MPhil / Ph.

D Thesis
Template / Guidelines

Institute of Molecular Biology and Biotechnology, (IMBB) /

Centre for Research in Molecular Medicine, (CRiMM)

The University of Lahore

i
 MPhil / PhD Thesis Template and Guidelines

Order of thesis contents

Thesis contents should be arranged in the following order:

Title page
Abstract
Certificate of Approval
Supervisor Declaration
Author’s declaration
Plagiarism undertaking followed by certification letter
Research completion certificate
Dedication
Acknowledgement
Table of contents
i. Text
ii. List of figures
iii. List of tables
iv. List of Abbreviations /Acronyms
Thesis Text
a. Chapter 1: Introduction
b. Chapter 2: Review of Literature
c. Chapter 3: Materials and Methods
d. Chapter 4: Results
e. Chapter 5: Discussion
Summary / Conclusion
Literature cited
Annexure/Appendices

1. Page format – adjust page borders at left margin - 1.5-inch, right

margin 1.0-inch, top and bottom 1.0-inch.
ii
1. University logo: High resolution with 1.25 x 1.25 box size
The University of Lahore - Copperplate Gothic Bold, Small Cap
Lahore, Pakistan – Times New Roman, Font Size 12

The University of Lahore

Lahore, Pakistan

iii
 POTENTIAL RISK ASSESSMENT OF EXTRAPOLATIVE
FACTORS AND ANTIOXIDATIVE CAPACITY IN
OSTEOPOROTIC INDIVIDUALS

2. Thesis title: Times New Roman Each

word Capitalized, Small Caps (Bold), font
size 18

In Partial Fulfillment for The Degree Of

Doctor Of Philosophy
In Biochemistry
3. “In Partial Fulfillment for-
-----”Times New Roman, Font
size 12
Degree title and discipline:
Times New Roman, font size 12
In Biochemistry – Each word
Capitalized Font size 12.0

4. Submitted by: Times

New Roman, font size 12

Submitted by
5. Registration No: Times New Students Name
Roman, Fontsize 12 6. Student’s name: Times New
Registration number Roman, Each word Capitalized 14,
Bold, followed by the Registration
Number font size 12

INSTITUTE OF MOLECULAR BIOLOGY AND BIOTECHNOLOGY

9. Author’s Name and year:
© Author’s Name, 2018 8. Department Name:
Times New Roman, fontsize 12, Times New Roman, ALL
not bold CAPS, fontsize 14
iv
10. Bismillah calligraphic box size no more than -
3.0 x 5.0 cms
Times New Roman Fontsize 12, May be italics

"Bismillah al rahman al rahim"

In the name of Allah the most gracious the most merciful

5
 11. Abstract Title: Times New Roman Bold,
Small Caps Font size 12,

ABSTRACT
Text Body

1. Times New Roman

2. Justified
3. 12 Font size
4. Paragraphs separated by paragraph spacing afterwards 6
5. Non indented
6. 1.15 line spacing
7. Length: 250 to 300 words
8. Without headings
_____________________________________________________________________
Keywords: AAA, BBB, CCC, DDD etc. (Time Roman, Font size 10)

Example
Cancer is the leading cause of death in economically developed countries and is
the second leading cause of death in developing countries. Breast cancer is the most
frequently diagnosed cancer in Pakistan especially, in young women. In this cancer,
signal transduction plays a critical role in promoting proliferation and survival
cascades.
Recent work on gene quantification in proliferative pathways has suggested that
Akt isoforms and kRaS significantly control the cancer transition. However, the exact
role of Akt isoforms (Akt 1, Akt 2 and Akt 3) remains to be elucidated in breast cancer.

6
 CERTIFICATE OF APPROVAL
12: Text Body - Times New Roman
Bold, Small Caps Font size 12, (Mandatory, Bold, Centered, Size 14, All caps)

This is to certify that research work presented in the thesis, entitled “Potential risk
assessment of extrapolative factors and antioxidative capacity in osteoporotic
females” was conducted by __________ under the supervision of ___________.

No part of this thesis has been submitted anywhere else for any other degree. This
thesis was submitted to the CoE-UOL in partial fulfillment of requirements for the
degree of Master of Philosophy / Doctor of Philosophy in the field of
Biochemistry, Institute of Molecular Biology and Biotechnology, The
University of Lahore.

Student Name: ____________________ Signature: _____________________

Supervisor I Name: ________________ Signature: ______________________

Supervisor II Name: ________________ Signature: ______________________

EXAMINATION COMMITTEE: (Times New Roman, Bold, Small Caps Lock, Font Size 12)
a) External Examiner: (Times New Roman, Bold, Font Size 12)
(Designation and Office Address)
Signature: _______________________
b) Internal Examiner: (Times New Roman, Bold, Font Size 12)
(Designation and Office Address) Signature: ______________________

DIRECTOR IMBB/CRIMM : (Times New Roman, Bold, Small Caps Lock, Font Size 12)
(Name and Office Address)

Dated: _____________________ Signature: _______________________

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Text Body – Times New Roman, SUPERVISOR (s) DECLARATION
Font size 12
(Mandatory, Bold, Centered, Size 14, All caps)

I certify that ________(Name)__________________, (Registration number)

research-work presented in the above-mentioned thesis was conducted under my

supervision. I certify that the thesis is complete, “Potential risk assessment of

extrapolative factors and antioxidative capacity in osteoporotic females” is her /

his original piece of work conducted by her / him. with no material omitted. Further, no

portion of the thesis has been plagiarized and any material used as reference is properly

referred/cited. This work has not been submitted previously by her / him for taking any

degree from The University of Lahore, Lahore, Pakistan or anywhere else in the

country/world.

