technique for phosphate from water because of the possibility of phosphorus recovery. Moreover, this approach has many advantages, such as efficient, easy operating conditions, low sludge production, and the possibility of regenerating the adsorbent. Numerous attractive low-cost adsorbents have been studied for phosphate removal, one of which is layered double hydroxides (LDH). Unfortunately, a high phosphate adsorption capacity of LDH can generally be achieved by calcination, which increases the preparation cost of LDH. In this study, LDH is functionalized with amorphous zirconium (hydr)oxide to obtain enhanced adsorption capacity and eliminate the high-temperature requirement during the synthesis process. Although different treatment techniques have been developed to eliminate phosphorus contamination, including for wastewater treatment, treated water often fails to meet quality regulations. Amorphous zirconium (hydr)oxide/MgFe layered double hydroxides composites (am-Zr/MgFe-LDH) with different molar ratios (Zr/Fe = 1.5 2) were prepared in two-stage synthesis by the combination of coprecipitation and hydrothermal methods. The synthesis of the composite could eliminate the requirement of high-temperature calcination in the LDH for phosphate adsorption. Moreover, the phosphate adsorption ability of the composite was higher than that of the individual LDH and amorphous zirconium (hydr)oxide. The presence of amorphous zirconium (hydr)oxide increased the phosphate adsorption ability of composite at low pH. The adsorption capacity was increased by decreasing the pH and increasing the temperature (from 290 to 324 K). The bicarbonate (HCO3 ) was the most competitive anion for phosphate adsorption. The pseudo-secondorder model provided the best description of the kinetic adsorption data. Furthermore, the adsorbed phosphate was easily desorbed by 1 N and reused 2 N of NaOH solutions. The results suggest that the am-Zr/MgFe-LDH composite is a promising material for phosphate removal and recovery from wastewater. A Fixed-bed column has been considered an industrially feasible technique for phosphate removal from water. Besides the adsorption capacity, the effectiveness of an adsorbent is also determined by its reusability efficiency. In this study, phosphate removal by a synthesized am-Zr/MgFe-LDH in a fixed-bed column system was examined. The results showed that the increased bed height and phosphate concentration, and reduced flow rate, pH, and adsorbent particle size were found to increase the column adsorption capacity. The optimum adsorption capacity of 25.15 mg-P g^{-1} was obtained at pH 4. The coexistence of seawater ions had a positive effect on the phosphate adsorption capacity of the composite. Nearly complete phosphate desorption, with a desorption efficiency of 91.7%, could be effectively achieved by 0.1 N NaOH for an hour. Moreover, the initial adsorption capacity was maintained at approximately 83% even after eight adsorption-desorption cycles, indicating that the composite is economically feasible. The am-Zr/MgFe-LDH, with its high adsorption capacity and superior reusability, has the potential to be utilized as an adsorbent for phosphorus removal in practical wastewater treatment. The possible adsorption mechanisms of phosphate by am-Zr/MgFe-LDH were investigated via X-ray diffraction (XRD), Fourier transform infrared (FTIR), X-ray photoelectron spectroscopy (XPS), and pH at the point of zero charge (pHPZC) analyses. It was suggested that the high phosphate adsorption capacity of the composite involves three main adsorption mechanisms, which are the electrostatic attraction, inner-sphere complexation, and anion exchange, where the amorphous zirconium (hydr)oxide on the surface of the layered double hydroxides likely increased the number of active binding sites and surface area for adsorption. This study provides insights into the design of am-Zr/MgFe- LDH for phosphorus removal and recovery in a practical system.

