Ehime University Institutional Repository

Ehime University (愛媛大学)博士(農学)thesi

PETER, Chivero

（様式５）(Style5) 
学位論文全文に代わる要約 
Extended Summary in Lieu of Dissertation 
 
 
氏名： 
Name 
           CHIVERO PETER  
 
学位論文題目： 
Title of Dissertation 
Physico-Chemical Properties of Soy Soluble Polysaccharide (SSPS) 
Oil-in-Water Emulsions: Effect of SSPS Type, Dispersed Phase Type 
and Addition of Stabilizing Polysaccharides 
(大豆水溶性多糖類 (SSPS) によるO/Wエマルションの物理化学的特
性 ーSSPSの種類、分散相の種類及び安定剤として添加した SSPS以
外の多糖類の影響ー) 
 
 
学位論文要約： 
Dissertation Summary 
Food and food product colloids and emulsions are considered as the most complicated systems whose 
microstructure and factors affecting stability are difficult to study. Natural foods are composed of many 
constituents including fats, oils, proteins, carbohydrates and small molecular weight molecules such as vitamins 
and enzymes, existing in complex mixtures and molecular structures. Each component plays a significant role in 
determining the long term stability and the overall food quality. Among the natural food components, amphiphilic 
compounds are of major interest in this thesis. An amphiphilic compound is described as a chemical compound 
possessing both hydrophilic (water-loving or polar) and lipophilic (fat-loving) properties. These molecules can be 
named emulsifiers, stabilizers, or dispersing agents since they possess surface-active characteristics. Many 
processed food systems contain emulsifiers. Food emulsions are therefore a very important sector of the food 
industry. Recently, more and more consumers have become more conscious about their health condition, giving a 
lot of pressure on the food manufacturers. Consumption of natural foods has been encouraged from all corners of 
the globe, resulting in an increased number of consumers who check the food label to see the ingredients. 
Synthetic food emulsifiers are henceforth becoming less popular in the food industry. The search for new viable 
natural emulsifiers is therefore an area of current interest to many food scientists and producers.   
In this regard, soy soluble polysaccharide (SSPS), extracted from the by-product of soy protein isolation 
(okara in Japanese), is a newly found natural emulsifier that has been proven to be an excellent emulsifier with 
comparable characteristics to other established emulsifiers. Hence, it is the aim of this thesis to fully characterize 
the physico-chemical characteristics of emulsions produced by different types of SSPS. Successful 
characterization of SSPS stabilized emulsions may lead to improved awareness of the existence of SSPS and its 
capabilities.   
The effect of SSPS type (i.e. SSPS-L; SSPS-M; and SSPS-H differentiated by their respective extraction 
conditions) and the type of oil (i.e. Perilla seed oil - PSO; palm kernel oil - PKO; and n-Hexadecane) was 
examined through analysis of the differences in average droplet diameter, droplet size distribution (DSD) and 
emulsion rheological properties. The three different types of SSPS where characterized in terms of their 
respective sugar and protein compositions and the values are as reported in Table 1 below. Minute differences in 
the extraction conditions resulted in the differences in the sugar and protein composition of the resulting SSPS 
sample. From this information, it was expected that the emulsification behavior of different types of SSPS can be 
different and the physico-chemical properties of their formed emulsions may differ as well. Given such a 
background, experiments were setup to reveal any differences if at all they existed.  
 
   
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Table 1: Monosaccharide compositions of different types of soybean polysaccharide (SSPS).      
Type of SSPS  Sugar composition (mol %) Protein 
(dry %) Rha Fuc Ara Gal Xyl Glc GalA 
SSPS-La 4.3 1.5 15.3 48.3 1.5 1.6 27.5 8.2 
SSPS-Ma 4.1 2.4 20.1 47.2 1.2 1.1 23.9 5.9 
SSPS-Hb 2.5 1.1 21.0 43.7 6.4 5.4 19.9 5.1 
Rhamnose (Rha), Fucose (Fuc), Arabinose (Ara), Galactose (Gal), Xylose (Xyl), Glucose (Glc), Galacturonic acid (GalA). aNakamura et al., 
2004; bNakamura et al., 2012. 
To begin with, the interfacial tension of the three different types of SSPS was evaluated and results are 
shown in Fig. 1. Oil/water interfacial tension strongly depended on SSPS type. SSPS-L was the most capable in 
reducing interfacial tension against PSO, PKO and hexadecane followed by SSPS-M and lastly SSPS-H. 
 