Supervisor I Signature_______________________ Date: ____________________

Name of Supervisor: ________________________ Designation: _______________

8
 AUTHOR’S DECLARATION
Text Body – Times New Roman,
Font size 12 (Mandatory, Bold, Centered, Size 14, All caps)

I ________ (Registration number) hereby state that my (Ph.D) thesis titled “Potential

risk assessment of extrapolative factors and antioxidative capacity in osteoporotic

females” is an original piece of work conducted by me. This work has not been

submitted previously by me for taking any degree from The University of Lahore or

anywhere else in the country/world.

At any time if my statement is found to be incorrect even after my post-graduation,

the university has the right to withdraw my (MPhil / Ph.D Biochemistry) degree.

Student Signature__________________ Registration Number: _________________

Student Name: ___________________

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Text Body – Times New Roman,
Font size 12
PLAGIARISM UNDERTAKING
(Mandatory, Bold, Centered, Size 14, All caps)
I solemnly declare that research work presented in the thesis titled “Potential risk
assessment of extrapolative factors and antioxidative capacity in osteoporotic
females” is solely my research work with no significant contribution from any other
person. Small contribution/help wherever taken has been duly acknowledged and that
complete thesis has been written by me.

I understand the zero-tolerance policy of the Higher Education Commission (HEC)

and the University “The University of Lahore” towards plagiarism. I,
________(Name)______________ as an Author of the above titled thesis declare that
the thesis is complete with no material omitted. Further, no portion of my thesis has
been plagiarized and any material used as reference is properly referred/cited.

I undertake that if found guilty of any kind of plagiarism in the above titled thesis, even
after award of MPhil / Ph.D Biochemistry, the university reserves the right to
withdraw/revoke my MPhil / Ph.D Biochemistry degree and that HEC and the
University has the right to publish my name on the HEC/University Website on
which names of students are placed who have submitted plagiarized thesis.

Name of Student: ______________________Reg No: _______________________

Signature: _______________________ Date: _____________________

10
 CERTIFICATION LETTER
(Mandatory, Bold, Centered, Size 14, All caps)

Name of Student:
Registration No.:
Thesis Title: “Potential risk assessment of extrapolative factors and
antioxidative capacity in osteoporotic females”

Name of Supervisor:

Department: Institute Of Molecular Biology And Biotechnology

Overall Similarity Index: _________%_____

Highest Similarity Index to One Single Source: ____________________________

Checked By:

____________________________________
Librarian
Pharmacy Library
The University of Lahore

CC: Director QEC

Director ORIC
Director Institute
dQEC
Office Record

11
 RESEARCH COMPLETION CERTIFICATE
(Mandatory, Bold, Centered, Size 14, All caps)

Text Body – Times New Roman, Fontsize 12

It is certified that this thesis titled, “Title of Thesis”, submitted by

______(Name _______, Registration No _______________ for
MPhil / Ph.D. degree at “Department name”, The University of Lahore, is an
original research work that has been conducted and completed under my
supervision.

Supervisor I Name
The University of Lahore

__________________
Signature

Supervisor II Name

_________________
Signature

Director’s Name
Department
University Name

____Signature_________

Date: _________________

12
 DEDICATION
(Bold, Centered, Size 14. ALL CAPS)

1. Times New Roman

2. Fontsize 12

3. 1.15 spaced

4. Make all text in the Centre of the Page

 ACKNOWLEDGEMENT

Text Body – Times New Roman, (Title - Bold, Centered, Size 14. ALL CAPS)
Font size 12

1. Times New Roman

2. Justified
3. Font Size 12
4. Each Paragraphs indented – one tab
5. 1.5 Spaced
6. Acknowledge supervisor, co-supervisors, mentors
7. Acknowledge collaborations/collaborators and/or organizations
8. Contribution of people who helped in conducting the study
and/or in preparation ofthe manuscript should be
acknowledged in order of their contributions
9. Acknowledge HEC if research has been carried out under an HEC
Scholarship

___________________________________
Student Name (Bold, Time New Roman)
Registration number
All Text Body – Times New LIST OF CONTENTS
Roman, Font size 12 (Time Roman, 14 Font size)

Bismullah ………………………………………………………………….......... ii
Title ……………………………………………………………………………… iii
Abstract ………………………………………………………………………….. iv
Board of Examiners ……………………………………………………………... vi
Certificate (Supervisory Committee)…………………………………………….. vii
Declaration …………………………………………………………………….... viii
Prayer ………………………………………………………………………….... ix
Dedication ……………………………………………………………………...... x
List of Contents …………………………………………………………………. xi
List of Figure…………………………………………………………………...... xiv
List of Tables ……………………………………………………………………. xvii
List of Abbreviation ……………………………………………………….......... xix
Acknowledgement ………………………………………………………………. xxi

