UNIVERSITI PUTRA MALAYSIA

ANTIMICROBIAL, IMMUNOMODULATORY AND TUMOR

CELLS- SELECTIVE CYTOTOXIC ACTIVITIES OF BRASSICA

OLERACEA L. AND VIGNA RADIATA L.

RAND R. HAFIDH

FSTM 2011 27

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ANTIMICROBIAL, IMMUNOMODULATORY AND TUMOR CELLS-

SELECTIVE CYTOTOXIC ACTIVITIES OF BRASSICA OLERACEA L. AND

VIGNA RADIATA L.

By

RAND R. HAFIDH

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfillment of the Requirements for the Degree of Doctor of Philosophy

May 2011

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DEDICATION

I dedicate the results of this work and my degree to the people

who without them i wouldn’t be able to reach what i have

reached, my beloved husband, my caring mother, my father, my

grandmother Adilaa and my aunt Lamiaa.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement of the degree of doctor of philosophy

ANTIMICROBIAL, IMMUNOMODULATORY AND TUMOR CELLS-

SELECTIVE CYTOTOXIC ACTIVITIES OF BRASSICA OLERACEA L. AND

VIGNA RADIATA L.

By

RAND R. HAFIDH

May 2011

Chair: Professor Dr. Fatimah Abu Bakar, PhD

Faculty: Food Science and Technology

The world today faces a great challenge in the emergence of drug-resistant microbes

and dangerous cancers against most of the commonly known antimicrobial and

anticancer drugs, respectively. Screening for effective, new, and cheap remedies

from natural resources has become the goal of many scientists all around the world.

The plants’ methanol extracts used in this study were red cabbage (RC) and mung

bean sprout (MBS) which both showed remarkable antiviral activities against herpes

simplex virus type 1 (HSV-1) and respiratory syncytial virus (RSV). The mode of

action of RC extract was shown to be prophylactic against RSV (the concentration

that inhibit 50% of virus cytopathic effect (IC50) = 13.9 mg/ml and selectivity index

(SI) = 17.67). Against HSV-1, RC extract showed a remarkable viricidal activity

(IC50 = 11.51 mg/ml and SI = 34.52) with less prophylactic activity (IC50 = 32.97

mg/ml and SI = 11.71). MBS extract had viricidal effect (IC50 = 15.62 mg/ml and SI

= 14.18), (IC50 = 7.62 mg/ml and SI = 18.23) and to a lesser extent prophylactic

(IC50 = 17.23 mg/ml and SI = 12.82), (IC50 = 12.72 mg/ml and SI = 10.9) against

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RSV and HSV-1 viruses, respectively. One of the good explaination on the effective

prophylactic activities of MBS and RC extracts was their excuisite ability to induce

production of high levels of IFNβ, TNFα, IL-1, and IL-6 cytokines (300-900%

higher levels when compared to untreated cells; P<0.0001) in virally infected cells.

These antiviral cytokines proved to play critical and synergistic role in the antiviral

resistance of human cells. For the cytotoxic effects of the tested extracts, RC extract

showed selective cytotoxic effect against human cervix adenocarcinoma cells

(HeLa), (SI = 10.88) while it showed less selectivity against human hepatocellular

carcinoma cells (HepG2), (SI = 8.93). On the contrary, MBS extract showed a

selective cytotoxic effect against both HeLa and HepG2 cells with SI values of 12.44

and 11.94, respectively. In an attempt to disclose part of the underlying mechansims

of cytotoxic effect of both extracts using concentrations lower than IC50, RC

induced the production of the anticancer cytokine TNFα (up to 600% increase;

P<0.0001) whereas MBS induced the production of both anticancer cytokines TNFα

(up to 700% increase; P<0.0001) and IFNβ (up to 300% increase; P<0.001). The

increase in the anticancer cytokine levels was dose-dependent. In addition, the IC50

of RC extract induced cell cycle arrest in G0/G1 phase in cancer cells increasing the

percentage of cells from 66.87 to 76.96% and from 57.83 to 70.41% in the treated

HeLa and HepG2 cells, respectively. On the other hand, the IC50 of MBS extract

induced cell cycle arrest in G0/G1 phase only in HeLa cells (62.87 to 80.48%). Both

extracts induced remarkably apoptosis in the treated human cancer cells in a dose-

and time-dependent manner. The induction of apoptosis in cancerous cells 12-16h

after exposure to RC and MBS extracts was significant via caspase-dependent

pathways including the extrinsic pathway by upregulating caspase 8 (about 8-32

folds) and the intrinsic pathway by upregulating both caspase 9 (about 32-256 folds)

