1. Your cells hates stress too: Muscle and Cell’s response
to stress (external vs. internal stress)
Emmanuel Adewa

2. Abstract
 Stress is the specific response by the body to a stimulus that disturbs or interferes with the ‘normal’
physiological equilibrium6. Exercise creates stress that affects the muscles in the body6. Reactive
oxygen species (ROS) creates stress that affects the cell. ROS at low concentrations are important
regulators of physiological processes, such as receptor-mediated cell signaling, cell proliferation and
transcription activation1. However, ROS are deleterious at high levels (DNA damage)1. The purpose of
this study is to observe how the body responds to exercise and how the cells responds to H2O2
(oxidaive damage). To observe the response of the muscle to exercise, a fitness experiment was
performed (squats) twice a week for 10 weeks. To observe the cell’s response to oxidative stress, a
microarray experiment was performed using HeLa cells treated with H2O2 (600 μM). The regulation of
TXNRD2, a gene known for H2O2 scavenging was especially observe from the microarray
experiment. The fitness experiment showed that stress results in the recruitment and strengthening of
muscle fibers, hence, improving fitness (jumped height increased by 10.5 cm). The microarray
experiment showed that cells upregulates and down regulates its gene to help in adapting to stress
produced by internal stressors (H2O2). The most upregulated genes has function for synthesis of
proteins involved in the oxidoreductase pathway while the most downregulated genes has function for
cell apoptosis and signaling (not involved in oxidoreductase activities). TXNRD2 downregulation
showed that the cells are undergoing H2O2-induced apoptosis due to the high levels of H2O2
7. In
conclusion, this results are interesting because is shows that the body is equipped to deal with
changes in the environment (external or internal). External stressors like exercise results in a
response like motor unit recruitment and muscle fiber stimulation and strecthing8,2. Internal stressors
like elevated H2O2, lead to cells protecting itself by upregulating genes that functions for protection
and downregulation of genes that do not. Too high levels of H2O2 would result in the initiation of H2O2-
induced apoptosis which in itself results in the upregulation and downregulation of genes.

3. Introduction
 Stress is the specific response by the body to a stimulus/stressor that
disturbs or interferes with the ‘normal’ physiological equilibrium6
 Stress induces many physiological changes and adaptations2
• hormone levels, cell structure and function2
 Exercise creates stress that affects the muscles in the body6
• the degree of stress it creates is influenced greatly by the intensity and
duration of the exercise6
 Reactive oxygen species (ROS) creates stress that affects the cell. ROS at
low concentrations are important regulators of physiological processes (cell
proliferation, cell signaling) however at high levels they are deleterious
(DNA, lipid damage, Cell apoptosis)1
• Cells regulate the level of ROS (H2O2) in mitochondria by upregulating
genes like TXNRD24
A great observation to make is the effect of exercise on fitness
(muscle) and the effect of H2O2 on cells?

4.  To observe the body's response to stress (exercise), a fitness
experiment was done using Jump squats. Jump squats is a
great fitness variable because it improves Vertical Jump &
Horizontal Jump by increasing the muscle density (lower body)
of the quadriceps, hamstring and calf muscle8,2
 To observe how cells respond to oxidative stress, a microarray
experiment was done on HeLa cells treated with H2O2 (ROS)
• HeLa cells gives us the ability to observe this response outside
the body (In Vitro)

5. Fitness Regimen
Jump Squat
Design: 5 Sets of 8 Repetition: Twice a week Duration: 10 weeks
Fitness Component
Anaerobic (Experimental) Flexibility (control)
• Vertical Jump
• Horizontal Jump
• Sit &Reach
• Groin Flexibility
• Trunk Rotation
• Shoulder Flexibility

6. Fitness Method
Horizontal Jump Test
• Equipment: Athletic Footwear,
Landing Mat, tape measure
• Procedure: Repeat 3 times, 1
min rest between trial
• Measurement: distance
travelled from take off point to
landing point
Vertical Jump Test
• Equipment: Timing Mat
• Procedure: Repeat 3 times, 1
min rest between trial
• Measurement: Takeoff Speed,
Jump Height & Flight time for
Leg power calculation