1. CHAPTER ONE – INTRODUCTION (Times New Roman, Small Caps Lock, Font Size 12) 1- 10
1.1 ajsnkaj ……………………………………………….………. 02
1.2 nsaiciisci ………………………………………………….…. 02
1.3 ahidjbaajscnq………………………………………………… 05 Regular Times New
Roman Font size 12
1.4 jancuascyb…………………………………………………… 07
1.5 Working Hypothesis, Aims and Objectives………………….. 10
2. CHAPTER TWO - LITERATURE REVIEW 11-58
(Times New Roman, Small Caps Lock, Font Size 12)
2.1 asx askah h a………………………………………………..... 12
2.1.1 Classification, gene structure and Substrates........... 14
2.1.2 Role of xxxx gene in physiological and pathological
conditions…………………..…………………..…...16
2.1.2.1 PDP1……………………………………..... 17
2.1.2.2 PDP2……………………………….……... 19
2.1.2.3 PDP3……………………….……….…….. 20
2.1.3 Regulation of PDP……………..…………………... 23
2.1.3.1 Positive Regulation…………………..…..... 23 Regular Times New
Roman Font size 12
2.1.3.1.1 Upstream to PDP...……..…….... 25
2.2.3.1.2 Downstream to PDP…………….26
2.1.3.2 Negative Regulation…….......……………... 26
2.1.4.1 PHLiPPs…………….....………….. 27
2.1.4.2 PTEN….…………….....………….. 30
2.1.5 PDP in embryonic Development.....………………. 33
2. 1.6 PDP tumor Formation …………………………….. 33
2.1.7 RCAS 1 gene………………………………………...34
 2.1.8 Bcl 2 family genes and PDP Activities…….……….35
2.2 RaS Gene………………………………………………………..37
2.2.1 Rafs………………………………………………….41
2.2.2 Extracellular signal Regulated Kinases 1/2.……….. 42
2.2.2.1 ERK 1…………………………………....... 45
2.2.2.2 ERK 2…………………………………..... 45 Regular Times New
2.2.2.3 ERK 1/2 in Cancer………………….…….. 47 Roman Font size 12

2.3 p53 Gene............................................................................... 48

2.3.1 p53 Molecule interacting with DNA molecule……..48
2.3.2 p53 Domains………………………………………..49
2.4 NFB Genes.......................................................................... 53
2.4.1 NFB1 / NFB2 in cancer....................................... 58
3. CHAPTER THREE - MATERIAL AND METHOD 74-95

3.1 Collection of Specimens……………………………………… 60

3.1.1 Criteria……………………………………………... 60
Regular Times New
3.1.2 Proforma………………………………………….... 60
Roman Font size 12
3.2 Specimen Storage…………………………………….…….…. 60
3.3 Sample Processing……………………………………………... 60

Section A – Genomic Work

3.4 Total RNA Isolation (Tissues)……………………………….. 61
3.5 Formalin Fixed Paraffin Embedded Tissues…………………. 61
3.5.1 Total RNA isolation…………………………………. 61
3.5.2 RNA isolation……………………………………...... 62
3.6 RNA Quantification…………………………………………… 62
3.7 Primer design…………………………………………………... 63
3.7.1Primer design for RCAS 1 and Bcl 2 family genes...... 63
3.8 Reverse Transcriptase PCR reaction…………………………... 64
3.9 Real Time PCR Reaction……………………………………… 69
3.9.1 LUX Primer PCR……………………………………. 69 Regular Times New
3.9.1.1 Optimization of Primer Concentration……… 69 Roman Font size 12
3.9.1.2 Optimization of Tm (Gradient PCR)……….. 70
3.9.1.3 Optimization of cDNA Concentration……… 70
3.9.1.1 Optimization of Multiplex PCR……………. 70
3.9.2 Eva Green Real Time PCR…………………………. 71
Section B – Proteomic Work
3.10 BioPlex Assay…………………………………………………71
3.10.1 Protein Extraction…………………………………. 72
3.11 Statistical Data Analysis Software…………………………….72
3.12 Bioinformatics Tools………………………………………….72
 3.12.1 Akt Isoforms Alignment …………………………...72
3.12.2 Unspecific Phosphorylation Sites…………………..72
4. CHAPTER FOUR - RESULTS 96-179
Section 4a Demographic Data
4.1 Sample Distribution…………………………………………..... 78
Section 4b Real Time Quantification
4.2. Quantification by LUX™ Primers……………………….……. 81
4.2.1 Normal Tissues…………………………………….. 81
4.2.2 Hyperplasia Tissues………………………………... 81
4.2.3 DISC Tissues………………………………………. 83
4.2.4 Grade I Tissues……………………………………. 83
4.2.5 Grade II Tissues……………………………………. 84
4.2.6 Grade III Tissues…………………………………… 84
4.2.7 Unclassified………………………………………....87
Layout of Statistical Analysis (Genomics).………………...…........89
4.3. The dynamics of each gene studied in various grades of
Breast cancer tissue………………………………………… 90
4.3.1 p53……………………………………………….… 90
4.3.2 PDP1……………………………………………… 91
4.3.3 PDP2……………………………………………… 93
4.3.4 PDP3……………………………………………… 94
Regular Times New
4.3.5 kRaS……………………………………………… 96 Roman Font size 12
4.4 Unclassified tissues………………………………………… 97
4.5 Statistical Analysis ………………………………………… 98
4.5.1 Overall analysis for 6 genes in Ovarian
tissues………………………………. ………….…. 98
4.3.2.1 ANOVA…………………………………... 98
4.3.2.2 Pearson’s Correlation ………………..…… 98
4.3.2.3 Tukey’s Pairwise analysis ……..…………. 98
Section 4c Real Time Quantification of ERK and NFB
4.6 Real time Quantification of ERK 1/2 by Eva Green Dye
Binding Assay……………………………………………… 100
4.7 Dynamics of ERK and NFB isoforms in different
grades of tissues……………………………………………… 103
4.7.1 ERK 1……………………………………………… 103
4.7.2 ERK 2……………………………………………… 105
4.7.3 NFB 1…………………………………………… 109
4.7.4 NFB 2…………………………………………… 110
Section 4d Real time quantification of RCAS 1 and Bcl 2 Family genes
4.8 Quantification of RCAS 1 and Bcl 2 genes by LUX primers… 114
4.8.1 PDP3 and RCAS 1 gene quantification ……………. 114
4.8.2 Bcl 2 antiapoptotic genes quantifications………… 116

