Defence Science Organization

Period: 3 Nov 14 – 20 Feb 15

Internship Final Report DIPLOMA IN BIOMEDICAL SCIENCE

BY

PRISCILLA CHONG SHI HUI S10122181G

COMPANY SUPERVISOR: DR PAMELA PUN BOON LINP SUPERVISOR: DR ZHU CONG JU

School of Life Sciences & Chemical TechnologyNGEE ANN POLYTECHNIC

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TABLE OF CONTENT

1. Abstract

2. Description of my role and contributions

a) Description of company

b) Specific roles and responsibilities

c) Training received

d) Accomplishments

3. Reflection and Analysis of Internship Experience

a) Workplace safety

b) Challenges and solutions

c) Applying prior knowledge

4. Acknowledgement letter to company

5. Curriculum Vitae (CV)

6. References

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1. Abstract

Major Depressive Disorder (MDD) is characterized as a mood disorder, diagnosed by 2 or

more major depressive episodes and each episodes lasting at least 2 weeks. Symptoms

of MDD include feeling dejected all the time, changes in appetite or sleep patterns, having

suicidal thoughts, etc (Center for substance Abuse, 2008).

Studies have shown that the prevalence estimates of a lifetime and 12 month MDD is

5.8% and 2.2 % respectively and the aged group with the highest risk of developing MDD

is 18 to 34 years old (Chong et al, 2012). Studies have revealed that there are certain

genetic markers, proteins and metabolites that are related to the onset of depression.

Thus the aim of this study is to identify and confirm a list of possible genetic as well as

physiological depression biomarkers found in the serum of the volunteers from MMI, in

order to assist and boost the effectiveness of detecting depression in patients.

2. Description of my role and contribution 2a) Description of the company

Defence Science Organization (DSO) is divided into seven divisions that concentrate

on Singapore’s defence and security. The division I am under is known as Defence

Medical & Environmental Research institute (DMERI). DMERI combat against

chemical, radiological and biological threats and undergoes R&D to develop human

science so as to improve the safety and performance of the troops. DMERI is divided

into 3 subdivisions- Chemical, Toxin, Radiology and Nuclear Defence (CTRN),

Biological Defence (BD), Combat Protection and Performance (CPP). Under BD, there

are 4 subgroups-Biosurveillance and Service Program (BSS), Host pathogen

Interaction Lab (HIL), Bio-defence Therapeutics Lab (BTL) and Microbiology lab. Under

CPP, there are also 4 subgroups- Screening and diagnostic lab (SDL), Combat Care

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Program (CBC), Training Effectiveness and Safety (TES) and WPA. For this project,

DSO is also recruiting voluntary patients from Military Medicine Institute (MMI).

2b) Specific Roles and Responsibilities

The objectives of my research project is to study depression as a disease so as to

identify and confirmed a list of genetic (Deoxyribonucleotide Acid, Single Nucleotide

polymorphism, etc) and physiological biomarkers (proteins, biochemical, etc) that are

associated to depression and thus improvise the screening of individuals with

depressive disorders. The initial stages of recruiting subjects, administering

questionnaires regarding demographics and lifestyle to distinguish between depressed

and normal subjects and obtaining blood samples from the subjects have been done.

Currently, the project is at its final stages where my role is to consolidate the responses

of the questionnaire as well as analyzing the selected analytes in the blood samples of

the different subjects using Multiplex or single analytes ELISA assay. I am also tasked

to do literature review of those physiological biomarkers listed for analysis.

I am also helping out in another project which is classified as confidential. Basically, my

role is to help out in the preparation like labeling Eppendorf tubes, Blood Vacutainer

tubes and Tissue pencil cassettes as well as inserting PE10 cannula tubings into PE50

ones. As we are also dealing with rats, we have to make sure the rats are all in the

desired condition for the trial. During the trial, I was tasked to check on the status of the

rats and to record what substances was injected into the rats at 2 time points. For the

preparation of the trial, I had to help fill over 200 syringes of Anesthesia.