7. Sit & reach Test
• Equipment: “Flex-Tester”
Unit
• Procedure: Repeat 3 times
(3 trials)
• Measurement: distance
between start point and end
point of finger plate
Trunk Rotation Test
• Equipment: Wall Space, Chalk, Tape
Measure
• Procedure: Repeat 2 time with both
right and left trunk
• Measurement: distance between
marked line and fingertips line
Groin Flexibility Test
• Equipment: Ruler
• Measurement: distance
between sole of feet and
groin
Shoulder Flexibility Test
• Equipment: Ruler
• Procedure: done with both left and right
shoulder
• Measurement: distance between
fingertips, if fingers touch record as
good.

8. Result - Fitness
 Anaerobic
Baseline 1
Baseline 2
450
500
550
600
650
0 2 4 6 8 10
Flighttime(ms)
Weeks
Flight time
Figure 2. Flight time of vertical jumps. Each data point indicates
the average of 3 trials. Increase of about 78.5 ms.
Baseline 1
Baseline 2
2.4
2.6
2.8
3
0 2 4 6 8 10
TakeoffSpeed(m/s)
Weeks
Takeoff Speed
Figure 3. Takeoff speed of vertical jumps. Each data
point indicates the average of 3 trials. Increase of about
0.38 m/s
Baseline 1
Baseline 2
28
33
38
43
48
0 2 4 6 8 10
JumpedHeight(cm)
Weeks
Jumped Height
Figure 4. Jumped height of vertical jumps. Each data point
indicates the average of 3 trials. Increase of about 10.5 cm
Baseline 1
Baseline 2
80
85
90
95
100
0 2 4 6 8 10
LegPower(Kg/m/s)
Weeks
Leg Power (Kg/m/s)
Figure 5. Leg power calculated using the maximum jump
height of 3 trails in each data point. Leg Power (kgm/s) =
2.21 x body mass (kg) x √ best jump (m). Increase of about
11.8 Kg/m/s
• The measured and calculated parameters of vertical jump increased
through the 10 week.
Vertical Jump Test

9.  Flexibility
Baseline 1
Baseline 2
2.5
2.55
2.6
2.65
2.7
0 2 4 6 8 10
HorizontalJump(m)
Weeks
Horizontal Jump
Figure 6. Distance travelled in horizontal jumps. Each data point
indicates the average of 3 trials. No observable increase
% Rank Females Males
91-100 2.94 - 3.15 metres 3.40 - 3.75 metres
81 - 90 2.80 - 2.93 metres 3.10 - 3.39 metres
71 - 80 2.65 - 2.79 metres 2.95 - 3.09 metres
61 - 70 2.50 - 2.64 metres 2.80 - 2.94 metres
51 - 60 2.35 - 2.49 metres 2.65 - 2.79 metres
41 - 50 2.20 - 2.34 metres 2.50 - 2.64 metres
31 - 40 2.05 - 2.19 metres 2.35 - 2.49 metres
21 - 30 1.90 - 2.04 metres 2.20 - 2.34 metres
11-20 1.75 - 1.89 metres 2.05 - 2.19 metres
1-10 1.60 - 1.74 metres 1.90 - 2.04 metres
Adapted from: Chu, D.A. (1996) Explosive Power and Strength.
Champaign: Human Kinetics. p. 171
Performance
rank
Horizontal Jump Test
• No observable increase in horizontal jump distance
• Procedure errors most likely the reason for the lack of increase (no Rest
time between trials at week 7) and not necessarily the lack of variable effect
• Performance rank shows a slight increase in % rank
Baseline 1
Baseline 2
12.5
13
13.5
14
14.5
15
0 2 4 6 8 10
Sit&Reach(inch)
Weeks
Sit and Reach Test
Figure 6. Distance travelled by finger plate on flex tester unit.
Each data point indicates the average of 3 trials. Increase of
about 0.65 inches
Baseline 1
Baseline 2
Baseline 1
Baseline 2
0
10
20
30
40
50
60
70
0 2 4 6 8 10
TrunkRotation(cm)
Weeks
Trunk Rotation Test Right Trunk
Left Trunk
Figure 7. Distance between fingertip line and marked line
in trunk rotation test. Each data point indicates the
average of 2 trials. Right trunk increase = 12.8 cm, left
trunk = 10.7cm
Table 1 % rank chart for horizontal jump