Section 4e Dynamic of 13 quantified genes

4.9 Dynamic of quantified genes summarized in each grade……. 120
4.9.1 Normal tissues…………………………………… 120
4.9.2 Hyperplasia……………………………………… 120
4.9.3 DISC………………………………………………. 121
4.9.4 Grade I……………………………………………. 122
4.9.5 Grade II……………………………………………. 122
4.9.6 Grade III………...…………………………………. 123
4.9.7 Unclassified………………………………………... 124
4.10 Dynamics of RCAS 1 and Bcl 2 genes in each grade
of breast tissue……………………………………………... 127 Regular Times New
4.10.1 Hyperplasia………………………………………… 127 Roman Font size 12
4.10.2 Grade II tissues .…………………………………… 127
4.10.3 Grade III……….…………………………………... 128
4.10.4 Statistical analysis of PDP–RCAS 1 – Bcl 2 family
genes with associated proteins……………………. 129
4.9.4.1 Pearson’s Correlation……………………… 129
4.9.4.2 Spearman’s Correlation……………………. 130
Section 4f BioPlex 200 protein quantifications
4.11 Statistical Analysis Layout (Proteomics) ………………….... 131
4.12 Statistical Analysis …………………………………………... 155
4.12.1 Analysis for 7 proteins by PDP-Kinases kit ………. 155
4.12.1.1 ANOVA ………………………………… 155
4.12.1.2 Pearson’s Correlation …………………... 155
Section 4g Bioinformatic Data
4.13.1 PDP3 – PTEN docking…………………………….. 162
4.13.2 PDP3 – ERK 1 docking……………………………. 162

5. Chapter Five – Discussion 161-184

5.1 Demographic information………………………………… 164

5.2 Dynamics of quantified genes in various grades of
breast cancer……………………………………………… 166
5.2.1 PDP Isoforms………………………………………. 166
5.2.2 RCAS 1……………………………………………. 169
 5.2.3 kRaS and ERK 1/2………………………………… 172
5.2.4 p53………………………………………………… 174
5.2.5 NFB 1 and NFB 2 Isoforms……………………. 175
5.2.6 Antiapoptotic Bcl 2, Bcl xL, Mcl 1………………... 177
5.3 proteomic analysis of PI3K – Akt – mToR pathway……… 178
6. Summary (3 Pages, Time New Roman Font Size 12)…………………………… 180-183
7. Conclusion (2 Pages, Time New Roman Font Size12)……………..,…………….184-185
8. References……………………………………………………………………...186-223
9. Appendices……………………………………………………………………..224-228
Regular Times New
Roman Font size 12 LIST OF FIGURES
(Mandatory if figures are used, Centered, Bold, Size 14, ALL CAPS.)

Figure 1.1: Figure legend (Chapter 1 Figure)…………………………………………... 25

Figure 3.1: Figure legend (Chapter 3 Figure)… ............................................................... 50

Figure 3.2: Figure legend (Chapter 3 Figure) ................................................................... 60

 LIST OF TABLES
(Mandatory if tables are inserted, Centered, Bold, Size 14, ALL CAPS.)

*See list of figures part for reference

 LIST OF ABBREVIATIONS/ACRONYMS
(Mandatory if abbreviations are used, Centered, Bold, Size 14, ALL CAPS.)