The reduction of excessive discharge of phosphate into water bodies is a dominant theme to combat the critical eutrophication issue and requires the development of high-performance materials for effective phosphate treatment. In this study, rice straw was used as a raw material for the synthesis of biochar functionalized with layered double hydroxides (BC-LDHs) as efficacious phosphate adsorbents, and their successful synthesis was corroborated via characterization analysis. Experimental investigations, including pH, coexisting anion, reaction time, and initial phosphate concentration effects were systematically performed with selected BC-LDHs 6 and pure LDHs. An optimum pH of 3.0 was observed in both samples. Kinetic and isotherm studies indicated that phosphate adsorption on these samples was controlled by the pseudo-second-order model and the Freundlich model. Comparative kinetic tests also demonstrated that BC-LDHs 6 and pure LDHs reached the equilibrium within 24 h and 3 h, respectively. Nonetheless, the maximum adsorption capacity of the composite was 192 mg/g, which was higher than that of pure LDHs (166 mg/g). The coexistence of various anions negligibly affected the removal efficiency of the composite; however, fluoride was the most competitive anion for adsorption on pure LDHs. The adsorption mechanisms of the composite involved electrostatic interaction, inner-sphere complexation, pore diffusion, precipitation, and reconstruction. Furthermore, phosphate adsorbed on both materials could be easily recovered by 0.1 M NaOH solution owing to the displacement reaction between phosphate and hydroxyl ions. Additional evidence from reusability experiments exhibited that the composite could maintain its good adsorption performance even after three adsorption-desorption cycles. The transformation of BC-LDHs 6 after its usage in phosphate treatment (P-BC-LDHs 6) into a fertilizer was further explored by using seed germination and early growth assays of lettuce through a comparison with phosphate-loaded LDHs (P-LDHs). Lettuce seeds germinated in all P-BC-LDH 6 treatments showed undesirable growth characteristics compared with the controls, while total germination failure was observed under high concentrations of P-LDHs. In the latter experiments, the optimal application rates for plant growth were 2.5% for P-BC-LDHs 6 and 1.0% for P-LDHs. The considerably greater biomass development and length of lettuce were visible in samples delivered from P-BC-LDHs 6 compared to those from P-LDHs. The results obtained suggest that BC-LDHs 6 is a promising adsorbent for phosphate treatment and post-adsorption BC-LDHs 6 has the application potential to serve as a fertilizer for horticultural crop production.

ダイアモンド・ブラックファン貧血 (Diamond–Blackfan anemia : DBA) は, リボゾーム蛋白異常によって生じる赤芽球癆で, 新生児期の貧血および一部で身体奇形を合併する. 臨床的特徴が多様で, 原因となるリボゾーム蛋白遺伝子も多数存在するため, 新生児期にDBAと確定診断し, 適切な治療を行うことが困難である. 本研究では, 全エクソーム解析 (whole-exome sequencing; WES) を用いて最終診断した3組の母子例について報告する. 貧血の重症度や治療反応性は各母子間で異なり, 低身長, 翼状頸, 母指球形成不全などの特徴的な身体奇形を認めた症例は, 母1名のみだった. この母はRPL11 (exon 2, c.58_59del) のフレームシフト変異があり, 子は一過性の新生児貧血を認めたがリボゾーム蛋白遺伝子の変異はなかった. 他の2組の母子では, それぞれRPS19 (exon 4, c.185G>A) のミスセンス変異とRPS7のスプライシング変異 (exon 3, c.76-1G>T) を同定した. それぞれの変異と別に, 貧血を来し得る遺伝子変異はなかった. 本研究は, WESがヒトリボゾーム病の迅速かつ正確な診断を得るために有用であることを示唆した.

With the deterioration of bridges as social infrastructure, appropriate maintenance and life extension are required. However, aging degradation of individual bridges is not the same. Since traffic volume and bridge environment are different for each bridge, the degree of deterioration of the bridge is also different. Therefore, it is necessary to identify and eliminate the cause of individual deterioration and to take appropriate measures. This paper focuses on weathering steel bridge that the formation of dense rust is greatly influenced by the environment. The purpose of this study is to clarify the effect of anticorrosion by the environmental improvement which covers the whole steel girder with the metal sandwich panel for weathering steel bridge. Since the space in the girder covered with the metal sandwich panel cannot be expected to have the effect of washing by rainwater or drying and wetting by the flow of wind, the adoption of the metal sandwich panel for weatherproof steel bridge has not been judged until now. In this thesis, the corrosion behavior of weathering steel and the effectiveness of corrosion protection are shown by exposure test, and the corrosion protection effect of environmental improvement by metal sandwich panel is clarified. In addition, this study examines the economical advantages by the metal sandwich panel installation by calculating the life cycle cost. This paper consists of six chapters. Chapter 1 describes the background and purpose of this research. Chapter 2 summarizes previous studies on corrosion protection methods used in steel bridges. In Chapter 3, exposure tests were conducted inside and outside the metal sandwich panel and the following finding were obtained. 1) In the girder covered with the metal sandwich panel, fluctuation range of temperature and humidity is small throughout the year, and it does not follow the sudden weather change of outside of the panel. Since the difference between the temperature and dew point in the panel is large, the wet time in the panel is suppressed to 1/5 or less of the wet time of outside the panel. 2) At the structure which has thin floor slab and low height girder, the temperature rise in the panel may be unavoidable depending on season. However, since the humidity in the panel is low and the wet time is also greatly reduced, the anticorrosive effect can be expected in the steel bridge including the weathering steel bridge. 3) Amount of air born salt into the panel after the metal sandwich panel installation was not detected. In Chapter 4, a small test specimen was placed in the inside space of the steel girder, and the transition of corrosion, anticorrosion effect by environmental improvement and inner surface painting were examined. 1) The untreated steel and the uncoated steel in the ingrown rust region evaluated by the ion permeation resistance method are kept almost same condition after five years. 2) It is also conceivable that the initial salinity of the steel material subjected to the substrate adjustment by blasting exists even after the substrate adjustment, and that the adhering salinity penetrates into the inside of the steel plate at the time of rust formation. However, the increase rate of rust thickness due to aging is slow, and a method of installing a metal sandwich panel after blasting is also effective. 3) Although the effect of environmental isolation from outside the girder was confirmed, it became clear that it was difficult to completely suppress the progress of rust. Chapter 5 examined the economic effects of installing metal sandwich panels on new girders from the beginning and installing them on overbridges 50 years after the star of service. 1) If repainting is required even 1 time during the during the design service period of 100 years, the anticorrosion method by environmental improvement of the metal sandwich panel is economically superior. 2) Accumulating the cost of close visual inspection (for 50 years) of the overbridge that has been in service for 50 years increases the maintenance cost. Chapter 6 summarizes this research and describes future issues.