There are 4 different groups of subjects for this research project. The MDD cases are

the Retrospectives and Prospectives groups while the normal controls are the Battalion 4

and Sedentary groups. The Retrospectives are those who previously had depression

but have recovered while the Prospectives are those are currently referred by MMI for

having depression. The Battalion normal controls are those who do not suffer from

depression are actively participating in vigorous training while the Sedentary are those

who do not suffer from depression but are not actively participating in vigorous training.

The subjects comprises of Chinese National Service (NS) men aged 18-25 and each

subjects are assigned to an identity number.

After obtaining blood samples from each subject, we allow the blood to clot first before

spinning down the samples at 1000g at 4 degree celsius for 10 min to separate it into

plasma, serum and red blood cell layers. We extracted the serum samples and plasma

samples into respective 1.5ml tube. 200 μL of each samples is aliquoted to the

respective wells of the microplates. There are a total of 8 different microplates which

consists of all the subject’s samples. The serum samples are then analyzed at 6

different time points.

In order to analyze the selected analytes, we made use of Multiplex ELISA assay or

single analyte ELISA assay. The principles of Multiplex ELISA assay differs from the

single analyte ELISA assay.

For the multiplex ELISA assay, we added Antibody Magnetic Bead Mixture to each well

of the 96-Well Flat Bottom Plate and use a Magnetic Plate Washer to attract the

magnetic beads to the bottom of the wells. Since it analyzes 22 different analytes, the

beads mixture contain 22 different types of beads specific to each analyte. Washing is

carried out to remove excess antibody magnetic beads. We then added the serum

samples or standards into the respective well, followed by incubation to allow the

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samples to bind to the primary antibody. Washing is carried out to remove samples not

bound to the antibody. Detection Antibodies Mixture is then added to bind to the

analytes that are bound to the magnetic beads follow by incubation and Washing. Biotin

is conjugated to the detection antibodies. Streptavidin, bound to a fluorescent probe-

PE, has a strong binding affinity to Biotin. After incubation and washing off excess

Streptavidin-PE that is unbound, we use the Bioplex machine to detect the fluorescence

intensity and differentiate between different analyte components bound to each specific

bead. Basically, fluorescence intensity only accounts for the beads bound to a protein

analyte. From there, the Computer will help to plot a logistic-5 parameter graph of the

fluorescence intensity against the concentration of analytes. Results from the analysis

include total bead count, percentage of beads aggregation, the deviation of results the

standards are from the expected and fitprob. Total bead count is the total number of

beads either bound or unbound. A low bead count of <50 may mean that the beads are

aggregated or may be due to low sample concentration and this would mean the

accuracy of the readings are affected and very often, lower bead count would slow

down the readings. The ideal bead count would be 100. The percentage of bead

aggregation would allow us to know if low bead count is due to beads aggregation or

low sample concentration. If the both the bead count and percentage of bead

aggregation is low, it would mean that there low sample concentration. Fitprob tells us

how well the data fits to the standard curve. The closer Fitprob is to 1, the better the

data fits to the curve.

Whereas for the single analytes ELISA assay, we use plates that are already pre-

coated with polyclonal antibodies which target the specific analytes. We then add the

plasma samples or standards into respective wells to allow the antibodies to capture

the analytes. Incubation is done to allow samples to bind to the polyclonal antibodies,

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follow by washing to remove unbound samples. Subsequently, we add biotinylated

secondary antibody that will target the analytes bound to the primary antibody during

incubation and wash off the excess. Streptavidin-Peroxidase Conjugate is added and

washed followed by Chromogen Substrate. Once Chromogen Substrate is added, the

solution turns blue. Lastly, a stop solution is added to turn the solution from blue to

yellow. Absorbance is measured based on the intensity of the yellow solution in each

well. The intensity of the yellow solution is proportional to the concentration of the

analytes in each well. However for HDL ELISA assay, the intensity of yellow solution is

inversely proportional to the concentration of analytes in each well.