10. Weeks Right Left
1 Good Good
2 Good Good
3 Good Good
4 - -
5 Good Good
6 - -
7 Good Good
8 - -
9 - -
10 Good Good
Baseline 1
Baseline 2
0
2
4
6
8
10
12
14
0 2 4 6 8 10
Groin(cm)
Weeks
Groin Flexibility
Lower = Better
Figure 8. Distance between groin and sole of feet. Lower
flexibility means better flexibility. Decrease of about 3cm
Table 2. Shoulder flexibility test for left and right
shoulder
Good = Left and right fingertips are touching
• Anaerobic fitness (vertical jump) improved with Jump squats regimen.
HOW?
• Increased stress elicits high degree of motor unit recruitment and muscle
fiber stimulation(quadriceps, hamstring and calf muscle), which means
increased metabolic rate (oxidation of glycogen and triacylglycerol) as
reflected by increasing rates of chemical reactions, oxygen consumption,
and substrate depletion8,2. More exercise (stress) = more muscle strength.
• Flexibility was slightly improved, a result that can be described as an
indirectly effect of jump squat.
WHY?
• With the stimulation of muscle fiber, stretching of the muscle fiber also
occurs which means improvement in the flexibility of the lower body

11. Microarray Methods
Control PopulationTreated Population
Cy 5 Cy 3
Control = HeLa cellsTreated = HeLa cells + H2O2
Figure 1. Microarray experiment method. Treated population = 600 μM H2O2 + HeLa cells incubated for 24 hours. Control population = untreated
HeLa cells incubated for 24 hours. Cy 5 & 3 = cyanine fluorescent dye.

12. Gene Name Expression
Value
Biological Process
PHKB_rep1 1.98 Protein Phosphorylation
UBE2C_rep1 0.83 Protein Ubiquitination
WASL_rep3 0.77 Regulation of protein localization
C9orf114_rep4 0.67 Unknown function
C17orf70_rep3 0.44 DNA repair
C1orf126 0.44 Unknown function
C2orf56 0.44 DNA methylation for the locking
of gene in on or off position
(Oxidoreductase activity)
IFITM3 0.41 Defense response to virus
CD22_rep3 0.38 Cell surface receptor signaling
pathway
WT1_rep1 0.38 Positive regulation of
transcription
CPSF2_rep3 0.34 RNA splicing, processing and
polyadenylation
FHL5_rep2 0.34 Positive regulation of
transcription
TMEM25_rep2 0.34 Unknown function
PFN1_rep3 0.32 Actin cytoskeleton organization
AHCY 0.31 DNA methylation for the locking
of gene in on or off position
(Oxidoreductase activity)
GTF2H4 0.31 DNA repair
TMEM205 0.31 Unknown function
ZNF74_rep1 0.31 Positive Regulation of
transcription
ACTB 0.3 Post translational protein folding
CPNE1_rep5 0.3 Vesicle mediated transport
 Top 20 Upregulated genes
• Genes grouped by biological process(gene ontology)
Protein
Modification &
Degration
Cell Defence
Unknown
Signalling
Activity
DNA
Modification &
Repair
Transport
Activity
Transcription
Factors
RNA
modification
Cytoskeleton
Organization
Table 3. Description of top 20 upregulated genes
Figure 10. Gene ontology of the top 20 upregulated gene. Genes
grouped based on biological process.
• The most upregulated genes are
genes involved in the repair and
modification of damaged DNA
(effect of H2O2)
Result - Microarray