1. Times New Roman

2. 12 Font size
3. Line spacing 1.15
4. In alphabetical order

CAFs Carcinoma associated fibroblasts

Casp Caspase
COX-2 Cyclooxygenase-2
CXCR4 CXC chemokine receptor 4
DAMPs Damage-associated molecular patterns
ECM Extracellular matrix
EMT Epithelial to mesenchymal transition
Erk Extracellular signal-regulated kinases
FGF Fibroblast growth factor
H2O2 Hydrogen peroxide
HIF-1α Hypoxia inducible factor-1α
ICAM Intercellular adhesion molecule
IFN Interferon
IL Interleukin
IL-6 Interleukin-6
ILs Interleukins
iNOS Inducible nitric oxide synthase
IRAK interleukin-1 receptor-associated kinase
IRF interferon regulatory factor
MAPK Mitogen Activated Protein Kinase
MDA Malondialdehyde
MIP1 Macrophage inflammatory protein 1
MMPIs Matrix Metalloproteinase Inhibitors
MMPs Matrix metalloproteinases
MyD88 Myeloid Differentiation Protein 88
NFAT Nuclear factor of activated T cells
 Chapter 1

(Times New Roman, Italics, Font size 12, Centered, )

INTRODUCTION

(Times New Roman, Small Caps, Bold, Underlined, Font size 14)

1.0 Heading
(First level heading. Times New Roman, Font size 14, line spacing 1.5)

1.1 Subheading
(Second level heading. Times New Roman, Bold, Font size 12, line spacing 1.5)

1.1.1 Sub-Subheading
(Third level heading. Times New Roman, Bold, Font size 12, line spacing 1.5)
 Example
The deficiency of vitamin D causes severe effects on the bones which ultimately
lead to osteoporosis in elder women. The concentrations of vitamin D are also related to
calcium absorption. The active form of vitamin D is called as 1, 25-dihydroxyvitamin
D,that is essential for the bone and calcium homeostasis (Holick, 1995). It is a steroid
hormone that maintains the levels of calcium, phosphorous and other minerals in the body
system (How et al., 1994). The main sources of vitamin D are fish oils (as it contains higher
fat components) and dairy products which are enriched with micronutrients (Maclaughlin
et al., 1982). Vitamin D is synthetically produced in skin after the exposure of ultraviolet
rays (wavelength 290-315nm) that triggers the activation of 7-dehydroxycholestrol ending
with pre-vitamin D (Bouillon et al 1998). There are decreased concentrations of vitamin D
in older (Holick, 2003). The decrease levels of vitamin D are also present in people with
darker skin shade as they have sufficient amount of melanin which protects the skin against
UV light exposure. The significance of ultraviolet rays is to enhance the activation of pre-
vitamin D which is inhibited under the action of melanin. Therefore, darker skin is more
vulnerable to vitamin D deficiency (Martin et al., 2016). The ingestion of food enriched
with vitamins is recommended for these patients (Zhou et al., 2006). Aging is not the only
reason for the deficiency of vitamin D but other contributing factors such as diet, ethnicity
and rate of sunlight exposure have significant importance to maintain its concentrations
(Need et al., 1993). The reduced levels of vitamin D increase the vulnerability of bone
rupture to many folds. The condition may be termed as hypovitaminosis that advances with
age and may serve as a major causative factor in the development of hyperthyroidism
(Holick et al., 2005 and Sahota et al., 2006).

Vitamin D acts as an important mediator to maintain the phosphorous homeostasis.

It has a specific receptor from which it binds and triggers the absorption of calcium in the
small intestine (Holick, 1999). 50% of women and 30% men are tolerating the osteoporosis
associated fractures (Chrischilles et al., 1991 and Anonymous, 1997). In order to maintain
the bone mass, awareness as well as the best nutrition would be important (Reid, 1996).
Low levels of vitamin D are responsible to high chance of osteoporosis related to rupture
of the bones. There is a significant effect of 1, 25-dihydroxyvitamin D on the bones and
skeletal muscles (Venning, 2005). Clinically, it has been proven that high concentration
of vitamin D in serum has linked to muscle capacity. The increase age and poor diet both
are directly involved to enhance body mass
Chapter Two Review Of Literature
Student name, Year

Chapter 2

(Times New Roman, Italics, Font size 12, Centered, )

REVIEW OF LITERATURE

(Times New Roman, Bold, Small Caps, Font size 14)