Data are reported as means from three independent replications (n = 3) for each sample. Means which have different letter superscripts are 
significantly different (p < 0.05). 
Figure 1: The interfacial tension between three types of oil (PKO; PSO; & hexadecane) and 
water or 1 %wt. SSPS solution (SSPS-L; -M; or -H) measured at 25 0C.  
Irrespective of oil type, at 20 % oil content, SSPS-L and M were better emulsifiers for achieving small 
droplets probably due to their high protein content as shown in Table 1. Generally, the emulsifying power of 
SSPS depended on the composition of the polysaccharide and the type of lipid being emulsified. Irrespective of 
lipid type, emulsions droplet size (EDS) of no less than 0.7 µm could be achieved with SSPS-L and -M as sole 
emulsifiers at considerably low concentrations compared to most surface active polysaccharides. Higher 
homogenization pressures could not achieve smaller EDS, but rather produced bigger EDS, especially with 
SSPS-H, due to “over-processing”. Both SSPS (type L and M) solutions and their respective emulsions 
regardless of oil type exhibit Newtonian flow behavior while SSPS-H solution and the respective emulsions 
show shear-thinning behavior. Therefore, products that do not focus on particularly small EDS but rather 
designed for high viscosity and shear-thinning characteristics should employ SSPS-H as an emulsifier at either 
conventional or higher homogenization pressures. Emulsion viscosity was influenced by both SSPS and oil types 
with SSPS-H and hexadecane producing high viscosity emulsions. Due to high Laplace pressure of hexadecane, 
resulting emulsions had high viscosity and large EDS compared to emulsion systems of PKO and PSO. 
Obtaining a stable oil-in-water (O/W) emulsion remains to be a great challenge for the food industry and 
it is here that the non-adsorbing hydrocolloids find their role. Various researchers have reported the use of 
（様式５）(Style5) 
non-adsorbing hydrocolloids as stabilizers in food emulsions so as to improve creaming stability. Substantial 
increase in the aqueous phase viscosity is the primary key to arrest emulsion droplet movement, thereby 
improving creaming stability. With this idea in mind, the effect of incorporating xanthan gum (XG) or guar gum 
(GG) in SSPS stabilized O/W emulsions was studied. The emulsions contained 6 %wt. of SSPS, 20 %wt. Perilla 
Seed Oil (PSO), an omega-3 vegetable oil, and variable amounts of XG or GG ranging from 0.03 to 0.3 %wt. 
The presence of minute amounts of XG or GG in fresh emulsions significantly decreased the emulsion droplet 
size (EDS) although such low concentrations did not provide enough continuous phase viscosity to arrest 
creaming. Emulsion creaming stability, droplet polydispersion, and droplet sizes were improved by the presence 
of polysaccharide dispersions. The high continuous phase viscosity in XG or GG incorporated emulsions was 
responsible for improved homogenization efficiency and inhibition of emulsion droplet movement resulting in 
promoted stability. Emulsion microstructure indicated the presence of flocculation even at high concentrations of 
XG or GG caused by a depletion mechanism. All emulsions with XG or GG exhibited pseudoplastic behavior 
while the control emulsions showed an almost Newtonian behavior. Emulsion droplet polydispersion generally 
decreased with increase in the continuous phase viscosity indicating the importance of continuous phase viscosity 
in the dissipation of shear energy throughout the emulsion during homogenization. Therefore, manipulation of 
the emulsion continuous phase viscosity can be used to produce stable emulsions of different droplet sizes for 
different applications.  
Application of SSPS and other hydrocolloid emulsifiers in real food products was attempted. The aim of 
this study was to assess the possibility of employing hydrocolloid emulsifiers as sole emulsifiers in combination 
with XG as a thickener and stabilizer to formulate mayonnaise-like O/W emulsions. Moreover, different 
rheological models were tested to find an appropriate model that can be used to correlate the shear rate and shear 
stress data. Hydrocolloid emulsifiers, SSPS, gum arabic (GA), and octenyl succinate starch (OSA-S), were 
investigated for their ability to produce O/W mayonnaise-like emulsions. XG was used as a stabilizer and 
thickener. All the three types of hydrocolloid emulsifiers were capable of emulsifying huge amounts of oil 
although contents above 60 %wt. could not be completely emulsified. Some floating oil could be observed on top 
of the emulsion samples immediately after preparation. Accelerated emulsion stability depended on the amount 
of dispersed phase with higher dispersed phase contents conferring more stability due to a strong network of 
droplets formed in the system. The formation of Pickering emulsions was confirmed in OSA-S stabilized 
emulsions. SSPS and GA stabilized mayonnaise-like emulsion systems satisfied the Power Law rheological 
model while OSA-S stabilized mayonnaise-like emulsions satisfied the Herschel-Bulkley model and showed an 
apparent shear thinning behavior similar to the traditional mayonnaise. The results obtained in this study open 
new avenues into the replacement of egg yolk in production of mayonnaise. 
The results obtained in this thesis may contribute to the knowledge of the physico-chemical properties of 
food emulsions produced by different types of SSPS. This information might be used as a reference by food 
producers who may find interest in adopting SSPS as a legitimate emulsifier for formulating various food 
systems like ice creams, yoghurts and mayonnaise among others. 
 