　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。

Cardiovascular diseases are the leading cause of mortality and disability worldwide. We have previously found that sphingosylphorsphorylcholine (SPC) is the key molecule leading to vasospasm. We have also identified the SPC/Src family protein tyrosine kinase Fyn/Rho-kinase (ROK) pathway as a novel signaling pathway for Ca^{2+}-sensitization of vascular smooth muscle (VSM) contraction. The present study aimed to investigate whether hesperetin can inhibit the SPC-induced contraction with little effect on 40 mM K^+-induced Ca^{2+}-dependent contraction and to elucidate the underlying mechanisms. Hesperetin significantly inhibited the SPC-induced contraction of porcine coronary artery smooth muscle strips with little effect on 40 mM K^+-induced contraction. Hesperetin blocked the SPC-induced translocation of Fyn and ROK from the cytosol to the membrane in human coronary artery smooth muscle cells (HCASMCs). SPC decreased the phosphorylation level of Fyn at Y531 in both VSMs and HCASMCs and increased the phosphorylation levels of Fyn at Y420, myosin phosphatase target subunit 1 (MYPT1) at T853 and myosin light chain (MLC) at S19 in both VSMs and HCASMCs, which were significantly suppressed by hesperetin. Our results indicate that hesperetin inhibits the SPC-induced contraction at least in part by suppressing the Fyn/ROK pathway, suggesting that hesperetin can be novel drug to prevent and treat vasospasm.

The hippocampal dentate gyrus has been identified to play a critical role in maintaining contextual memory in many mammalian species. To evaluate learning-induced synaptic plasticity of granule cells, we subjected male rats to an inhibitory avoidance (IA) task and prepared acute hippocampal slices. In the presence of 0.5 µM tetrodotoxin, we recorded miniature EPSCs in male rats experiencing four groups: untrained, IA-trained, unpaired, and walk-through. Compared with the untrained, IA-trained, unpaired, and walk-through groups, the unpaired group significantly enhanced mean mEPSC amplitudes, suggesting the experience-induced plasticity at AMPA receptor-mediated excitatory synapses. For inhibitory synapses, both unpaired and walk-through groups significantly decreased mean mIPSC amplitudes, showing the experience-induced reduction of postsynaptic GABA_A receptor-mediated currents. Unlike the plasticity at CA1 synapses, it was difficult to explain the learning-specific plasticity at the synapses. However, overall multivariate analysis using four variables of mE(I)PSC responses revealed experience-specific changes in the diversity, suggesting that the diversity of excitatory/inhibitory synapses onto granule cells differs among the past experience of animals include the learning. In comparison with CA1 pyramidal neurons, granule cells consistently showed greater amplitude and frequency of mE(I)PSCs. Fluctuation analysis further revealed that granule cells provide more postsynaptic AMPA receptor channels and greater single-channel current of GABA_A receptors of than CA1 pyramidal neurons. These findings show functional differences between two types of principal cells in the hippocampus.