The analytes we are currently analyzing in the 22-plex ELISA assay are Leptin,

Resistin, BDNF, EGF,G-CSF, VEGF-A,TNF-α, MIP-α,IFN-α, IFN-γ, MCP-1, TNFRII,

SCD40L, IL-1-ß, IL-1RA, IL-2R, Il-6, IL-7, IL-8, IL-10, IL-13, IL-15. For the 4-plex ELISA

assay, the analytes to be analyze are Adiponectin, RANTES, MMP,PAI-1, while for the

2-plex ELISA assay, the analytes to be analyzed are ApoA and ApoE. Lastly, for the

single analytes to be analyzed are DHEA, DHEA-S, Cortisol, High Density Lipoprotein

(HDL), Insulin, Folate, CRP, MMP9, BDNF, PGP, SBDP, Human GFAP and Gold Dot.

There are currently no kits for the analysis of certain analytes such as Folate, thus I am

responsible for aliquoting the plasma samples into 1.5ml tube to be sent to SGH for

High Performance Liquid Chromatography-Mass Spectrometry (HPLC-MS) analysis.

Other roles include sending blood samples to other laboratories in NUH or SGH and

ensuring that the blood samples are delivered to the right person for testing.

For literature review, the physiological biomarkers are already classified into Tier 1, Tier

1B and Tier 2. Biomarkers from Tier 1 and 1B are those with concrete evidence of their

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capability as MDD diagnosis biomarkers. Biomarkers from Tier 2 are those with certain

evidence that there are altered expressions in MDD patients as compared to normal

controls. I am required to find and read up on other published research paper to

validate and support the findings of these depression biomarkers as well as look for

other plausible novel depression biomarkers that are not stated in the current list. In

addition, I have to classify the different biomarkers according to its involvement in the

different biological mechanisms leading to depression.

Tier 1 physiological markers Tier 2 physiological markers

- DHEA- Cortisol- High Density Lipoprotein- Folate- CRP- Insulin- BDNF- IL-6- TNF- α

- sIL2R- S100 β- Homocysteine- MMP-9- α 2 macroglobulin- Alpha 1 Antitrypsin- Apolipoprotein A1- Serum Acylation Stimulating

protein- Insulin-like growth Factor-1- Prolactin

Tier 1b physiological markers

- IL-1β- Il-7- IL-8- Il-10- IL-13- IL-15- IL-1PA- EGF- CCL2- PAI-1

- RANTES- VEGF-A- TNFRII- CD40-LIGAND- Leptin- MIP-1 α- Adiponectin- IFN- γ- Resistin- G-CSF

2c) Trainings received

Before I officially help out for all the experiments, I was asked to observe how ELISA

assays was done with the actual blood samples from the volunteers. What I have learnt

from the process of ELISA is that the volume of the primary antibodies and detection

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antibodies must not be lesser than the stated volume as we do not know if the

concentration of antibodies would be sufficient for the binding of the analytes in the

wells. Initially when I start helping out for the ELISA assay, my supervisors will first

observe my skills and once I’m capable of handling ELISA, I am allowed to carry out all

ELISA assay myself.

I have been given an opportunity to attend a cognitive scales training workshop which is

a formal training to help diagnose patients with Traumatic Brain Injuries (TBI) as well as

to assess their state of cognitive function. Patients with TBI may suffer from depression

and anxiety because part of their brain controlling emotions has been damaged. Some

patients’ vision may also be affected. Thus tests like Stroop Colour-Word or Colour

Trails tests will help assess the patients’ vision and coordination while questionnaire

like the Hospital Anxiety and Depression (HADS) and World Health Organization

Quality of Life (WHOQOL-BREF) will help to assess their emotional and mental state of

health. It is very important to standardize the test results of the assessment among the

assessor to ensure that results should not vary too much from the expected.

2d) Accomplishments

Based on the literature research, I have really gained much knowledge of depression

as a disease. Initially, I thought that depression was a controllable emotion-sadness.

