Ehime University Institutional Repository

Ehime University (愛媛大学)博士(医学)thesi

能美, 幸信

NOUMI, Yukinobu

（第７号様式） 
 
学位論文審査結果の要旨 
 
   氏     名 能美 幸信 
 
 
 
 
 
   審 査 委 員 
 
 
 
 
 
 
 
 
 
                                      
 
                                      
 
                                      
 
                                      
 
                                      
 
 
      論  文  名 「日本人において、n-3 多価不飽和脂肪酸摂取量と血中レジスチンと 
          の負の関連は SNP-420 G/G 型において最も強い：東温ゲノムスタディ」 
 
学位論文の要旨 
【背景】 
 レジスチンは、マウスにおいて脂肪細胞から分泌され、インスリン抵抗性を惹起するサイトカ
インである。ヒトでは、主に単球やマクロファージに発現し、インスリン抵抗性及び炎症や虚血
性心疾患などの動脈硬化症とも関連する。発表者らは、レジスチン遺伝子の転写調節領域に存在
する一塩基多型(single nucleotide polymorphism: SNP)-420 が G/G 型の場合に、2 型糖尿病の
リスクが高まることを見出している。機能的には、SNP-420 が G の場合に、転写因子 Spl/3 が特
異的に結合し、レジスチンの転写活性、単球 mRNA、及び血中濃度を上昇させた。一般住民にお
いて、血中レジスチンは SNP-420 C/C 型＜C/G 型＜G/G 型の順に高かった。一方、各群、特に G/
G 型において、血中レジスチン値のレンジは幅広く、SNP-420 以外の遺伝子や環境因子の影響が
示唆された。 
 そこで、日本人の一般住民において、環境因子としての栄養摂取量と SNP-420 が、血中レジス
チンに及ぼす影響を解析した。 
【方法】 
 愛媛県東温市における観察型疫学研究である東温ゲノムスタディの参加者のうち、遺伝子解析
の同意が得られた 1,981 名(30-79 歳、平均 58 歳、男性 714 名、女性 1,267 名)を対象とした。
血中レジスチンは ELISA 法で測定した。SNP-420 は TaqMan 法でタイピングした。各栄養摂取量
は食事摂取頻度調査票（food frequency questionnaire: FFQ）により算出した。総エネルギー
摂取量は、残差法を用いて調整した。 
【結果】 
 各栄養摂取量を 4 分位で 4 群に分け、血中レジスチンとの関係を解析した。血中レジスチンは、
総エネルギー、炭水化物、脂質、タンパク質摂取量とは関連しなかったが、魚、食物繊維、緑黄
色野菜の摂取量と負に、肉の摂取量と正に関連した。これらの関連は、性、年齢、BMI、総エネ
ルギー摂取量で多変量調整後は有意ではなかったが、血中レジスチンは魚の摂取量と負に、肉の
摂取量と正に関連する傾向がみられた。 
 魚には n-3 多価不飽和脂肪酸、肉には飽和脂肪酸、一価不飽和脂肪酸、n-6 多価不飽和脂肪酸
が多く含まれていることから、次に血中レジスチンと脂肪酸摂取量との関係を解析した。血中レ
ジスチンは、性、年齢、BMI、総エネルギー摂取量で調整後、n-3 多価不飽和脂肪酸摂取量と負
に関連した。この関連は、多変量調整後（性、年齢、BMI、総摂取エネルギー、CRP、インスリン
抵抗性指数（HOMA-IR）、HDL コレステロール、推算糸球体濾過量（eGFR））も有意であった。
血中レジスチンと飽和脂肪酸、一価不飽和脂肪酸、多価不飽和脂肪酸、n-6 多価不飽和脂肪酸と
の関連は認められなかった。 
 最後に、対象者を SNP-420 の C/C 型、C/G 型、G/G 型の 3 群に分け、血中レジスチンと n-3 多
価不飽和脂肪酸摂取量との関係を検討した。血中レジスチンと n-3 多価不飽和脂肪酸摂取量との
負の関連は、G/G 型において最も強く、次いで C/G 型、C/C 型の順であり、有意な交互作用を認
めた。これらの関連は、多変量調整後も変わらなかった。 
【結論】 
 日本人において、n-3 多価不飽和脂肪酸摂取量と血中レジスチンとは負に関連し、この関連は
レジスチン SNP-420 G/G 型において最も強かった。 
 