 
 
 
 
 
(注) 要約の文量は，学位論文の文量の約１０分の１として下さい。図表や写真を含めても構いま
せん。(Note)  The Summary should be about 10％ of the entire dissertation and may include 
illustrations 


English

Physico-Chemical Properties of Soy Soluble Polysaccharide (SSPS) Oil-in-Water Emulsions : Effect of SSPS Type, Dispersed Phase Type and Addition of Stabilizing Polysaccharides

愛媛大学Ehime University博士(理学)thesi

土佐, 靖彦

TOSA, Yasuhiko

Development of neuronal circuits in the reptilian and amphibian forebrain: with reference to the vertebrate brain diversification

Ehime University (愛媛大学)博士(学術)thesi

Siriporn, Lunprom

（第３号様式）(Form No. 3) 
学  位  論  文  要  旨  
Dissertation Abstract 
 
 
氏名：  
Name 
Siriporn Lunprom 
 
 
学位論文題目：  
Title of Dissertation 
Character ization of vacuolar amino acid transporters from 
Fusarium oxysporum  and Schizosaccharomyces pombe 
 
(フザリウム菌および分裂酵母における液胞アミノ酸トラン
スポーターに関する研究 )  
 
 
学位論文要旨：  
Dissertation Abstract 
 
 
 