However, I realized that depression was caused by changes in the concentration of

certain biomarkers in the brain which lead to inflammation of the brain and changes in

the brain structures and only by taking anti-depressant medications would the patient

be able to control depression. There are also many who despite taking anti-depressants

have not been able to recover from depression because depression may also be a

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psychological cause due to severe trauma or certain events that trigger depression.

Thus these patients would only be able to recover with the help of cognitive and

behavior therapy.

I’ve found that the depression biochemical markers are linked to many major pathways

like the Monoaminergic system, Hypothalamic-Pituitary-Adrenal Axis, Neurotrophins,

Biochemical, Immunological and Neurotransmission.

From the results I’ve obtained from the ELISA assay, we can conclude that certain

components in human’s serum may be affected by the activity level of a person. From

the graphs below, sample type 1, 2, 3 are the SAF’s population while sample type 4, 5,

6, 7 are the civilians’ population. When we compare these 2 groups of samples, we can

conclude that there is a significant difference in the concentration levels of components

like BDNF, EGF, SCD40I, etc. Whereas there is no significant difference in the

concentration levels of components such as IFN- α, IFN- γ, IL-10 and etc. Among the

SAF, there are the depressed and control group and we can clearly see a significant

difference in the concentration levels of certain components such as BDNF, EGF and

VEGF- α. Based on literature research, these 3 biomarkers are found to have a lower

concentration in patients with major depressive disorder as compare to controls.

For the civilians’ population, there are also the depressed and control group and we can

clearly see a significant difference in the concentration levels of certain components

such as TNF- α, GCSF and IL-15.

Furthermore, as I was also helping out in the other project, I was able to experience a

small scale Blast trial held at Rifle range. During the preparation for the trial, I was

taught how to measure the concentration of several components such as glucose,

sodium, chloride, etc found in the blood of the rats using an I-STAT machine and I was

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able to observe how surgery was done on the rats by my colleagues and how to insert

cannulas in the small blood vessels of the rats as well as drawing the blood from the

cannula. Not forgetting to mention, I get to see the different organs of the rats during

the dissection to collect the different tissues of the rats.

Initially, I always needed help with the calculations of diluting samples because I’m

rather weak in that topic. However after much practice from both the previous

laboratory I work in and DSO, I’m finally able to calculate the dilutions for each

experiments.

I’ve also matured so much after this internship period by experiencing what it is like to

work in an actual research laboratory with advanced technology and experiencing the

reality of a working life. Working with so many elite researchers has also taught me

team building and proper communication with the rest of my team members to ensure

all experiments proceed accordingly and smoothly. This internship has allowed me to

gain future insights of what it is like to be a researcher and allowed me to consider if

this career path is suited for me. In fact, this project not only provide me an insight of

science research but also provide me an overview in psychology because for this

project we are dealing with people of different mental state and this project has deeply

intrigued me on how science and a mental state of a person can intersect.

In addition, I’ve learnt to be more independent in my work. I realized that the workplace

is rather different from school. In school, we have lecturers to guide us all the time.

However at work, although there will be someone to guide me on the first few weeks,

subsequently, most of the things are to be done myself and sometimes I even had to

overcome my boundaries and deal with things on my own because it is impossible to

rely on others as they are also busy with their own work. Also, because everyone is

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busy with their own work, I cannot solely depend on my supervisors to give me work to

do and there are times I have to be proactive and ask them for some work to do. There

are also times where my work requires me to interact and communicate with people

from other laboratories or companies and I have to send blood samples or other

products to the companies by my own. So this work experience has really trained my

people’s skills.

3. Reflection and Analysis of Internship Experience

3a) Workplace safety

Overall the laboratory is a safe place to work in due to the safety practices conducted.