審査会のまとめ 
本論文の公開審査は平成 29 年 3 月 14 日に開催された。申請者は研究内容について英語をまじ
えて明確に発表し、以下の内容を含む多くの質疑に対して的確に応答した。  
 1）レジスチンに対する人種、性、年齢、さらに女性では閉経の影響について、2）研究対象の
バイアス（年齢や性の分布）について、3）対象における基礎疾患の影響について、4）レジスチ
ンの一塩基多型は日本人の代表としてとらえてよいか、5）食事摂取頻度調査票（FFQ）の信頼性
について、6）FFQ から求めた n-3 多価不飽和脂肪酸摂取量の信頼性について、7）総エネルギー
摂取量を調整した残差法について、8）n-3 多価不飽和脂肪酸摂取量を 4 分位にして検討した理
由について、9）血中レジスチンと血中の EPA や DHA との関係について、10）n-3 多価不飽和
脂肪酸摂取量と血中 EPA や DHA との関係について、11）飽和脂肪酸や n-6 多価不飽和脂肪酸
摂取量のレジスチンへの影響について、12）レジスチンとその他のマーカー（HOMA-IR や高感度
CRP）との関係について、13）今後の研究の方向性や介入研究（食生活の改善や薬剤投与）につ
いてなどの質問がなされた。 
 審査委員は、申請者が本論文関連領域に対して学位授与に値する十分な見識と能力を有するこ
とを全員一致で確認し、本論文が学位授与に値すると判定した。 
 
 


English

An inverse association between serum resistin levels and n-3 polyunsaturated fatty acids intake was strongest in the SNP-420 G/G genotype in the Japanese cohort: The Toon Genome Study

大橋, 淳史

隅田, 学

中原, 真也

林, 秀則

Japanese

科学教育事業への参加が理工系学生の学習意識に及ぼす影響に関する調査

樫木, 暢子

中野, 広輔

苅田, 知則

八木, 良広

薬師神, 裕子

堀内, 史枝

吉松, 靖文

インクルーシブ教育システム下において、児童生徒の健康問題へ適切に対応できる教員養成カリキュラムの開発 : 医教連携による学際的講義・実習の充実を目指して

兼平, 裕子

兼平, 麻渚生

英国ヒンクリーポイントC 原発支援国家補助に対する欧州委員会承認 : 委員会決定およびEU 一般裁判所決定[T-382/15]に示された比例性原則と市場の失敗

金子, 省子

養育行動を描いた絵本について : 家庭科保育学習教材としての検討

TSUYUGUCHI, Kenji

学校におけるソーシャルキャピタルと主観的幸福感 : 「つながり」は子どもと保護者を幸せにできるのか?

高橋, 幸造

福田, 隆

バレーボール競技における中学生高校生用のスカウティング方法の開発

Ehime University (愛媛大学)博士(工学)thesi

HERIANSYAH, PUTRA

1 
 
（第３号様式）(Form No. 3) 
学 位 論 文 要 旨 
Dissertation Summary 
 
氏   名 (Name): Heriansyah Putra 
論 文 名： Optimization of Enzyme-Mediated Calcite Precipitation for Soil 
Improvement Technique 
 
 
 
 
 
 
 
 
 
 
 
 
 
Enzyme-mediated calcite precipitation (EMCP) as a soil-improvement technique has 
been studied as an alternative method for improving the engineering properties of soil. The 
increase in unconfined compressive strength ranging from 400 kPa to 1.6 MPa depending 
upon the amount precipitated calcite are achieved and the permeability of the improved 
samples is reduced by more than one order of magnitude (Neupane et al., 2015, 2013, 
Yasuhara et al., 2012, 2011). This technique employs enzyme of urease to dissociate urea into 
ammonium and carbonate ions. The produced carbonate ions are precipitated as calcite 
crystals in the presence of calcium ions. The reaction of the urea hydrolysis and the calcite 
formation are shown in Equations (1)-(3).  
 