In Saccharomyces cerevisiae , vacuoles are the largest organelles that function as 
digestive compartment and also serve as a major storage compartments for various amino 
acids. They contain about 70-90% of the basic amino acids in whole cells, whereas around 
90% of acidic amino acids are found in the cytosol. This difference in the distributions of 
amino acids suggests the presence of specific transport systems that operate across the 
vacuolar membrane. So far, several genes for vacuolar amino acid transporters have been 
identified and characterized in S. cerevisiae using reverse genetics and measurement of 
transport activities by isolated vacuolar membrane vesicles. These include Avt3p ( YKL146w,  
ScAvt3p) and Avt4p (YNL101w, ScAvt4p), which are involved in the extrusion of neutral 
amino acids, and both neutral and basic amino acids, respectively, from the vacuolar lumen 
into the cytosol. These genes must play important biological roles, amino acids homeostasis 
or recycling of amino acids . Thus the aim of this study was to identify and characterize 
vacuolar amino acid transporters from the organisms other than S. cerevisiae .    
By searching genome databases, interesting proteins homologous to ScAvt3p were 
found. One is in Schizosaccharomyces pombe (SPAC3H1.09c , SpAvt3p) and the other one is 
in Fusarium oxysporum (FOXG_11334 , FoAvt3p). To understand the function of these genes,  
SpAVT3 and FoAVT3  were cloned, heterologously expressed in S. cerevisiae , and 
characterized. The results showed tha t the GFP-SpAvt3p and also GFP-FoAvt3p localized to 
the vacuolar membrane in S. cerevisiae , as GFP-ScAvt3p and GFP-ScAvt4p. The analysis of 
amino acid content of the vacuolar fraction indicated that the amounts of neutral amino 
acids in vacuoles were greatly decreased by overproduction of SpAvt3p and FoAvt3p, and 
（第３号様式）(Form No. 3) 
interestingly, basic amino acids were also decreased by SpAvt3p overproduction, which was 
similar to ScAvt4p overproduction. The transport assay of amino acid using vacuolar  
membrane vesicles isola ted from S. cerevisiae heterologously expressing SpAVT3 
demonstrated that alanine and tyrosine were exported from vesicles in an ATP -dependent 
manner. In addition, uptake activities of arginine was strongly inhibited in the vesicles 
isolated from S. cerevisiae  cells overexpressing SpAVT3 , suggesting that SpAvt3p was also 
involved in the export of basic amino acids, not only the neutral ones. Furthermore,  
GFP-fusion of SpAvt3p overproduced in S. pombe cells reduced various neutral and basic 
amino acids in vacuoles. Together, these results suggest that SpAvt3p is a vacuolar  
transporter involving in the export of amino acids from S. pombe vacuoles. Similarly, the 
ATP-dependent export activities of FoAvt3p  for proline, alanine,  threonine and valine were 
detected using isolated vacuole membrane vesicles from S. cerevisiae  cells heterologously 
expressing FoAVT3 . The analysis of the amino acid contents of the vacuolar fraction and 
amino acid transport activities revealed that FoAvt3p function s as a vacuolar amino acid 
transporter, exporting mainly neutral amino acids.  
Another vacuolar membrane protein in S. cerevisiae , namely Atg22p (ScAtg22p), has 
been reported to be involved in autophagy, a degradation process which is induced under  
nutrient starvation condition t o supply amino acids for protein synthesis. In atg22∆ cells,  
the degradation of autophagic bodies, inner membrane-bound structures of autophagosomes, 
which are released into vacuoles, is impaired. It has been suggested that ScAtg22p is 
involved in the release of vacuolar amino acids generated by autophagy from vacuole to the 
cytosol. Although ScAtg22p belongs to a transporter family, it is still not clear whether it  
transports amino acids from the vacuole to the cytosol. Two ScAtg22p homologues,  
FOXG_00164 and FOXG_09714, which are encoded in F. oxysporum genome, were 
characterized. Subcellular localization analysis showed that only GFP-fusion of 
FOXG_00164 localized to the vacuolar membrane like ScAtg22p dose in S. cerevisiae . Thus,  
only FOXG_00164 was further studied and was named “FoAtg22p”. To investigate  whether  
FoATG22  is involved in autophagy pathway, two experiments were carried out, i.e.  
observing the accumulation of the autophagic bodies and analysis of Ape1 maturation, 
which occurs in a manner dependent on the degradation of autophagic bodies. The r esults 
showed that the number of autophagic bodies decreased when FoATG22 was expressed in 
S. cerevisiae atg22Δ mutant cells. Moreover, under nutrient starvation condition, precursor  
Ape1 was processed to the mature form in FoATG22-expressing strain. From these, it can be 
concluded that  FoATG22 complements the atg22Δ phenotype of S. cerevisiae . FoAtg22p is 
the first homologue of ScAtg22p, which is functional in autophagy process.  


Characterization of vacuolar amino acid transporters from Fusarium oxysporum and Schizosaccharomyces pombe

Ponyadira, Kabwadza-Corner

（様式５）(Style5) 
学位論文全文に代わる要約 
Extended Summary in Lieu of Dissertation 
 
 
氏名： 
Name 
 
Ponyadira Kabwadza-Corner 
学位論文題目： 
Title of Dissertation 
 
ADSORPTION MECHANISM AND SELECTIVITY OF HEAVY METALS 
AND PESTICIDES ON ZEOLITES AND MONTMORILLONITE 
（重金属及び殺虫剤のゼオライト及びモンモリロナイトへの吸着選択性
と吸着機構） 
 