2.0 Heading
(First level heading. Times New Roman, Bold, Small Caps, Font size 14, , indented 1
tab)

Paragraph spacing with 8

2.1 Subheading
(Second level heading. Times New Roman, Bold, Font size 12, indented 2 tabs)

2.1.1 Sub-Subheading
(Third level heading. Times New Roman, Bold, Font size 12, , indented 3 tabs)

8
Chapter Two Review Of Literature
Student name, Year

Example
2.1 Overview of Vitamin-D
Vitamin-D gets great importance because it involves the absorption of
calcium and maintaining the normal activity of bones. Reduced levels of vitamin D are
linked with the abnormality in the absorption of calcium ions and ultimately give rise to
the levels of parathyroid hormone that initiates the bone remodeling process. According to
the survey of National Health and Nutrition Examination, observed 26 million to 38 million
US adults suffer from osteoporosis and more prone to hip fractures. This vitamin has also
acted as an essential mediator during muscle functioning, normal bone metabolism and
immune response (Deluca, 2004). Some food contains significant amounts of vitamin D.
Major sources of vitamin D are cod liver oils and oily fishes such as salmon mackerel and
sardines. Intake of these fishes helps in maintaining the proper concentration of vitamin D.
Some dietary elements such cereals, orange, juices are rich in vitamin D (Tangpricha et al.,
2003). In the milk, low contents of vitamin D are found and thus the influence of vitamin
D is changeable. For the purpose to get more vitamin D in a casual routine, sunlight
exposure is the best main source. In the spring season, the vitamin D3 is produced in the
skin that is stored in body fat. The skin has high ability to synthesize vitamin D3. With the
passage of time, the bone becomes weak and losses its fragility as low level of 7-
dehydrocholestrol synthesize in skin (Chuck et al., 2001). The ergocalciferol (vitamin D2)
is a secondary form, which produces from yeast and plant sterol. The absorption pattern of
calciferol is good as compared to vitamin D2, which is less absorptive capacity.

2.2 DEFICIENCY OF VITAMIN-D AND ITS RELATED CONSEQUENCES

The previous study has showed that the deficiency of vitamin D directly presents
clinical importance of rickets as well as metabolic disease. About 60% of older people and
70-100% of healthy people have lower levels of 25-hydroxyvitamin D and instantly
increase the levels of ALP in the serum as well as parathyroid hormone (Burckhardt et al.,
1996). This is basically caused by inadequate contact with sunshine. The recommended
value of vitamin D in U.S is 400 IU and recent medication of vitamin D for aged person is
800 IU. The low levels of 25-dehydroxy vitamin D may cause high risk of osteopathy. In
the pathological conditions, lower levels of vitamin D directly affect the bone mineral level
to flop and causes osteomalacia as well as rickets in children and adults respectively.
9
Chapter Two Review Of Literature
Student name, Year

Osteoporosis is the secondary form to increase the levels of parathyroid hormone (PTH).
Defective part of bone such as proximal femoral fractures

10
Chapter Three Materials And Methods
Student name, Year

Chapter 3

(Times New Roman, Italics, Font size 12, Centered)

MATERIALS AND METHODS

(Times New Roman, Bold, Small Caps, Font size 14)

3.0 Heading
(First level heading. Times New Roman, Bold, Small Caps, Font size 14, , indented 1
tab)

Paragraph spacing with 8

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2.1.1 Sub-Subheading
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29
Chapter Three Materials And Methods
Student name, Year

Example
3.1 Source Of Data
The current study substitutes randomly selected women with
postmenopausal women age in between 49 to 57 years from the province Punjab-Pakistan
on the criteria as follow: women with reported postmenopausal for more than one year
of time period, all of the subjects were obtained with informed consent able to
communicate and were actively mobile. Whereas, exclusion criteria remained women
with any acute infection, with disease of kidneys or pancreas, diabetes, lung or liver
malignancies and those who were on hormonal medication for the period of three or more
years. The respondents were interviewed from 2012-2013, using a questionnaire that
included items on age and duration of menopause. All participating women signed an
informed consent form. Among 300 subjects, 28 did not meet the inclusion criteria of
whom 17 subjects were on biochemical testing revealed to have diabetes mellitus and 11
did not show up for laboratory tests. The said experimental protocol was approved by the
Institutional Review Board (IRB), The University of Lahore. For the biochemical
analysis of several markers about five (5ml) venous blood was extracted and centrifuged
for the separation of serum which was later stored at -70oC for their future analysis.

3.2 Chemicals

All of the reagents and chemicals were of analytical grade obtained from
the Sigma/Invitrogen Chemical Co. (St. Louis, Mo, USA).

3.3 Blood Separation

Blood serum was separated by centrifuging it for ten minutes at 3000 rpm
and stored at -70oC until the analysis is performed.

3.4 Biochemical Assays

3.4.1 Estimation Of Malanodialdehyde (Mda)

MDA was measured by spectrophotometric method of Okawa et al.,
(1979). MDA is often performed to check the degree of lipid peroxidation and production
of oxidants within the cell. Increased lipid peroxidation basically means the degree of
damage to the cell by the peroxidation of lipid membrane under the action of free radicals.

30
Chapter Three Materials And Methods
Student name, Year

It generally comprised of three basic steps that are initiation, elongation and termination in
proper sequence (Catala 2006). Lipid peroxidation initiation scopes the abstraction of
hydrogen atom. There are several different species which can abstract first H atom and
form radicals such as., hydroxyl (∙OH), alkoxyl

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Chapter 4

(Times New Roman, Italics, Font size 12, Centered, )

RESULTS

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4.0 Heading
(First level heading. Times New Roman, Bold, Small Caps, Font size 14, , indented 1
tab)
Paragraph spacing with 8

2.1 Subheading
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2.1.1 Sub-Subheading
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Example
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4.1 Hematological Profile In Women Suffering From Osteoporosis