CO(NH2)2+2H2O
urease
→   2NH4
++CO3
2-
 (1) 
CaCl2→Ca
2++2Cl
-
 (2) 
Ca2++CO3
2-
→CaCO3↓(precipitated) (3) 
 
An enzyme-reagent mixed solution (i.e., purified urease and CaCl2-urea), which 
produces the precipitated calcite after the chemical reaction, is injected into the soil. The 
precipitated calcite may provide bridges between the grains of sand, restricting their 
movement, and hence, improving the stiffness and the strength of the soil (Yasuhara et al., 
2011). A schematic of the whole process listed above and grouting mechanism expected are 
illustrated in Figure 1. 
(Dissertation Title) 
 
2 
 
 
Figure 1: Schematic of calcite precipitation process on EMCP technique 
In this study, the precipitation of calcite is evaluated directly in the transparent 
polypropylene (PP) tubes. Various combinations of reagent and enzyme are mixed thoroughly 
and allowed to react. As seen in the schematic in Figure 2, reagent and urease are prepared 
separately. Various combinations of urease are thoroughly mixed in distilled water for 2 mins 
and filtered using a filter paper (pore size of 11 m) to remove the undissolved particle of 
urease. The purified urease and the reagent solution (i.e., urea and CaCl2) are mixed 
thoroughly in the PP tubes, in a total solution volume of 30mL.  
 
Figure 2: Schematic of precipitation test 
Test tubes are kept in a box without shaking at a room temperature of 20
o
C. After the 
curing time, the grout solution is filtered through filter paper (pore size of 11 m). The 
particles deposited on the filter paper and the particles remaining in the tubes are dried at 
60
o
C for 24 hrs, and then the total mass of the precipitated minerals is evaluated by 
combining the precipitated minerals deposited in the test tubes with the materials remaining 
3 
 
on the filter paper. Two identical tests are performed for each condition to check the 
reproducibility. The mass of the precipitated minerals is shown as the precipitation ratio, 
namely, the ratio of the actual mass of the precipitated minerals to the theoretical mass of the 
maximum precipitation of CaCO3. The theoretical mass of CaCO3 (g) is evaluated as CVM, 
where C and V represent the concentration of the solution in moles per liter and the volume of 
the solution in liters, respectively, and M is the molar mass of CaCO3 of 100.087 g/mol. The 
actual mass is the mass of the precipitated materials in grams evaluated from the tests.  
In order to evaluate applicability of EMCP as a soil-improvement technique, the 
evolutions in the strength and permeability of the treated sample are examined through 
unconfined compressive strength (UCS) tests and permeability test, respectively. Soil 
specimens are prepared in PVC cylinders and treated with concentration-controlled solutions 
composed of reagent-enzyme. Polyvinyl chloride (PVC) cylinders (5 cm in diameter and 10 
cm in height) are used to prepare the sand samples. The fixed volume of the solution is 
injected into the prepared sand specimens. The injected volume is controlled by the number of 
pore volumes (PV), one PV being ~75 mL. Firstly, dry silica sand is poured into the PVC 
cylinders to obtain a relative density of 50%. Secondly, one PV of the grout solution is poured 
into the PVC cylinders from the top. After the curing time, the treated specimens are removed 
from the PVC cylinders. The surface of the treated samples is flattened before the UCS tests 
are conducted. The schematic of UCS test is illustrated in Figure 3.  
 