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and Bax genes (about 8-32 folds). Furthermore, both extracts upregulated highly

caspase 7 (about 32-512 folds) which could induce apoptosis via caspase-

independent pathway. More to the point, Cdk-inhibitor proteins were upregulated

(p21: about 8-512 folds, p27: about 4-16 folds, and p53: about 16-32 folds) 12-16h

after exposure to both extracts and their upregulation might be the main mechanism

used by both extracts to exert G1 cell cycle arrest and ultimately the final fate of

cells, apoptosis. For the immunomodulatory effect of tested extracts, RC extract

showed potent anti-inflammatory activity by inhibiting the production of the pro-

inflammatory cytokine, IFNγ (about 80% decrease; P<0.01) in peripheral blood

mononuclear cells (PBMC). On the other hand, MBS extract was effective

immunomodulator agent as TNFα and IFNβ cytokines are also potent

immunomodulators. In addition, MBS extract showed an immunopolarizing effect by

inducing IFNγ (about 300% increase; P<0.01) and inhibiting IL-4 production (about

75% decrease; P<0.01) in PBMC. The immunopolarization effect of MBS extract

was dose-dependent. For the antibacterial and antifungal potential of the tested

extracts, antibacterial and antifungal activities against multiple drug resistant (MDR),

non-MDR bacteria, and fungi were shown to be significant for both extracts. The

antibacterial and antifungal activities were dose-dependent. Remarkably,

antibacterial activity of RC extract was evident particularly against highly infectious

microorganisms such as Methicillin-resistant Staphylococcus aureus (MRSA),

Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumoniae,

Staphylococcus aureus, and Salmonella enterica serovar Typhimurium as well as

against human fungal pathogens, Trichophyton rubrum and Aspergillus terreus. MBS

extract revealed potential antibacterial and antifungal activities against MRSA,

Escherichia coli O157:H7, Pseudomonas aeruginosa, Klebsiella pneumonaie,

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Staphylococcus aureus, and Salmonella Typhimurium as well as against human

fungal pathogens, Trichophyton rubrum and Trichoderma harzianum. The findings

of the electron microscopy together with phenotype microarray (PM) supported these

results for the antibacterial activity. PM screening was a valuable tool in the search

for compounds that can inhibit bacterial growth by affecting certain metabolic

pathways like peptidoglycan synthesis pathway. This pathway appeared to be

targeted by both RC and MBS extracts at metabolic points differ from that of other

available drugs. These points could be targets for discovering new therapeutic agents

for the currently studied microbes. The considerable results of this study could be

explained by the powerful antioxidant activity of RC and MBS which is most likely

due to the phenolic and antioxidant compounds present in these plants which act

individually or synergistically to bring to light the importance of these plants as

candidates for new therapeutic agents.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan ijazah dari doktor falsafah

AKTIVITI ANTIMIKROB, IMUNOMODULATOR DAN SEL-SELEKTIF

TUMOR SITOTOKSIK DARIPADA BRASSICA OLERACEA L. DAN VIGNA

RADIATA L.

Oleh

RAND R. HAFIDH

Mei 2011

Pengerusi: Profesor Dr. Fatimah Abu Bakar, PhD

Fakulti: Sains dan Technologi Makanan

Dunia saat ini menghadapi cabaran besar dengan munculnya mikrob resistan

terhadap ubat dan kanser berbahaya terhadap sebahagian besar antimikrob umumnya

dikenali dan ubat antikanser, masing-masing. Penyaringan untuk Penemuan remedi

yang berkesan, baru, dan murah daripada sumber alam semulajadi menjadi matlamat

ramai saintis di seluruh dunia. Ekstrak metanol yang digunakan dalam penelitian ini

adalah kubis merah (RC) dan kacang hijau tunas (MBS) yang keduanya

menunjukkan aktiviti antivirus yang luar biasa terhadap jenis virus herpes simpleks 1