前身に投与された治療薬が脳組織実質に到達するには、神経組織の血管により形成される血液脳関門を通過する必要がある。そのため、組織への損傷を最小限に抑えて血液脳関門を開くことができれば、難治性神経疾患の治療法開発に大きな進展をもたらすことが期待される。本研究では、血液脳関門を形成する血管内皮細胞に発現するBasiginに着目し、その内因性リガンドであるCyclophilin A (CypA) を用いて、血液脳関門機能を人為的に制御することを目的とした。マウス脳血管内皮細胞株を用いたin vivo解析により、CypAの投与がBasiginを介して血液脳関門機能を低下させること、それにより脳実質へ効率的に薬物を送達できることを示した。単層培養された血管内皮細胞において、CypAはタイト結合構成分子の一つであるClaudin-5を一過性かつ可逆的に細胞膜から消失させて、バリアー機能を低下させることを見出した。また、マウスへのCypAの単回静脈内投与では血液脳関門が一定期間開いた後、自発的に元の状態へ回復することが示され、そしてその限定された機関において、全身投与された水溶性薬物Doxorubicinが脳組織実質へ送達されることが明らかとなった。本研究の結果は、CypAの静脈内投与によって、脳実質への薬物送達を自在にコントロールできることを示しており、難治性神経疾患に対する治療法確立に向けた重要な成果であると考えられる。

【背景】近年、歯周炎や歯肉炎に関連する嫌気性グラム桿菌であるFusobacterium nucleatum (F. nucleatum) は大腸がんの発生や進行に関与することが報告されている。この菌の制御が大腸がんの予防につながる可能性があると考え、深紫外線発光ダイオード (DUV-LED) によるF. nucleatumの殺菌効果を検討した。【方法】DUV-LEDのF. nucleatumに対する殺菌効果を定性的、定量的に評価した。ピーク波長が265nmと280nmの2種類のDUV-LEDを使用した。F. nucleatumのDNAに対するダメージは、シクロブタンピリミジン二量体(CPD)とピリミジンピリミドン光生成物 (6-4PP) の生成で評価した。【結果】DUV-LEDでの265nmまたは280nmの波長を3分間照射したところ、コロニーの成長は観察されなかった。265nmのDUV-LED光照射下におけるF. nucleatumの生存率は10秒照射で0.0014%、20秒照射で0%に低下した。同様に、280nmのDUV-LED光照射では，10秒照射で0.00044%、20秒照射で0%に低下した。DUV-LEDから35mmの距離での放射照度は、265nmのLEDで0.265mW/cm^2、280nmのLEDでは0.415mW/cm^2であった。従って、致死量を示す放射エネルギーは265nm LEDは5.3mJ/cm^2、280nm LEDは8.3mJ/cm^2であった。265nmと280nmのDUV-LED光をF. nucleatumに照射した際のCPDと6-4PPの量はそれぞれ6.548ng/μg、1.333ng/μgであった。【結論】DUV-LED光は、F. nucleatumに対して、ピリミジン二量体を形成することにより殺菌効果を発揮した。

【背景】全層性治癒（Transmural healing healing; TH）は、クローン病の新たな治療標的として注目されているが、日本ではTHに関する臨床データはほとんどない。我々は、クローン病のモニタリング法として低被曝線量CTエンテログラフィ（CTE）を導入し、CTEによるTHの評価をレトロスペクティブに検討した。【方法】2009年 1月から2021年3月までに当院で低被曝線量CTEを施行したクローン病患者のうち、2週間以内に大腸内視鏡検査またはバルーン内視鏡検査を施行した122例を対象とした。放射線検査と内視鏡検査の結果は、それぞれ放射線科医と消化器内視鏡医が独立して検討した。CTEと内視鏡検査の診断の一致率を算出した。【結果】26名（21.3％）のクローン病患者がTHを達成し、カッパ係数は0.743と2人の放射線科医の間でかなりの一致が見られた。TH群と非TH群の比較では、クローン病活動指数（Crohn’s Disease Activity Index ; CDAI ）（P値 = 0.02)、内視鏡的治癒率（P値 ＜ 0.001）、血清アルブミン（P値 = 0.043）、血清C反応性蛋白（P値 = 0.018）に有意差が認められた。122名の患者のうち、69名（56.5％）はCTEの診断と内視鏡検査が一致し、22名（18.0％）はTHと内視鏡の両方の治癒を達成した。【結論】本研究は、日本における低被曝線量CTEによるクローン病のリアルワードデータを示すものである。本研究で用いたTHの基準はカッパ係数が高く、多くの施設で再現性を持って用いることができると考えられる。