 
Figure 3: Schematic of UCS tests 
The permeability tests are conducted to examine the effect of the precipitated minerals 
on the hydraulic conductivity of the treated sand. An acrylic cylinder (5 cm in diameter and 
10 cm in height) is used to conduct the tests. The procedure of the sample preparation for the 
permeability tests is equivalent to that for the UCS test. It is the same for the sample 
preparation, density of sand, the grouting solution and number of pore volumes (PV). The 
schematic for the permeability test is showed in Figure 4.  
4 
 
 
Figure 4: Schematic of permeability tests 
The acid leaching method is used to evaluate the amount of precipitated calcite between 
the sand samples (Neupane et al., 2013; Yasuhara et al., 2012, 2011). In this process, the 
treated sand is washed with distilled water to dissolve the salt materials and then dried in an 
oven at a temperature of 100
o
C for 24 hrs. The dried sand is weighed and washed with 0.1 
mol/L of HCl several times until air bubbles no longer appeared. Filter paper (pore size of 11 
m) is used to minimize the lost mass of sand during the ishing process. The sand is dried 
again, and the final weight is taken. The dry weight lost during the acid leaching is evaluated 
and assumed to be the weight of the precipitated materials. The reactions taking place are 
expressed by Equation (4). 
OH+CO+CaCl→2HCl+CaCO 2223
 
(4) 
 
Finally, by comparing the relation between the numbers of injections, the mineral mass, and 
the UCS within the treated specimens obtained in this study, the effects on the mechanical 
properties are explicitly investigated.  
A series of experiments is conducted in this study. First series of experiment is 
conducted to evaluate the applicability of magnesium chloride as the substituted material in 
EMCP technique. Magnesium chloride was newly added to the grouting solutions composed 
urea, urease and CaCl2 to control the reaction rate and to enhance precipitation amount. The 
second series of experiments is conducted to examine the applicability of magnesium chloride 
as delaying agent to control the precipitation process and improve the homogenous 
distribution of the precipitated minerals within soil. Sand specimens are prepared in 1-m PVC 
cylinders and are treated with the obtained grouting materials. Thus, the distribution of the 
precipitated minerals within the sand samples is evaluated. The third series of experiments is 
conducted to evaluate the effectivity of magnesium sulfate as a substitute material in EMCP 
technique. Magnesium sulfate is added to the grouting solutions composed urea, urease and 
CaCl2 to increase the precipitated amount and promote the formation of aragonite and gypsum. 
The fourth series of experiments is performed to evaluate the applicability an ammonium 
removal method of ion exchange using natural zeolite to reduce the concentration of 
ammonium ion in EMCP technique. The natural zeolite of mordenite is added to the prepared 
grouting solutions, and the evolution of concentration of ammonium ion is evaluated. The 
effects of the utilization of natural zeolite during the grouting preparation on the EMCP 
parameters such as amount, pH, mineralogical substance of the precipitated minerals, and the 
improvement in the strength of treated soil are also evaluated.  
Whole series of the experiment has the specific and valuable finding in order to 
5 
 
optimize the application EMCP as a soil improvement technique. The findings in this study 
are listed below. 
1. Application of magnesium chloride as the substituted material in the EMCP technique is 
indeed found to increase the precipitated amount and delay the reaction rate of the 
precipitation process.  
2. The presence of magnesium chloride in the grouting material is found to be able to delay 
the reaction rate and hence, improves the uniformity distribution of the precipitated 
materials within 1-m column sample.  
3. Utilization of magnesium sulfate in the grouting materials is found to significantly 
increase the efficiency of precipitated ratio in a high concentration of reagent (1.0 mol/L) 
and promotes the formation of aragonite and gypsum in addition to calcite. The 
production of the high amount of precipitated materials causes a sufficient strength, which 
can be obtained by a limited of number of injections. 
4. Application of ammonia removal method of ion exchange using zeolite in EMCP 
technique is found as a potential method to be a solution for the environmental issue in the 
application of EMCP as a soil improvement technique. A significant reducing in the 
ammonium ion is obtained without compromising in the efficacy of EMCP as a soil 
improvement technique. 


Optimization of Enzyme-Mediated Calcite Precipitation for Soil Improvement Technique

寺内, 浩

四国遍路縁起と西国巡礼縁起

松野尾, 裕

賀川豊彦と宮澤賢治 : 新しい人づくり・新しい郷づくり

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