When I first enter the company, I had to go through a 2 days laboratory safety and

laboratory standard Operation Procedure briefing and was even brought on a laboratory

tour by Ms Lili Tan. Every staff in DSO also has to view the safety slides and complete a

short quiz. We are required to pass the quiz before starting on any experiments. In

addition, I had to practice how to clear up a chemical spill on the spot. Of course, the

chemical was replaced with tap water. Also, most staffs do know where to discard the

used reagents properly. One good practice seen in DSO is that whenever we discard any

reagents into a waste container, we have to write down all reagents discarded to ensure

that the reagents are compatible for mixing. In addition, our lab-in-charge, Ms Lili would

always inform us beforehand if the laboratory exhaust is shutdown so that we will not

enter the laboratory to do experiments that day. Occasionally, there will be a fire drill to

prepare the staffs in case of any fires caused due to experimental errors.

When entering the animals’ room, we have to ensure we were properly dress whereby we

not only do we have to wear lab coats and long pants, we also have to wear head caps

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and shoe covers and in most laboratories in DSO, there will always be a dirty and clean

washing area to prevent contamination.

However, there are certain practices where some staffs failed to comply. Firstly, I’ve

observed that some staffs do not wear their lab coats when handling blood samples or

doing any other experiments. Secondly, some staffs are not properly attired and could be

seen wearing short skirts. These are certain practices to take note of to prevent or

minimize any experimental injuries from occurring.

To improve work environment, I suggest all staffs should first practice proper attire for

laboratory work. I also feel that the reagents should be stored in a more organized manner

for easier access to the reagents.

3b) Challenges and solutions

Before the start of my internship, I was told to be proactive in my work, going the extra

mile. There was once I thought they would be fine if I help them continue the ELISA assay

procedure that I was helping out as it was past the time of incubation, but it turns out they

were not very happy when I didn’t ask before I started doing. Thus it made me confused

as to whether I am supposed to go ahead and do things without constantly asking them or

should I follow their orders? In the end I realized that some things we have to gauge if we

should ask our supervisors or do on our own accord because the project most of the staffs

in DSO are handling are large scale and every experiments are extremely important to

them, so any little errors are required to be accounted for.

Our laboratory session at Ngee Ann was always fun and we were allowed to make

mistakes. However after coming to DSO, research was taken at a serious setting and 13

mistakes are seldom seen because we are handling actual blood samples. Thankfully, I

had an opportunity to work in such setting during my Final Year Project at the Anatomy

Laboratory in NUS thus I was able to adapt well in DSO.

3c) Applying prior knowledge

The immunology module during year 2 plays a huge role in teaching me about the

process of ELISA assay based on the binding affinity of a specific antibody to an antigen.

We were also taught of the applications of ELISA assay and the differences between

direct and indirect ELISA. Also, the immunology module has provided me a learning

platform to handle animals and thus despite not going for the formal animal handling

course, I’m confident in handling the rats. In fact, the Translational medicine module has

also taught me the uses of biomarkers and how it can be used in a drug development

process. Even though our current research is primary and the biomarkers we found may

not necessary be beneficial for future drug development, these biomarkers are important

indicators of depression and we will thus be able minimize the occurrence of depression

by measuring the changes in the levels of biomarkers in the blood samples of patient

relative to the brain since it is an invasive procedure to obtain biomarkers from the brain.

We also had IS modules that helped enhanced my learning experiences in DSO

4. Acknowledgement letter to Company

I would like to express my deepest gratitude to my supervisors Dr Pamela Pun Boon Li,

Dr Teo Ai Ling Melissa and Dr Zhu Congju for their constant supervision and support

over my course of 16 weeks internship at Defence Science Organization and also

provide me a great leaning opportunity in DSO

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I would also like to thank Dr Wujian, Mr Ng Kian Chye and Dr Yong Chiat for providing

me with invaluable advice and guidance throughout the whole course of different

project.

Lastly, I would like to Chan Ying Hui Janlin, for guiding me during the first few weeks of

internship.

I will definitely recommend this internship because DSO is a rather prestige place for

students who are interested in research and students would be able to gain bountiful

knowledge throughout their course of internship in DSO which will be applicable for

their future careers in the life science industry.