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。

中等度の冠動脈狭窄病変において、血行再建治療を延期（defer）し薬物療法を行った場合、薬物療法中に心血管イベント発症抑制効果を評価する代用評価法があれば有用である。冠動脈イメージングでプラーク退縮と安定化を調べることで薬物治療効果を評価するのと同様に、定量的冠血流比（QFR）の経時的変化は、deferした中等度狭窄に対する薬物治療効果を評価する代用評価法として有用な可能性がある。本研究では、経皮的冠動脈インターベンション（PCI）後に未治療血管に冠動脈中等度残存狭窄を有し二次予防として薬物治療を受けた患者で、deferした中等度残存狭窄のQFRの経時的変化を調査した。山口大学医学部附属病院と萩市民病院でPCIを施行され、PCI（ベースライン[BL]）時に未治療血管に中程度狭窄を有し、6～18ヶ月後にフォローアップ（FU）の冠動脈造影が施行された患者を対象とした。52人の患者でBLとFUの両方で未治療血管の中程度狭窄のQFRの解析が可能であった。BL時にQFRの中央値は0.83（IQR, 0.69, 0.89）、FU時にQFRの中央値は0.80（IQR, 0.70, 0.86）であった。QFRの増加した患者（QFR増加群）は21人で、QFRの減少した患者（QFR減少群）は31人であった。経時的なQFR変化の中央値はQFR増加群で0.05（IQR, 0.03, 0.09）、QFR減少群で-0.05（IQR, -0.07, -0.03）であった。単変量および多変量解析でQFRの増加に影響する因子を解析したところ、FU時のLDLコレステロール値と相関を認めた（OR 0.95, 95% CI [0.91, 0.98], p=0.001）。QFRの経時的変化の評価は、deferした中程度狭窄を有する患者に対する薬物治療効果を判定する代用評価法として有用性が示唆された。

心臓突然死（sudden cardiac death: SCD）は心サルコイドーシス患者における主要な死亡原因であり、SCDの大部分は心室性不整脈が原因である。これまでのところ、心サルコイドーシス患者の致死的不整脈とSCDを予測するバイオマーカーは報告されていない。本研究では心サルコイドーシス患者における持続性心室頻拍（sustained ventricular tachycardia: sVT）およびSCDを予測する因子は何であるかを調査した。連続89症例において心サルコイドーシス患者の炎症活動性を反映する酸化的DNA損傷のマーカーである尿中8-hydroxy-2'-deoxyguanosine（尿中8-OHdG）、他のバイオマーカー、心機能の指標、腎機能を測定した。追跡期間中、15人の患者はsVT（N = 12）またはSCD（N= 3）を示した。COX比例ハザードモデルを用いた多変量解析では、尿中8-OHdG濃度および心室瘤（ventricular aneurysm: VA）の存在がsVT/SCDの独立した予測因子であることが示された。尿中8-OHdGおよびVAの存在は、心サルコイドーシス患者の初回のsVT/SCDの強力な予測因子であり、心臓イベントのリスクの層別化に有用である。さらに、これらはVT基質についてのさらなる情報を与えるものであることが示唆された。

背景と目的：同種造血幹細胞移植ではgraft-versus-leukemia (GVL) 効果と呼ばれる抗腫瘍免疫を適切に誘導することが成績向上の鍵となる。しかしGVL効果の増強は移植片対宿主病の重症化にも繋がるため、 同種免疫応答の指標となるバイオマーカーの開発が望まれる。我々は以前、移植処置の前と好中球生着時の血清可溶性インターロイキン-2受容体の比である「sIL-2R index」を定義し、骨髄移植における移植片対宿主病の発症予測マーカーとして有用であることを示した。しかし、骨髄移植とは異なる免疫特性を有する臍帯血移植ではsIL-2R indexも異なる挙動を示す可能性があり、臍帯血移植におけるsIL-2R indexの有用性を検討した。 対象と方法：当院で初回同種造血幹細胞移植として臍帯血を施行した31症例を対象とした。sIL-2R indexを算出し、患者背景や移植成績との関連を後方視的に解析した。 結果：移植後3年の再発率は、sIL-2R index 3.7以上の群で有意に低下した（12.8% vs 50.0%; p = 0.04）。それに伴い移植後3年の全生存率はsIL-2R index 3.7以上の群で有意に良好であった（79.8％ vs 20.0％; p < 0.01）。sIL-2R indexには移植後1日目から好中球生着日までの累積ステロイド投与量が影響しており、ステロイドの使用理由は生着前免疫反応に対する治療であった。なお骨髄移植とは異なり、臍帯血移植ではsIL-2R indexと急性移植片対宿主病の発症率との有意な関連性は認めなかった。 結語：sIL-2R indexは臍帯血移植後の予後予測マーカーになり得る。sIL-2R indexはGVL効果を反映する可能性があるが、更なる検証が必要である。