13.  Top 20 Downregulated genes
• Genes grouped by biological process(gene ontology)
Gene Name Expression
Value
Biological Process
YARS2_rep2 -2.13 RNA amino-acylation
LSM2 -1.75 RNA processing
KRT4 -1.65 Negative regulation of epithelial
proliferation
GPRC5B -1.62 Signal transduction
AURKC -1.56 Protein phosphorylation
ITPR3 -1.53 Second messenger that
mediated the release of
intracellular calcium
YARS2_rep1 -1.51 RNA amino-acylation
PLP1 -1.5 Cell death
ZNF212_rep1 -1.5 Regulation of transcription
COPS2_rep1 -1.47 Signal transduction
PLA2G7 -1.46 Cellular protein metabolic
process
TAF9 -1.46 Regulation of apoptotic process
OSBPL6_rep1 -1.45 Lipid transport
THBS3 -1.45 Cell adhesion
COLEC12_rep2 -1.44 Carbohydrate mediated
signaling
ABI3BP -1.43 Regulation of cell substrate
adhesion
C5orf22_rep1 -1.43 Regulation of RAS protein signal
SLCO1A2 -1.43 Transmembrane transport
activity
KSR1 -1.41 RAS protein signal transduction
RPS27A -1.41 Regulation of apoptotic
Table 4. Description of top 20 downregulated genes
RNA
Processing
Transcription
Activity
Apoptotic
Process
Cell
Signalling
Transport
Activity
Protein
Modification
Cell
Differentiatio
n & Adhesion
Figure 11. Gene ontology of the top 20 downregulated gene. Genes
grouped based on biological process.
The most downregulated genes are
genes involved in signaling of
processes like cell apoptosis (does
not contribute t the protection of the
cell from H2O2 induced damages

14.  Comparative analysis of top 20 induced and repressed genes
-3 -2 -1 0 1
YARS2_rep2
LSM2
KRT4
GPRC5B
AURKC
ITPR3
YARS2_rep1
PLP1
ZNF212_rep1
COPS2_rep1
PLA2G7
TAF9
OSBPL6_rep1
THBS3
COLEC12_rep2
ABI3BP
C5orf22_rep1
SLCO1A2
KSR1
RPS27A Rep2
Rep1
Pilot
Experiment
-1.5 -1 -0.5 0 0.5 1 1.5 2 2.5
PHKB_rep1
UBE2C_rep1
WASL_rep3
C9orf114_rep4
C17orf70_rep3
C1orf126
C2orf56
IFITM3
CD22_rep3
WT1_rep1
CPSF2_rep3
FHL5_rep2
TMEM25_rep2
PFN1_rep3
AHCY
GTF2H4
TMEM205
ZNF74_rep1
ACTB
CPNE1_rep5 Rep2
Rep1
Pilot
Experiment
Log2 (gene Expression Relative to Control) Log2 (gene Expression Relative to Control)
Figure 9. Comparative analysis of the top 20 upregulated and downregulated genes. A = top upregulated genes, B= to downregulated
genes. Expression values of individual experiment were compared to 2 fellow student and a previously performed pilot data. Expression
value of 0 means that the gene was missing in the data
A B
• Several genes are oppositely regulated amongst data. Microarray experiment is a
highly variable experiment
• Genes that are similarly regulated have biological process associated with cell
response to H2O2 stress (Oxidoreductase activity, DNA repair, Cell defense, Cell
apoptosis regulation)

15. TXNRD2 Function
 Gene Name: Thioredoxin reductase 2
• NADPH-dependent members of the pyridine nucleotide-disulfide
oxidoreductase family, form homodimers, possess two interacting
redox-active centers and located in mitochondria5
 Chromosome location: 22 (q11.21)3
 Function: codes an enzyme that catalyzes the reduction of the active site
disulfide of thioredoxin and other substrates3
• Maintains thioredoxin in reduced state for defense against oxidative
stress (cellular redox homeostasis)
-1.1 -0.9 -0.7 -0.5 -0.3 -0.1 0.1
TXNRD2
Rep2
Rep1
Pilot
Experiment
Log2 (gene Expression Relative to Control)
Figure 12. Comparative analysis of the TXNRD2. . Expression values of individual experiment were compared to 2 fellow student and a
previously performed pilot data
Why is it downregulated?