 
学位論文要約： 
Dissertation Summary 
 The adsorption mechanism and selectivity of heavy metals and an organophosphate 
pesticide on zeolites and montmorillonite was studied with the objective of assessing the capacity of 
zeolites and montmorillonite to remove heavy metals (Cu, Pb and Cd) and pesticides (Diazinon). The 
selectivity sequence of the removal and the factors affecting the removal capacity were studied in detail. 
The study further analyzed the mechanism of pollutant removal by the aforementioned adsorbents. 
Various types of zeolites, with variations in their cation exchange capacities (CEC), pore structures, 
Si/Al ratio and surface area were used in the study. Zeolite A4, faujasite X, faujasite Y, mordenite, 
Na-P1, clinoptilolite and montmorillonite were used for the heavy metal adsorption experiments. For 
the diazinon adsorption experiments, iron-modified montmorillonite was used. 
The adsorption of Cu onto zeolites and montmorillonite as a reference was conducted at an 
initial Cu concentration range of 0-0.60 mM in the presence of 100 mM NH4NO3 at an initial pH of 5. 
The zeolites used were A4, Faujasite X, Faujasite Y, mordenite, Na-P1 and clinoptilolite. The samples 
were analyzed before commencement of the adsorption experiments: CEC measurement, Si/Al ratio 
determination, and XRD analysis. Table 1 is an illustration of the CEC and Si/Al ratio of the samples. 
Zeolite A4 was the sample with the highest CEC of 6150 mmol kg-1 followed by the rest of the 
samples.   
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Table 1: CEC and Si/Al ratio of the samples 
Sample CEC (mmol kg-1) Si/Al ratio 
A4 6150 1.00 
Faujasite X 5560 1.24 
Faujasite Y 3470 2.66 
Na-P1 4500 1.67 
Modernite 2457 4.88 
Montmorillonite 1200 - 
Clinoptilolite 2112 5.04 
 
The samples for the experiments were then saturated with Na in order to obtain samples that 
had uniform exchangeable cations. Langmuir and Freundlich models were used in analyzing the 
equilibrium data and a selectivity sequence derived from the Langmuir calculation was A4 > faujasite 
X > modernite > Na-P1 ≈ montmorillonite ≈ faujasite Y > clinoptilolite.  Zeolites A4 and faujasite X 
had high adsorptive capacities of 1429 mmol kg-1 and 909 mmol kg-1, respectively. Zeolite A4 has the 
highest CEC among all the samples (6150 mmol kg-1), and the adsorption capacity of Cu was largely 
influenced by the CEC of the samples.  
The adsorption mechanism was based on the exchange of Cu from solution with mostly Na 
which was the main exchangeable cation available. Figure 1 shows the adsorption isotherms of copper 
on the studied zeolite samples. The adsorption isotherm for A4 was very steep in the lower 
concentration range compared to the other samples. This resulted in A4 having the highest Langmuir K 
constant compared to all other samples. The steep nature of the isotherm further indicated that Cu was 
more strongly attached to zeolite A4 than the rest of the samples, indicating that A4 would be used 
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efficiently for the adsorption of Cu in nature at low concentrations. This was followed by faujasite X 
and the rest of the samples. 
 
 
Figure 1: Copper adsorption isotherms on zeolites A4, faujasite X, modernite, Na-P1, montmorillonite, 
faujasite Y and clinoptilolite. 
  Following the Cu adsorption experiments, Pb adsorption experiments were conducted. The 
adsorption of Pb on zeolites A4, faujasite X, faujasite Y and mordenite was studied at various initial 
pH with the purpose of assessing the pH dependence of Pb adsorption. The samples were also 
saturated with Na as a uniform exchangeable cation. The adsorption was conducted using 0-0.6 mM 
PbNO3 in the presence of 100 mM NH4NO3 and pH adjustment done using HNO3. The coexisting 
NH4NO3 served as a representative of other cations available in nature. The study was conducted at 
initial solution pH ranging from 3-5. Adsorption results were analyzed using Langmuir isotherm 
analysis.  
（様式５）(Style5) 
Figure 2 shows the Lead adsorption isotherms on the studied samples. Adsorption was noted 
to be dependent on pH with increasing adsorption as pH increased from 3-5 for zeolites A4, faujasite X 
and faujasite Y. It was observed that adsorption was highest at an initial pH of 5 among the samples 
except mordenite whose adsorption was almost constant across this pH range. The uptake capacity of 
metal ions and the adsorption mechanism involved are known to be dependent on the pH of the 
solution, which affects the degree of ionization, surface charge of the adsorbent and the speciation of 
the adsorbate (Hasan, 2008 and Stefanova, 2000). As the pH increased, more negatively charged 
surfaces became available, therefore leading to a greater metal uptake.  
 