脳由来神経栄養因子Brain-derived neurotrophic factor (BDNF)は, 中枢神経内での恒常性や神経発達に重要な役割を果たしており, 神経変性疾患や神経免疫疾患の治療薬への応用が期待されている. しかし, BDNF自体は分子量が大きく, 末梢に投与したBDNFは血液脳関門Blood-brain barrier (BBB)を通過して中枢神経内に作用することができない. 一方で脂溶性化合物はBBBを通過しやすいとされている. そこで, BBBを超えてアストロサイトに作用し, アストロサイトからBDNF分泌を促進する脂溶性化合物の同定を試みた. 温度条件不死化ヒト脳血管内皮細胞 (EC), ペリサイト (PCT), アストロサイト (AST)のBBB in vitroモデルに20種類の脂溶性化合物を反応させ, 48時間後にBDNFの分泌量をELISAで測定した. 脂溶性化合物をEC/AST co-cultureに72時間反応させながら電気抵抗値を測定した. その結果, prostaglandin E2 receptor 4 agonist (EP4) とsphingosinesphingosine-1-phosphate receptor 5 agonist (S1P5)がEC, PCTの有無にかかわらずASTからのBDNFを有意に促進させ, 電気抵抗値の低下は伴わなかった. このことからEP4とS1P5はBBBへ影響を与えずにASTからのBDNF分泌を促進したと考えられた. S1P5は進行型多発性硬化症治療薬であるシポニモドの標的の1つである. シポニモドの神経保護作用はS1P5を介したASTからのBDNFが関与している可能性が考えられた. ASTでのEP4の機能は未だ不明な点が多いが, BDNF分泌を促進することで神経保護に関与する可能性がある. いずれの化合物も治療薬への発展が期待される.

術後膵液瘻は膵臓手術後の重篤な合併症である。これまで多くの予防法が検討されたが、充分な効果は得られておらず、発症率は減少していない。我々の研究室では長年、難治性皮膚潰瘍に対する細胞シート移植を研究し、有効な創傷治癒効果を報告してきた。細胞シート移植は他臓器の創傷治癒にも有効である可能性が予想され、膵液瘻に対する予防法になりうると着想した。本研究では動物の術後膵液瘻モデルを用いて、積層繊維芽シートの自家移植による膵液瘻の予防を検証した。 ラットを全身麻酔下に開腹し、膵管とその周囲の膵組織を切開してラット膵液瘻モデルを作製した。ラット尾より線維芽細胞を単離し、培養して積層繊維芽シートを作製した。自家積層線維芽細胞シートを膵管とその周囲の膵組織切開部に移植し、細胞シート移植による膵液瘻の予防効果を経時的な腹水及び血清中膵酵素値の測定、膵組織の免疫組織化学、定量的PCR法を用いて評価した。 ラット膵液瘻モデルでは術後に腹水中膵酵素値が上昇し、病理組織学的には広範囲の膵組織に炎症と壊死所見を認めた。膵液瘻の発症と膵組織の損傷が示唆された。積層線維芽細胞シートの自家移植により腹水中膵酵素の上昇と膵組織の炎症性変化は有意に抑制され、正常な構造を保つ膵組織が広範囲に温存された。対照群である細胞活性を持たないシートを移植した群と比較して、細胞シート移植群では切開部周辺に線維化と血管新生が惹起されていた。特に切開部付近の膵管はコラーゲン線維で充填、被覆されており、膵液の漏出を抑制する上で重要な機序であったと示唆された。これら線維化と血管新生を介して膵臓への障害が抑制されたと考えられた。 以上から、動物モデルにおいて積層線維芽細胞シートの自家移植は膵液瘻を充分に予防し、膵組織を保護することが示された。上記細胞シートの自家移植は術後膵液瘻を予防する有効な方法となり得ることが示唆された。