(HSV-1) dan virus RSV (RSV). Mod tindakan RC ekstrak terbukti profilaksis

terhadap RSV (kepekatan yang menghalang 50% daripada kesan virus sitofatik

(IC50) = 13.9 mg / ml dan indeks selektivitas (SI) = 17.67). Terhadap HSV-1, RC

ekstrak menunjukkan aktiviti virisidal luar biasa (IC50 = 11.51 mg / ml dan SI =

34.52) dengan aktiviti profilaktik kurang (IC50 = 32.97 mg / ml dan SI = 11.71).

Kesan virisidal ekstrak MBS (IC50 = 15.62 mg / ml dan SI = 14.18), (IC50 = 7.62

mg / ml dan SI = 18.23) dan ksan profilaksis tahap yang lebih rendah (IC50 = 17.23

mg / ml dan SI = 12.82), (IC50 = 12.72 mg / ml dan SI = 10.9) terhadap RSV dan

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HSV-1 virus, masing-masing. Salah satu alasan yang baik menjelaskan kegiatan

profilaksis berkesan ekstrak MBS dan RC adalah kemampuan mereka untuk

menginduksi pengeluaran peringkat tinggi IFNβ, TNFa, IL-1, dan IL-6 sitokin (tahap

300-900% lebih tinggi bila dibandingkan dengan yang tidak dirawat sel; P <0.0001)

pada sel yang dijangkiti virus. Sitokin ini terbukti memainkan peranan kritikal dan

sinergis dalam kesan rintangan sel antivirus manusia. Untuk antikanser ekstrak, RC

ekstrak menunjukkan aktiviti anti-kanser selektif terhadap sel adenokarsinoma

serviks manusia (Hela), (SI = 10.88), sementara kurang selektiviti terhadap sel

karsinoma hepatoseluler manusia (HepG2), (SI = 8.93). MBS ekstrak masing-masing

menunjukkan aktiviti anti-kanser selektif terhadap kedua HeLa dan sel-sel HepG2

dengan nilai SI 12.44, dan 11.94. Dalam usaha untuk mendedahkan sebahagian

daripada mekanisma yang mendasari aktiviti anti-kanser daripada kedua-dua ekstrak

dengan kepekatan yang lebih rendah daripada IC50, RC mengaruh pengeluaran

sitokin TNFa antikanser (meningkat hingga 600%, P <0.0001) sedangkan MBS

mengaruh pengeluaran antikanser sitokin TNFa (sehingga 700% meningkat; L

<0,0001) dan IFNβ (hingga mencapai 300%, P <0,001). Peningkatan kadar sitokin

antikanser adalah dos-bergantung. Selain itu, IC50 dari ekstrak RC mengaruh sel

penangkapan kitaran sel pada fasa G0/G1 dalam sel kanser dengan meningkatkan

peratusan sel 66.87-76.96% dan 57.83-70.41% dalam sel HeLa dan HepG2, masing-

masing. Selain itu IC50 dari ekstrak MBS mengaruh penangkapan kitaran sel pada

fasa G0/G1 hanya dalam sel Hela (62.87-80.48%). Kedua-dua ekstrak mengaruh

apoptosis dalam sel kanser manusia dengan cara-dos dan masa-dependen. Induksi

apoptosis dalam sel kanser selepas pendedahan 12-16h untuk ekstrak RC dan MBS

yang signifikan melalui laluan caspase-dependent termasuk laluan ekstrinsik

menggunakan upregulating caspase 8 (sekitar 8-32 lipatan) dan laluan intrinsik

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dengan upregulating kedua-dua caspase 9 (tentang 32-256 lipatan) dan gen Bax

(sekitar 8-32 lipatan). Seterusnya, kedua-dua ekstrak terselaras tinggi terhadap

caspase 7 (sekitar 32-512 lipatan) yang boleh mengaruh apoptosis melalui laluan

bebas-caspase. Lebih penting lagi, protein Cdk-inhibitor terselaras (p21: sekitar 8-