Word count: 3515 words (excluding headings or sub-headings)

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Results from 22-plex ELISA assay

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5. Curriculum Vitae (CV)

OBJECTIVEA laboratory researcher position in academic institutions

PERSONAL PARTICULARSName Priscilla Chong Shi Hui

NRIC S9535389D

Nationality Singaporean

Date of Birth 27 September 1995

Residential address Blk 276 Choa Chu Kang Ave 2 #06-301 Singapore 680276

Contact no. 91460408

Email prischongsh@hotmail.com

EDUCATIONQualifications/Name of institution Duration

Diploma in Biomedical Science 2012 - Current

GCE 'O' Level certificate at Dunearn Secondary School

2008 - 2011

PSLE at South View Primary School 2002 - 2007

MAJOR ACHIEVEMENTS/ PARTICIPATION Participated in MOE excel fest 2006

Participated in Intra School Activities

2008

Represented school at West Zone inter school netball C Division Championships

2009

Attended Youth Leader’s camp and achieved a certificate of participation

2009

Participated in University of New South Wales Science Camp

2009

Attained 2010 Edusave Scholarship 2010 Represented school in Pesta Sukan Netball carnival 2010 An executive member of the Student councilor 2010-2011 Participated in Outward Bound Singapore 2011

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Represented school at National Inter School Netball B division Championships

2011

Participated in Overseas Immersion Programme in Wuhan, China

2013

SKILLS & ABILITIESTechnical skills

2D-PAGE Polymerase chain reaction Gel Electrophoresis Western Blot Migration, invasion, proliferation, adhesion assays HPLC ELISA Handling animals DNA and Protein extraction

Language ability Fluent in English writing and speaking. Fluent in Chinese writing and speaking

IT literacy Competent in the use of Microsoft word. Competent in the use of Microsoft powerpoint Able to use softwares like graphpad, biorad-CFX manager

Others Research interests – Breast cancer research, Neuroscience research,

heart transplant research Hobbies – Reading, play basketball, badminton, netball

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WORK AND RESEARCH EXPERIENCE DURATIONFinal Year Project under the supervision of Dr. Zhu Congju from Ngee Ann Polytechnic and Professor George Yip from National university of Singapore

Title: Roles of ECT2 oncoprotein in breast cancer invasiveness

Brief description of project:ECT2 is a guanosine nucleotide exchange factor which regulates several cellular functions such as cell motility, cell adhesion, etc. However, research have shown that ECT2 is overexpressed and mislocalized in many cancers such as lung, glioma and esophagus but have yet to be established in breast cells. Thus our project is to downregulate ECT2 in breast cancer cell line and find out whether it will affect breast cancer invasiveness and migration through cell based assays.

Techniques learnt includes: Western blotting, Quantitative Polymerase Chain Reaction, Agarose Gel Electrophoresis, Transfection, Cell seeding, Cell Based Assay

Intern experience under the supervision of Dr. Zhu Congju from Ngee Ann Polytechnic, Dr Pamela Pun and Dr Melissa Teo from Defence Science Organization

Title: Biomarker for Detection of Depression susceptibility

Brief description of project:Major Depressive Disorder (MDD) is a mental disorder due to several major depressive episodes lasting for more than 2 weeks. As research on certain depression biomarkers have not been fully established, the objective of this research is to identify and thus confirm a list of physiological and genetic biomarkers to boost the efficiency of depression screening in patients.

Techniques learnt includes: ELISA, DNA extraction

Mar 2014 - Sep 2014

Job Title/ Company:- Part time sales assistant at Tango Global

Selling food items Ability to interact with others

- Defence Science Organization Conduct scientific research on biomarkers of

depression

Nov 2008- current

Nov 2014- Feb 2015

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6. References

1. Center for Substance Abuse Treatment. (2008). Appendix D—DSM-IV-TR Mood Disorders.

2. Chong, S. A., Vaingankar, J., Abdin, E., & Subramaniam, M. (2012). The prevalence and impact of major depressive disorder among Chinese, Malays and Indians in an Asian multi-racial population. Journal of affective disorders, 138(1), 128-136.

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