【方法】 Wister/STラットに左肺全摘を行うことで気管支断端モデルを作製した。口腔粘膜組織から線維芽細胞を単離し、24wellプレートに5.0×10^5個/wellを播種して72時間培養することで積層線維芽細胞シートを作製した。積層線維芽細胞シートの移植による気管支断端の補強効果を検討するため、術後7、14、28日目に標本を摘出し、細胞シート移植の有無による2群間での気管支断端の変化を肉眼的、組織学的、力学的に比較した。 また、細胞シートによる組織修復のメカニズムを検証するために、創傷治癒に関わる成長因子、サイトカインを細胞シート作製時の培養液上清を用いたEnzyme-linked immunosorbent assay (ELIZA)法でin vitroに測定した。更に、Green fluorescent protein (GFP)遺伝子を導入した線維芽細胞で作製した細胞シートを移植し、移植した細胞の残存性を検証した。 【結果】 作製した積層線維芽細胞シートは直径6mmであった。線維芽細胞が4-5層に積層し、コラーゲン線維を含む細胞外マトリックスを保持した状態で回収された。 非移植群では、術後7、14、28日目のいずれのタイミングでも、気管支断端の閉鎖に用いた縫合糸が露出していた。一方で、移植群の気管支断端は新生組織で広く被覆されていた。なお、細胞シートの移植の有無に関わらず、標本摘出までの期間に気管支断端瘻が生じた個体はなかった。摘出した標本を組織学的に観察すると、非移植群の気管支断端周囲にはごく僅かな結合組織が形成されたのみであったが、移植群では気管支断端周囲に多量の結合組織が形成されていた。また、移植群に生じた気管支断端周囲の結合組織が徐々に成熟すること（Azan染色）、その組織に多くの血管構造が含まれていること（抗CD31抗体による免疫染色）が観察された。新生組織を含む気管支壁の厚み、それに含まれる血管構造を定量化したところ、いずれのタイミングでも移植群の気管支壁が有意に厚く、より多くの血管構造を含んでいた。また、気管支断端の補強効果を力学的に検証するために、気管支断端の耐圧能を評価したところ、非移植群では全ての標本で気管支断端からエアリークが生じた。一方、移植群では300mmHgまでの加圧によってエアリークが生じた標本はなく、その耐圧値は移植群で有意に高かった。 細胞シート作製時の培養液上清を用いたELIZA法では、VEGF、Hepatocyte growth factor (HGF)、C-X-C motif chemokine ligand 1 (CXCL1)、Angiopoietine-2、Monocyte chemoattractant protein-1 (MCP-1)、Transforming growth factor beta 1 (TGF-β)が細胞シートから分泌されていることが示された。 GFP遺伝子導入積層線維芽細胞シートを移植したところ、術後3日目の時点ではGFP遺伝子導入線維芽細胞が気管支断端に残存していることが確認されたが、7、14 日目にはGFP遺伝子が発現した細胞は確認されなかった。 【考察】 気管支断端は僅かに新生された結合組織でのみ覆われることが報告されており、本研究においても、非移植群の気管支断端の周囲には僅かな結合組織が形成されたのみであった。一方、積層線維芽細胞シートを移植することにより、気管支断端の周囲に多くの血管構造を含んだ結合組織が形成され、力学的にも気管支断端の強度が増していることが示された。気管支断端瘻は、術後1週間から3ヶ月、特に10日目前後に多く発生するとされていることから、細胞シートの移植後7日目の時点で、気管支壁がより厚く、血管構造に富み、優れた耐圧性を獲得していることは、気管支断端瘻の予防にとって十分に効果的な可能性がある。 積層線維芽細胞シートにより気管支断端が補強される機序を解明するため、細胞シートが分泌する成長因子やサイトカインの測定、GFP遺伝子導入細胞シートを用いた検証を行ったが、移植したGFP遺伝子導入細胞は術後3日目には残存していたが、7日目の時点では確認できなかった。このことは、移植した線維芽細胞が生存、増殖して結合組織を形成するのではなく、細胞シートの移植が宿主の組織治癒を促進させている可能性を示している。VEGF、HGFやTGF-βなどの成長因子、サイトカインが細胞シートから分泌され、これらに宿主に働きかけることで、気管支断端周囲での組織形成や血管新生が促進されたと推察される。 積層線維芽細胞シートの移植後7日目の時点で、気管支断端周囲に血管新生を伴った結合組織が形成され、力学的な補強効果があることが示された。積層線維芽細胞シートの移植は、局所の血流低下による組織治癒遅延が原因とされる気管支断端瘻に対して有効な予防法となる可能性がある。 【結語】 積層線維芽細胞シートの移植による気管支断端の補強効果が示された。本法は、気管支断端瘻の有効な予防法となる可能性がある。