512 lipatan, p27: sekitar 4-16 lipatan, dan p53: sekitar 16-32 lipatan) 12-16h setelah

terdedak kepada untuk kedua-dua ekstrak dan upregulation mungkin merupakan

mekanisme utama yang digunakan oleh kedua-dua ekstrak untuk mengerahkan

penangkapan kitaran sel G1 dan akhirnya, apoptosis. Untuk kesan imunomodulator

dari ekstrak diuji, RC ekstrak menunjukkan aktiviti anti-inflamasi tinggi dengan

menghalang pengeluaran sitokin pro-inflamasi, IFNγ (sekitar 80% menurun, P

<0.01) pada sel mononuklear darah periferi (PBMC). MBS ekstrak sebagai agen

imunomodulator berkesan sitokin TNFα dan IFNβ. Selain itu, MBS ekstrak

menunjukkan kesan immunopolarizing dengan mengaruh IFNγ (sekitar 300%

meningkat; P <0.01) menghalas dan pengeluaran IL-4 (penurunan sekitar 75%, P

<0.01) pada PBMC. Kesan immunopolarization ekstrak MBS adalah dos-

bergantung. Untuk potensi antibakteria dan antikulat dari ekstrak diuji, aktiviti

antibakteria dan antikulat terhadap beberapa dadah rintang bakteria (MDR), non-

MDR, dan kulat menunjukkan kesan signifikan bagi kedua-dua ekstrak. Aktiviti

antibakteria dan antikulat adalah bergantung dos. Hebatnya, aktiviti antibakteria

ekstrak RC adalah jelas terutama terhadap mikroorganisme yang sangat berjangkit

seperti Staphylococcus aureus resistan methicillin-(MRSA), Escherichia coli O157:

H7, Pseudomonas aeruginosa, Klebsiella pneumoniae, Staphylococcus aureus, dan

Salmonella enterica serovar Typhimurium serta terhadap kulat patogen manusia,

Trichophyton rubrum dan Aspergillus terreus. Ekstrak MBS menunjukkan aktiviti

antibakteria dan antikulat berpotensi terhadap MRSA, Escherichia coli O157: H7,

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Pseudomonas aeruginosa, Klebsiella pneumonaie, Staphylococcus aureus, dan

Salmonella Typhimurium serta kulat patogen manusia, Trichophyton rubrum dan

Trichoderma harzianum. Penemuan menggunakan mikroskop elektron bersama-

sama dengan microarray fenotipe (PM) menyokong keputusan untuk aktiviti

antibakteria. Penyaringan PM adalah alat yang berharga dalam mencari bahan yang

boleh menghalang pertumbuhan bakteria dengan mempengaruhi pusat metabolisme

tertentu seperti pusat sintesis peptidoglikan. Laluan ini ampaknya menjadi sasaran

oleh kedua-dua ekstrak RC dan MBS pada titik metabolisme yang berbeza dari ubat

lain yang sedia ada. Titik-titik ini boleh menjadi sasaran untuk mencari agen terapi

baharu bagi mikrob yang dihaji. Keputusan kajian ini menunjukkan aktiviti

antioksidan yang kuat dari RC dan MBS berkemungkinan besar disebabkan oleh

sebatian antioksidan fenolik yang wujud dalam tanaman yang bertindak secara

individu atau secara sinergis tik untuk menjadikan pentingnya tanaman ini sebagai

sumber baru agen terapi.

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ACKNOWLEDGEMENTS

This work was highly supported by the superior guidance and encouragment of Dr.

Fatimah Abu Bakar, the supervisor committee. The finanical support from Univeristi

Putra Malaysia was highly helpfull to full all the requirments of this study. I want to

appreciate and thank Dr. Farid Azizi Jalilian for his kind advices and teaching which

he has provided to push this work forward.

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I certify that an Examination Committee has met on 3rd

May 2011 to conduct the

final examination of Rand R. Hafidh on her PhD thesis entitled “Antimicrobial,

Immunomodulatory and Tumor Cells-Selective Cytotoxic Activities of Brassica

oleracea L. and Vigna radiata L.” in accordance with Universiti Pertanian Malaysia

(Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree)

Regulations 1981. The Committee recommends that the student be awarded Doctor

of Philosophy degree.