16. TXNRD2 Downregulation Hypothesis
 Hypothesis
• TXNRD2 is upregulated for the reduction of H2O2, however excessive
H2O2 would result in H2O2-induced apoptosis4
• The downregulation of TXNRD2 is part of the Apoptotic pathway
(Apoptosis regulation)7. A-C pathway is active(figure 13)
Protection against oxidative stress
catalyze
Oxidative stress
Downregulation
NADP+
NADPH
H2O2-induced cell Apoptosis
x
Reduce
thioredoxin
Oxidized
thioredoxin
E-
TXNRD2
H+
Inactive active
H2O2
Figure 13. Pathway of upregulation and downregulation of
TXNRD2. 1 – 5 = the pathway of oxidoreductase activity of gene
in response to oxidative stress. A – c = the pathway for H2O2
induced apoptosis
X – inactivation
– Acts on
Activation/upregulation
1 A
2
3
4
B
C
5

17. TXNRD2 and Cell Apoptosis
catalyze
Oxidative stress
Downregulation
NADP+
NADPH
x
Reduce
thioredoxin
Oxidized
thioredoxin
E-
TXNRD2
H+
active
H2O2
Activation/upregulation
1 A
2
3
4
B
C
5
Protection against oxidative stress
H2O2-induced cell Apoptosis
Inactive
H2O2
• The knock out of TXNRD2 gene linked
to cell apoptosis
• The downregulation of TXNRD2 is a
process involved in cell apoptosis

18. Discussion/Conclusion
 This results are interesting because is shows that the body is equipped
to deal with changes in the environment (external or internal)
 External stressors like exercise results in a response like motor unit
recruitment and muscle fiber stimulation and strecthing8,2.
 For internal stress like elevated H2O2, the cells protects itself by
upregulating genes that functions to protect it and downregulation of
genes that do not.
• Too high levels of H2O2 would result in the initiation of H2O2- induced
apoptosis which in itself results in the upregulation and
downregulation of genes.
 Future directions:
• Observing the effect of other ROS on cells
• The relationship between strengthening of muscle and flexibility

19. Reference
1. Conrad M et al. Essential Role for Mitochondrial Thioredoxin Reductase in Hematopoiesis,
Heart Development and Heart Function. Molecular and cellular biology. 2004; 24(21): 9414–
9423
2. Coyle EF. Physical Activity as a Metabolic Stressor. The American Journal of Clinical
Nutrition. 2000; 72: 512S–520S
3. Ingenuity target explorer. [Cited 2013 Nov 20]. Available from:
https://targetexplorer.ingenuity.com
4. Sibbing D et al. Mutation in the Mitochondrial Thioredoxin Reductase Gene TXNRD2 cause
Dilated Cardiomyopathy
5. Conrad M and Bornkamm GW. Selenium: Its Molecular Biology and Role in Human
Health. Springer, 2007; chap 18 - 195
6. Hackney AC. Exercise as a stressor to the human neuroendocrine system. Medicina
(Kaunas). 2006; 42(10)
7. Whyte G. The role antioxidants play in preventing exercise-induced damage to your muscles
[Cited 2013 Nov 20]. Available from: http://www.pponline.co.uk/encyc/0888.htm
8. Fernandez CB, Tejero-Gonzalez CM, Curiel DA, The Effects of a Maximal Power Training
Cycle on the Strength, Maximum Power, Vertical Jump Height and Acceleration of High-
Level 400-Meter Hurdlers. Journal of Human Kinetics. 2013; 36:119-126