The data compiled in table 01 demonstrate the hematological profile of women

suffering from osteoporosis as compared to control individuals e.g., neutrophils,
lymphocytes, monocytes, RBCs, WBCs, PLTs and Hb. There are significant differences
between the values of osteoporosis groups and control subjects. The mean value of
neutrophils, lymphocytes, RBCs, platelets, haematocrit and Hemoglobin were noted to be
decreased (38.17±8.12 %, 25.69±2.367 %, 4.30±0.31 million/mm3, 179.75±9.12 109 /L,
34.93±3.50 % and 10.57±1.87 g/dl) in women suffering from osteoporosis as compared to
control individuals (56.11±3.47 %, 35.49±3.87 %, 4.64±0.13 million/mm3, 307.86±5.31
109 /L, 41.26±1.53 % and 14.13±0.89 g/dl) respectively. On the other hand, the mean value
of monocytes and WBCs were significantly raised (7.39±3.75 % and 9.27±1.63
million/mm3) in osteoporosis subjects verses normal women (3.65±5.24 % and 7.58±0.40
million/mm3) respectively. Highly significant increased levels of creatinine and uric acid
(1.56±0.29 mg/dl Vs. 0.72±0.03 mg/dl) and (4.93±1.02 mg/dL Vs. 3.87±0.86 mg/dL) were
observed in women suffering from osteoporosis as compared to control individuals.

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TABLE 4.1 Hematological Profile In Women Suffering From Osteoporosis

Variables Control (N=92) Patients (N=272) P ≤0.05

Mean±Sd Mean±Sd
Neutrophils % 56.11±3.47 38.17±8.12 0.003

Lymphocytes % 35.49±3.87 25.69±2.367 0.021

Monocytes % 3.65±5.24 7.39±3.75 0.032

RBCs (million/mm3) 4.64±0.13 4.30±0.31 0.031

WBCs (million/mm3) 7.58±0.40 9.27±1.63 0.035

PLTs 109 /L 307.86±5.31 179.75±9.12 0.021

Hct,% 41.26±1.53 34.93±3.50 0.023

Hb g/dl 14.13±0.89 10.57±1.87 0.017

Creatinine mg/dl 0.72±0.03 1.56±0.29 0.010

Uric A mg/Dl 3.87±0.86 4.93±1.02 0.018

Legend: Use Normal Standard Refence values as per test performed

Author can use both face –

portrait / landscape format for
tables and figures

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Chapter 5

(Times New Roman, Italics, Font size 12, Centered, )

DISCUSSION
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5.0 Heading
(First level heading. Times New Roman, Bold, Small Caps, Font size 14, , indented 1
tab)
Paragraph spacing with 8

2.1 Subheading
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2.1.1 Sub-Subheading
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Example
Osteoporosis is said to be a porous bone disease, in other words, it is characterized
as a weak bone mass. It is believed to be a major health problem related to bone disorder
which affects hundreds of people mainly postmenopausal women. Clinical symptoms of
this disease found to be fractured bone. Hip and spine fractures are two serious types of
fractures that are linked with substantial pain, disability and even death. Furthermore,
osteoporosis creates the significant burden on both the individual and society. In this
disorder, low bone mass and disruption have found in bone architecture, resulting in higher
risks of fractures (Armas et al., 2004). There is a mechanism which termed as bone
remodeling in which removal of old bone and the formation of new bone can take place
(Fernandez-Tresguerres-Hernandez-Gill et al., 2006). This mechanism of action can be
takes place with the help of two bone cells, osteoclast and osteoblast (Fraher, 1993). A
healthy skeleton is maintained by bone forming osteoblasts and bone resorbing osteoclasts
which regulate mineral contents and physiological structure. The bone remodeling
mechanism has completed within 5-6 months, which initiated from non-targeted area of
old bones and trigger bone resorption by osteoclast. Regulation of several bone cells during
the bone remodeling process has arranged by several pathways, including receptor
activator of nuclear factor NF-kB ligand (RANKL). Osteoporosis mostly occurs due to
uncoupling of bone resorption from bone formation. However, peak bone mass has taken
place in early childhood and with the passage of time bone mass reduction will be initiated.
Osteoporosis may occur at age under 45 years in women who have undergone
hysterectomy and oophorectomy. Osteoporosis is a common disorder of old age, creating
a worldwide health problem and the age tend to suffer from bone fractures from mild injury
or even without injury. These fractures increase the morbidity and mortality rates and
health funding as well as reduce the quality of life of the patients (Lopes et al., 2008).

Bones act as dynamic tissues renew constantly during the bone remodeling process
which carried out by functional and anatomical structures and also called as a basic multi-
cellular unit (BMU). It needs coordination of three major types of cells osteoclasts,
osteocytes and osteoblasts. In this process major factors that are associated includes
hormones, growth factors, cytokines, and other multiple molecular agents (Harada and
Rodan, 2003). After six months, old bones are removed from the osteoclast cells and new

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Chapter Five Discussion
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bone comes into that place under the action through osteoblast cells (Boulpaep and Boron,
2005). Thereby, the osteocytes activity in signal

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 Summary
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SUMMARY / CONCLUSION