低汗性外胚葉形成不全症 (hypohidrotic ectodermal dysplasia: HED) は、低汗症、乏歯症乏毛症を特徴とする遺伝性疾患である。本疾患の家系のほとんどがX連鎖劣性 (X-linked recessive: XR) の遺伝形式を示すが、稀に常染色体優性 (autosomal dominant: AD) または常染色体劣性 (autosomal recessive: AR) の遺伝形式を示す家系も存在する。XRのHEDはEDA遺伝子の変異で発症し、AD/ARのHEDはEDARまたはEDARADD遺伝子のいずれかの変異で発症する。現在までに、EDAおよびEDARの変異に関してはHEDの発症機序が明らかにされてきたが、EDARADDの変異がどのようにHEDを引き起こすかについての情報は乏しかった。 本研究では、過去にHEDの家系に同定されたEDARADD遺伝子変異のうち、ADの遺伝形式を示すp.D120Y、p.L122R、p.D123Nと、ARの遺伝形式を示すp.E152Kに着目し、培養細胞レベルでさまざまな解析を行った。EDARADDは、シグナル伝達の主要分子であるTRAF6と結合し、最終的に下流のNF-κBを活性化させるが、ADの変異型EDARADDはNF-κBの活性化能を著しく喪失していた。一方で、ARの変異型EDARADDの同活性化能の低下は軽度だった。また、解析した全ての変異型EDARADDは、EDARおよび野生型EDARADDとの親和性を維持していたが、ADの変異型EDARADDは、EDARと野生型EDARADDとの相互作用をdominant negative効果によって阻害することを明らかにした。さらに、ADの変異型EDARADDはTRAF6との結合能を完全に失い、ARの変異EDARADDも野生型に比べてTRAF6との結合能が低下することを示した。 HEDにおける臨床型と遺伝子型の相関関係は未だ明らかではないが、本研究で得られた知見は、EDARADD遺伝子変異とHEDの発症メカニズムの関連性の一端を解明したといえる。

遺伝性血管性浮腫（hereditary angioedema：以下HAE）は、全身の様々な部位に突発性、一過性の浮腫を生じる稀な常染色体優性遺伝性疾患である。HAEは、C1 inhibitor（C1INH）をコードするserpin family G member 1（SERPING1）遺伝子の変異により生じるHAEI型およびII型、SERPING1遺伝子以外の遺伝子異常を認めるHAEIII型（HAE with normal C1INH）の3つに分類される。これまでに、SERPING1遺伝子においては多数の病的変異が同定されているが、各変異によるHAEの発症機構については未だ十分に解明されていないのが現状である。 本研究では、以前に我々が報告したHAE I型の患者に同定されたSERPING1遺伝子のミスセンス変異c.449C>T（p.S150F）に関して、詳細な発現・機能解析をin vitroレベルで行った。まず、p.S150F変異型C1INHは細胞内では安定して発現するが、細胞外には全く分泌されないことが示された。次に、変異型C1INHが野生型C1INHの分泌を強力に阻害することが明らかになった。さらなる解析で、野生型C1INHは変異型C1INHとの相互作用によって細胞質内に留め置かれてしまうだけでなく、分解も誘導されることが示唆された。本研究によって、p.S150F変異型C1INHは野生型C1INHに対してdominant-negative効果を発揮することが証明され、それが本遺伝子変異によるHAE I型の主要な発症メカニズムと考えられた。

前立腺癌細胞と骨芽細胞の相互作用は、前立腺癌骨転移の発生に不可欠である。近年、新規のアンドロゲン受容体標的薬（ARAT）が、転移性去勢未治療前立腺癌（mCNPC）や非転移性去勢抵抗性前立腺癌（nmCRPC）に対して承認されているが、これらの薬剤と骨微小環境や腫瘍との関係を調べることは極めて重要である。今回、我々は去勢抵抗性前立腺癌（CRPC）の骨微小環境を模倣した新規のin vitro 3次元微小環境モデルを確立し、アンドロゲン受容体標的薬の薬剤感受性と、アビラテロンとデュタステリドの併用療法の有効性を評価した。キトサンナノ繊維で表面をコートした細胞培養基材を用いて、GFPを導入したC4-2細胞（CRPC細胞株）とRFPを導入したヒト骨髄由来間葉系幹細胞から分化した骨芽細胞を共培養し、去勢抵抗性前立腺癌の骨微小環境を模倣した。骨芽細胞とC4-2細胞の増殖はCell3 iMager duos（SCREEN）を用いて生細胞のまま解析し、骨芽細胞を維持したまま、最大30日間C4-2細胞の持続的な増殖を観察できた。また、骨芽細胞と共培養したC4-2細胞では、TGF-βの発現が増加し、上皮間葉転換（EMT）が促進されたことから、アンドロゲン受容体標的薬に対する抵抗性が示された。このモデルを用いて、各薬剤のIC50と、アビラテロンとデュタステリドの併用効果を評価した。アビラテロンとデュタステリドの併用療法は、それぞれの単剤投与と比較して、C4-2細胞の増殖を相乗的に抑制し、これは、ARアゴニストである3-keto-5α-abirateroneの減少によるものと考えられた。我々の新規骨微小環境モデルは、前立腺癌の薬剤感受性の評価に有用であり、今後このモデルは、前立腺癌の微小な骨転移から臨床的な骨転移に至るまでの未知のメカニズムの解析に役立つ可能性がある。