 
 
 
 
 
 
 
 
 
Figures 2 : Lead adsorption isotherms on (a)A4, (b)Faujasite X ,(c) Faujasite Y and (d) Mordenite 
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It is conceivable that at low pH values, there were more H3O+ ions in the solution, and 
competition existed between the positively charged hydrogen ions and metal ions for the available 
adsorption sites on the zeolite surface. As the pH increased, more of the positively charged metal ions 
in solution were adsorbed on the negative surface and thus the percentage removal of the metal ions 
increased. The adsorptive capacities were 2500, 2000, 588 and 179 mmol kg-1 for A4, faujasite X, 
faujasite Y and mordenite, respectively. 
Figure 3 is the plot of the amount adsorbed and equilibrium pH at initial concentration of 600 
µmol L-1. Zeolite A4 and faujasite X have steep isotherms which are spread over a narrow range of pH 
compared to faujasite Y and modernite whose isotherm is spread over a wider pH range. The figure 
highlighted the high dependence on pH, of the adsorption of Pb within this pH region. In the pH range 
 
Figure 3: pH dependence of the samples for Pb adsorption at initial Pb concentration of 600 µmol L-1 
and pH of 5. 
of 2–6, the sorption percentage increases gradually with increasing pH. Adsorption is mainly 
dominated by ion exchange as H+ ions are competitive to Pb ions for ion exchange sites. The figure 
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further highlights the fact that the adsorption of Pb is strongly affected by the increasing concentration 
of proton (H+), in other words, decreasing pH. 
In the subsequent experiments, the adsorption of Cd onto zeolites A4, faujasite X, faujasite Y 
and mordenite was conducted at an initial Cd concentration range of 0-0.60 mM in the presence of 100 
mM NH4NO3 at initial pH of 5. The samples for the experiments were initially saturated with Na in 
order to obtain samples that had uniform exchangeable cations. Similar to the experiments mentioned 
above, the selectivity sequence was A4 > faujasite X > faujasite Y > modernite. Cation exchange was 
also the main factor that influenced adsorption. Figure 4 indicates the adsorption isotherms of Cd on 
the studies zeolite samples. 
 
Figure 4: Adsorption isotherms of Cd on Zeolites A4, Faujasite X,Y ad Mordenite at initial pH of 5. 
Lastly, the adsorption of Diazinon, and organophosphate pesticide was conducted using 
montmorillonite as an adsorbent. Montmorillonite modified with iron was used. Two different types of 
iron-modified montmorillonite, each having different contents of iron and synthesized with different 
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pH and levels of Fe hydrolysis were used. One was denoted “Fe-modified” and the other denoted as 
“FeOH- modified”. The color of the samples changed from greyish green to light-reddish brown after 
the modification. X-ray diffraction and physical observations were used for characterization of the 
samples. The d-spacing of the samples was greater than 15Å, indicating the formation of iron 
hydroxides in the interlayer space of montmorillonite. The amount of adsorption was calculated from 
the difference between the initial and final concentration of diazinon. The adsorption data were 
analyzed using the Langmuir adsorption isotherms. Figure 5 indicates the adsorption isotherms of 
diazinon on the studied samples.  
 
Figure 5: Adsorption isotherms of Diazinon on Fe modified, FeOH modified and Ca 
montmorillonite 
The amounts of diazinon adsorbed were 58.8 and 54.1 mmol kg-1 for Fe-modified and 
FeOH-modified respectively. The steep rise in their adsorption isotherms indicated the possibility of 
adsorption for low levels of diazinon in polluted water. 
（様式５）(Style5) 
FeOH- modified sample 
 
 
 
 
Figure 6:  Mechanism for diazinon adsorption on FeOH-modifies sample 
Fe -Modified Sample 
 
 
 