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Summary – 3 Pages Maximum

Conclusion – 2 Pages Maximum
 Summary
Student name, Year

Example

Vitamin D acts as an important factor to maintain phosphorous homeostasis. The

active form of vitamin D has definite kind of receptor which induced absorption of calcium
in small intestine. Vitamin D is primarily obtained from food or can be synthesized in skin
in proper concentration by UV rays exposure. It is extensively widespread in nature and
photosynthesized from most plants and animals exposed to sunlight. In order to maintain
bone mass; best nutrition, sunlight exposure and awareness are essential to regulate bone
homeostasis. Approximately 50% women and 30% men are tolerating an osteoporosis
associated fractures. Moreover 100 million people suffered from osteoporosis in
worldwide. Although, there are various factors are involved in the deficiency of vitamin D
including low food ingestion, digestion and improper production in skin. If the amount of
vitamin D reduces from 20nmol/1, it will suggest a critical deficiency that ultimately
caused rickets or impairment of bones. Another observation shown that Arabian women’s,
found low level of 25 hydroxy-vitamin D, due to low contact of sunlight linked to their
dress getup and enclosed whole body. In women, decrease levels of vitamin D are
responsible to enhance the chances of bone fragility and bone ruptures.

The criteria based on nutritional category of vitamin D can be estimated by serum

25 hydroxyvitamin D concentrations. Initially it has produced in inactive form and can be
converted into active form in the regulatory part of body such as liver and kidney. Once
vitamin D converted into its active form, it shows its direct effect on skeletal muscle.
Clinically it has been proven that the high level of serum vitamin D has directly linked
muscle capacity. It has also concluded that medicated treatment of vitamin D possesses
good results during loss of bone components and fractures of bones in menopause.
Moreover, medication of vitamin D and calcium not only reduce hip and spine fractures
but also enhanced bone mineral density (BMD) in postmenopausal women. Osteoporosis
is a chronic hidden confused disease which is categorized by low bone density and bone
fragility. Several risk factors are involved in osteoporosis i.e, gender specification, age,
genetic predisposition, chronic inactivity, microgravity, low body weight, lifetime calcium
intake, medications, smoking and low ingestion of antioxidants. Through the remodeling
process old bones replace with the new one with the involvement of different bones cell
 Summary
Student name, Year

such as osteoblast and osteoclast. Reduce levels of Vitamin D can be analyzed by

concentration of 25-hydroxyvitamin D which has high affinity to bind with protein DBP
and responsible to store Vitamin D. Parathyroid hormone play important role to
 Student name, Year

REFERENCES

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Text
(Times New Roman, Font size 12, line spacing 1.15, each reference article line spacing 6)

Reference Style
(APA style)
 Student name, Year
Grady, J. S., Her, M., Moreno, G., Perez, C., Yelinek, J. (2019). Emotions in storybooks:
A comparison of storybooks that represent ethnic and racial groups in the United
States. Psychology of Popular Media Culture, 8(3), 207–
217. https://doi.org/10.1037/ppm0000185

• Parenthetical citation: (Grady et al., 2019)

• Narrative citation: Grady et al. (2019)

Book Reference
Jackson, L. M. (2019). The psychology of prejudice: From attitudes to social action (2nd
ed.). American Psychological Association. https://doi.org/10.1037/0000168-000
Sapolsky, R. M. (2017). Behave: The biology of humans at our best and worst. Penguin
Books.
Svendsen, S., Løber, L. (2020). The big picture/Academic writing: The one-hour
guide (3rd digital ed.). Hans Reitzel Forlag. https://thebigpicture-
academicwriting.digi.hansreitzel.dk/

• Parenthetical citations: (Jackson, 2019; Sapolsky, 2017; Svendsen & Løber, 2020)
• Narrative citations: Jackson (2019), Sapolsky (2017), and Svendsen and Løber
(2020)

Whole Book Reference

Torino, G. C., Rivera, D. P., Capodilupo, C. M., Nadal, K. L., Sue, D. W. (Eds.).
(2019). Microaggression theory: Influence and implications. John Wiley &
Sons. https://doi.org/10.1002/9781119466642

• Parenthetical citations: (Hygum & Pedersen, 2010; Kesharwani, 2020; Torino et

al., 2019)
• Narrative citations: Hygum and Pedersen (2010), Kesharwani (2020), and Torino
et al. (2019)

Chapter in a Book
Aron, L., Botella, M., Lubart, T. (2019). Culinary arts: Talent and their development. In R.
F. Subotnik, P. Olszewski-Kubilius, & F. C. Worrell (Eds.), The psychology of high
performance: Developing human potential into domain-specific talent (pp. 345–
359). American Psychological Association. https://doi.org/10.1037/0000120-016
 Student name, Year
Dillard, J. P. (2020). Currents in the study of persuasion. In M. B. Oliver, A. A. Raney, J.
Bryant (Eds.), Media effects: Advances in theory and research (4th ed., pp. 115–
129). Routledge.
Thestrup, K. (2010). To transform, to communicate, to play—The experimenting
community in action. In E. Hygum & P. M. Pedersen (Eds.), Early childhood
education: Values and practices in Denmark. Hans Reitzels
Forlag. https://earlychildhoodeducation.digi.hansreitzel.dk/?id=192

• Parenthetical citations: (Aron et al., 2019; Dillard, 2020; Thestrup, 2010)

• Narrative citations: Aron et al. (2019), Dillard (2020), and Thestrup (2010)
 Student name, Year

ANNEXURES / APPENDICES

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Annexure A / Appendix I

Annexure B / Appendix II