Members of the Examination Committee were as follows:

Mariana Nor Shamsudin, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Son Radu, PhD

Professor

Faculty of Food Science and Technology

Universiti Putra Malaysia

(Internal Examiner)

Alfi Khatib, PhD

Lecturer

Faculty of Food Science and Technology

Universiti Putra Malaysia

(Internal Examiner)

Peter John Houghton, PhD

Professor

Pharmaceutical Science Research Division

King’s College London

United Kingdom

(External Examiner)

___________________________________

SHAMSUDDIN SULAIMAN, PhD

Professor and Deputy Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 21th

June 2011

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Doctor of Philosophy.

The members of the Supervisory Committee were as follows:

Fatimah Abu Bakar, PhD

Professor

Faculty of Food Science and Technology

Universiti Putra Malaysia

(Chairman)

Faridah Abas, PhD

Lecturer

Faculty of Food Science and Technology

Universiti Putra Malaysia

(Member)

Zamberi Sekawi, PhD

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Member)

Fatemeh Jahanshiri, PhD

Lecturer

Faculty of Biotechnology and Biomolecular Sciences

Universiti Putra Malaysia

(Member)

___________________________________

HASANAH MOHD. GHAZALI, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date: 14th

July 2011

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or at

any other institution.

____________________________

RAND R. HAFIDH

Date: 2nd

June 2011

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TABLE OF CONTENTS

Page

DEDICATION ii

ABSTRACT iii

ABSTRAK vii

AKNOWLEDGMENTS xi

APPROVAL xii

DECLARATION xiv

LIST OF TABLES xix

LIST OF FIGURES xxiv

LIST OF ABBREVIATIONS xxx

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 7

2.1 The Natural Products and Phytochemicals 7

2.2 Red Cabbage (Brassica oleracea L. var capitata f.

rubra)

10

2.3 Mung Bean Sprout (Vigna radiata (L.) R. Wilczek) 13

2.4 The Current Drugs and their Problems 15

2.4.1 Antiviral Drugs 15

2.4.2 Antibacterial and Antifungal Drugs 17

2.4.3 Anticancer Drugs 21

2.4.4 The Immunomodulatory Drugs 24

2.5 Overcome the Problems of the Current Available

Drugs by Using Natural Products

27

2.6 The Notorious Microbes Addressed by the Current

Research

30

2.6.1 Respiratory Syncytial Virus (RSV) 30

2.6.2 Herpes Simplex Virus Type 1 (HSV-1) 31

2.6.3 Methicilline-Resistant Staphylococcus

aureus (MRSA)

33

2.6.4 Enterobacteriaceae 35

2.6.5 Pseudomonas aeruginosa 40

2.6.6 The Dermatophytes 41

2.6.7 Trichoderma harzianum 43

2.6.8 Aspergillus terrus 44

2.7

The Cancer Models Addressed by the Current

Research

45

2.7.1 Hepatocellular Carcinoma (HCC) 45

2.7.2 Cervical Cancer 46

3 ANTIVIRAL PROPERTIES OF RED CABBAGE

(BRASSICA OLERACEA L.) AND MUNG BEAN

SPROUT (VIGNA RADIATA L.)

3.1 Introduction 48

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3.2 Materials and Methods 49