 
                                                         Montmorillonite sheet          FeOH                     
                                                         Fe3+/Fe2+                             Diazinon                                                         
Figure 7: Mechanism for diazinon adsorption on Fe-modified sample 
Figures 6 and 7 are the mechanisms of diazinon adsorption by the samples. The degree of 
polymerization had a significant contribution to the adsorptive properties of the samples. Figures 6 and 
7 are illustrations of the mechanism for diazinon adsorption. The Fe-modified sample, which was 
prepared at a lower pH than the FeOH-modified sample was less polymerized and had a comparatively 
（様式５）(Style5) 
larger available room within its structure for diazinon adsorption. On the contrary, the FeOH-modified 
sample whose preparation pH was slightly higher, had a higher degree of polymerization. This implied 
that the available room for adsorption of diazinon was less hence lower adsorption. In other studies, 
FeOH-montmorillonite prepared at OH/Fe= 2 (similar to current study), practically all original 
exchangeable cations in the montmorillonite sample were replaced by ferric ions with a majority of the 
total present outside the interlayer space (Grygar, 2007). This was due to the large size of the 
polymerized Fe species which could into easily penetrate into the interlayer space. This is unlike the 
Fe-modified sample whose Fe species could easily penetrate the interlayer space. 
The results of this study can be used in designing or planning for the clean-up of polluted water 
using adsorption techniques. An important attribute of these findings was that the samples studied were 
shown to have the capacity of removing even a very low concentration of the studied pollutants, a 
property which is hardly achievable by most adsorbents. The results of this study are also important is 
assisting decision makers in choosing the most efficient, cost effective and environmentally friendly 
method of removing Cu, Pb, Cd and diazinon from polluted environments. These results can as well 
apply to other heavy metals and some organophosphate pesticides. 
Key words: Heavy metals; Pesticides; Cu adsorption; Pb adsorption, Cd adsorption, Selectivity; Cation 
exchange Capacity, Langmuir isotherm analysis; Freundlich isotherm analysis. 
References 
• Hasan, S., Ghosh, T.K., Viswanath, D.S., Boddu, V.M., (2008). Dispersion of Chitosan on 
Perlite for Enhancement of Copper(II) Adsorption Capacity. Journal of Hazardous Materials, 
(152), 826–837. 
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• Stefanova, R.Y., (2000). Sorption of Metal Ions from Aqueous Solutions by Thermally 
Activated Electroplating Sludge, Journal of Environmental Science and Health. Part A, (35). 
593–607. 
• Grygar,T., Hradil,D., Bezdicka,P., Dousova,P., Capek,L., Schneeweiss,O.,(2007). 
Fe(III)-Modified Montmorillonite and Bentonite: Synthesis, chemical and UV-Vis spectral 
Characterisation, Arsenic sorption and catalysis of oxidative dehydrogenation of propane, 
Clays and clay minerals, 55(2), 165-176 
 
 
 
 
 
 
 
(注) 要約の文量は，学位論文の文量の約１０分の１として下さい。図表や写真を含めても構いません。
(Note)  The Summary should be about 10％ of the entire dissertation and may include illustrations 


ADSORPTION MECHANISM AND SELECTIVITY OF HEAVY METALS AND PESTICIDES ON ZEOLITES AND MONTMORILLONITE

Ehime University (愛媛大学)博士(医学)doctoral thesi

岩本, 和樹

IWAMOTO, Kazuki

（第７号様式） 
 
学位論文審査結果の要旨 
 
   氏     名    岩 本 和 樹 
 
 
 
 
 