3.2.1 Preparation of the Extracts 49

3.2.2 Preparation of Stock Extracts 50

3.2.3

Specific Cells Propagation and

Cryopreservation

50

3.2.4 Virus Propagation (RSV and HSV-1) 51

3.2.5 Virus Titration 51

3.2.6 Cytotoxicity Assay 52

3.2.7 Virus Yield Reduction Assay 56

3.2.8 Virucidal Activity Assay 59

3.2.9 Prophylactic Activity Assay 63

3.2.10

Cytokine Production by Human Embryonic

Fibroblast Cell Lines (MRC-5) in Response

to HSV-1 Infection and Extracts’ Treatment

65

3.2.11 Data Analysis 69

3.3 Results 69

3.3.1

Estimating the Antiviral Activity by Virus

Yield Reduction Assay

70

3.3.2

The Direct Virus Inactivation (DVI) and the

Inhibition of Virus Replication (IVC) by RC

Extract

71

3.3.3

The Direct Virus Inactivation (DVI) and the

Inhibition of Virus Replication (IVC) by

MBS Extract

76

3.3.4

Comparison of the Virucidal and the

Prophylactic Properties of RC and MBS

Extracts Against RSV together, and with

Ribavirin

84

3.3.5 Comparison of the Virucidal and the

Prophylactic Properties of RC and MBS

Extracts against HSV-1 together, and with

Acyclovir

88

3.3.6

The Antiviral Cytokines Produced by the

Treated-Viral Infected Cells

92

3.4 Discussion 97

3.5 Conclusion 115

4 THE CYTOTOXIC AND IMMUNMODULATORY

PROPERTIES OF RED CABBAGE (BRASSICA

OLERACEA L.) AND MUNG BEAN SPROUT (VIGNA

RADIATA L.)

4.1 Introduction 117

4.2 Materials and Methods 119

4.2.1

Cancer Cells Propagation and

Cryopreservation

119

4.2.2

Isolation of Human Peripheral Blood

Mononuclear Cells (PBMC)

120

4.2.3 MTS Tetrazolium Assay 120

4.2.4 Cytokine Production by PBMC 123

4.2.5 Sandwich Enzyme-Linked Immunosorbent 124

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Assay (ELISA)

4.2.6 Cytokine Production by Human Cancer Cell

Lines

127

4.2.7 Analysis of Apoptosis 127

4.2.8 Data Analysis 134

4.3 Results 135

4.3.1 Cytotoxicity on Human Cancer Cells 135

4.3.2 Non-Specific Immune Response by PBMC 141

4.3.3

Specific Immune Response by Human

Cancer Cells

150

4.3.4

RC and MBS Extract Induced Apoptosis and

Cell Cycle Arrest in Human Cancer Cells

160

4.3.5

The Apoptosis- and Cell Cycle- Related

Genes in Human Cancer Cells Treated with

RC and MBS Extracts

174

4.4 Discussion 185

4.4.1

The Cytotoxic Effects of RC and MBS

Extracts

185

4.4.2

Immunomodulatory Activities of RC and

MBS Extracts

202

4.5 Conclusion 207

5 ANTIBACTERIAL AND ANTIFUNGAL PROPERTIES

OF OF RED CABBAGE (BRASSICA OLERACEA L.)

AND MUNG BEAN SPROUT (VIGNA RADIATA L.)

5.1 Introduction 210

5.2 Materials and Methods 211

5.2.1 Preparation of Stock Extracts 211

5.2.2 Microorganisms and Media 212

5.2.3

Selection of Multidrug Resistant (MDR)

Bacteria

213

5.2.4 Antimicrobial Sensitivity Tests 214

5.2.5 Scanning Electron Microscope (SEM) and

Transmission Electron Microscope (TEM)

217

5.2.6 Biolog Phenotype Microarray Technology 219

5.2.7 Data Analysis 222

5.3 Results 223

5.3.1 The MDR Bacteria 223

5.3.2 Disc Diffusion Assay for Bacteria 225

5.3.3 Microdilution Assay for Bacteria 228

5.3.4 Antimicrobial Assays for Fungi 235

5.3.5 Mode of the Antimicrobial Action 240

5.3.6

SEM and TEM Electron Microscope

Observations

241

5.3.7

Phenotype Microarray for E. coli and S.

aureus Bacteria Treated with RC and MBS

Extracts

244

5.4 Discussion 263

5.4.1

The Antimicrobial Activity of the Extracts

in the Current Research

263

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5.4.2 Antimicrobial Activity of RC Extract 265

5.4.3 Antimicrobial Activity of MBS Extract 274

5.5 Conclusion 282

6 SUMMARY, GENERAL CONCLUSION AND

RECOMMENDATIONS FOR FUTURE RESEARCH

284

REFERENCES/BIBLIOGRAPHY 293

APPENDIX 339

BIODATA OF STUDENT 343

LIST OF PUBLICATIONS 349