   審 査 委 員 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
      論  文  名：リボヌクレオチドリダクターゼ M2 は口腔扁平上皮癌治療における有
力な治療標的分子である 
   審査結果の要旨（2,000 字以内） 
口腔癌のみならず様々な癌腫において癌関連遺伝子の異常が癌細胞の増殖能や遊走能、浸潤能
といった悪性形質を支持していることが知られている。また頭頸部扁平上皮癌において epiderm
al growth factor receptor (EGFR) の高発現が予後不良因子である報告がある。局所進行症例
あるいは再発・転移症例に EGFR を治療標的とした Cetuximab (Cmab) を用いた化学放射線併用
療法あるいは白金製剤との併用化学療法により局所制御率および全生存率の改善が報告されてい
る。しかしながら、現在口腔扁平上皮癌患者に適応のある分子標的薬は Cmab のみであり、今後
新たな治療標的分子の探索が急務となっている。岩本氏は、ヒト正常表皮角化細胞とヒト口腔扁
平上皮癌細胞株 9 種を用いて遺伝子発現マイクロアレイ解析を行い、発現亢進する癌関連遺伝
子として Ribonucleotide Reductase M2 (RRM2) を同定した。そこで、本研究では RRM2 が、口
腔扁平上皮癌の治療標的分子として有用であるかどうか検討を行った。まず、ヒト口腔扁平上皮
癌細胞４株とヒト正常口腔粘膜由来初代培養細胞、ヒト口腔扁平上皮癌腫瘍組織と隣接正常口腔
粘膜組織における RRM2 タンパク質の発現を比較解析した。その結果、ヒト口腔扁平上皮癌細胞
とヒト口腔扁平上皮癌腫瘍組織において RRM2 の高発現を認めた。続いて、RRM2 に対する特異
的な合成 small interfering RNA (siRRM2) と RRM2 を標的分子とする代謝拮抗薬である Gemci
tabine (GEM) を用いて RRM2 の機能阻害を行い、ヒト口腔扁平上皮癌細胞４株およびヒト口腔
扁平上皮癌由来初代培養細胞の細胞増殖における影響を評価した。その結果、いずれの細胞にお
いても、siRRM2 導入群はコントロール群と比較して RRM2 タンパク質の著明な発現低下を認め、
54.8% 以上の細胞増殖抑制効果を認めた。また、GEM で処理すると濃度依存的に細胞増殖抑制効
果を認め、1000 nM では 83.0% 以上の増殖抑制を認めた。さらに、口腔扁平上皮癌患者由来腫
瘍組織 25 検体を用いて GEM および口腔扁平上皮癌の治療に一般的に用いられている抗癌薬で
ある Cisplatine (CDDP)、5-fluorouracil (5-FU)、Docetaxel (DOC) の抗腫瘍活性を Collagen
 gel droplet embedded culture drug sensitivity test (CD-DST) 法による抗癌剤感受性試験
で評価した。抗癌剤感受性試験の結果、GEM は DOC と同程度かつ、CDDP と 5-FU より高い抗腫
瘍活性を認めた。しかしながら、GEM 感受性と RRM2 mRNA の発現量の間に相関関係は認めず、
口腔扁平上皮癌において GEM は RRM2 の発現量に関係なく高い感受性を示した。以上の結果か
ら、RRM2 は口腔扁平上皮癌の増殖を支持しており、GEM に代表される RRM2 を標的とする分子
標的治療が口腔癌の新規治療戦略となる可能性が示唆された。 
  
発表後審査員より、 
① 解析対照群として、正常口腔扁平上皮細胞を用いていないのはなぜか、 
② GEMの正常細胞に対する抗増殖効果は癌細胞に比べてどうか、 
③ RRM2遺伝子の発現レベルとGEMの効果に相関性が無いのはなぜか、 
④ 頭頚部癌に対するGEMの臨床試験は現在どこまで進んでいるのか、 
⑤ GEMの放射線治療に対する増感作用が口腔扁平上皮癌細胞にも期待できるのか、 
⑥ GEMのRRM1、RRM2に対して阻害活性を持つが、口腔扁平上皮癌細胞においてはどちらの効
果が優位なのか、 
⑦ 膵癌や肺癌では、RRM2遺伝子の発現レベルとGEMの効果が相反するとの報告があるが、こ
れについての意見は、 
⑧ RRM2に対するsiRNAのデザインはどの配列を標的として行われたのか、 
⑨ 患者由来組織のprimary cultureで線維芽細胞の増殖を抑えるためにはどのような方法を
とったのか、 
⑩ RRM2に対するsiRNAがウエスタンブロットで検出できる二本のバンドのうち、高分子量の
バンドのみを抑制したのはなぜか、 
 
 などの活発な質疑がなされた。岩本氏はこれらの質問に対して明確に返答し、審査委員会は
一致して本論文が学位授与に十分値すると判断した。 
 
 尚、岩本和樹氏の学位論文公開審査会は、平成 27 年 2 月 10 日 14 時より第 １ ゼミナ
ール室で行われた。 
 
 
 
 


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