PhD defense

1. Characterization and identification of hypotensive,
immunomodulatory and metabolic disorder benefiting peptides from
Atlantic mackerel (Scomber scombrus) hydrolysate separated based
on molecular weight, charge and hydrophobicity
Dissertation Defense by
Soheila Abachi
June 2021
Supervisor: Prof. Lucie Beaulieu
Co-supervisor: Prof. Laurent Bazinet

2. Outline of presentation
Introduction
Hypothesis and objective
Design and analysis
Methodology, results and conclusion
(objectives 1, 2 & 3)
General conclusion
Future directions
Acknowledgment & references
2

3. Introduction: global issues
3

4. Non communicable diseases: >50% of deaths
CVD: >30% of deaths
Introduction: global issues
4

5. Non communicable diseases: >50% of deaths
CVD: >30% of deaths
Diabetes: 592 million by 2035
Overweight: >1.9 billion adults
Obesity: 13% of population
T2D costs France:
Indirect €8.5 billion/y
Direct €19.5 billion (2003)
Inflammation cause or effect of metabolic diseases
Introduction: global issues
5

6. Introduction: treatment
Synthetic treatments
Side effects & non-compliance issue
Usually mono-functional
6

7. Introduction: treatment
Synthetic treatments
Side effects & non-compliance issue
Usually mono-functional
Natural treatments
Commonly pose no adverse effects
Food-derived biomolecules
Non-cytotoxic, generally dual- & multi-functional
Could be incorporated into diet or treatment regimen
In demand by health-conscious customers
7

8. Introduction: fish biopeptides
Byproducts, discards &
Underutilized species (e.g., Atlantic mackerel)
Different compositional features & properties
Some marketed as therapeutics
Beneficial on immunity, MetS & risk factors
Derived, separated & purified by various techniques
Mainly enzymatic hydrolysis, filtration & chromatography
Newly designed EDUF technique
8

9. Introduction: mackerel biopeptides
Horse, blue, Indian,
Spanish mackerel:
Antioxidant
Horse, chub mackerel &
heshiko:
Anti-hyperlipidemic
Atlantic mackerel:
Antioxidant, antihypertensive,
antibacterial, antithrombotic
& anti-inflammatory effects
9

10. Hypothesis
Atlantic mackerel (Scomber scombrus) bioactive-peptide fraction(s)
isolated and separated by different techniques based on various
molecular characteristics possess beneficial biological activity on
hypertension, inflammation and other metabolic syndrome (MetS)
factors.
10

11. Main objective
Fractionation and identification of hypotensive,
immunomodulating and anti-MetS peptides with different
molecular weight, charge and hydrophobicity characteristics using
chromatographic, membrane filtration & electro-separation
techniques
11

12. Design and analysis
Experimental design
Completely randomized design
Conducted in triplicates, 2-7 independent studies
12

13. Design and analysis
Experimental design
Completely randomized design
Conducted in triplicates, 2-7 independent studies
Statistical analysis
Anderson-Darling Normality test (P > 0.05)
One way-ANOVA, Tukey’s multiple comparison (P < 0.05)
Student t-test for determination of significant differences between
control and treatment (P < 0.05)
13

14. OBJECTIVE 1
Isolation of immuno-modulatory biopeptides from Atlantic mackerel
(Scomber scombrus) protein hydrolysate based on molecular
weight, charge, and hydrophobicity
would be submitted for publication to the Journal Of Food Bioactives
14

15. Objective 1
Separation of anti- and pro-inflammatory peptides from hydrolysate using:
I- EDUF: MWCO 20 kDa (pH 3, 6 and 9)
II- Pressure driven UF: MWCO 1 kDa
III- SPE: hydrophobic peptides
15

16. Obj.1- Methodology: hydrolysis
Mackerel lysate provided by Merinov, QC, Canada
Enzymatic hydrolysis: protamex
E: S ratio 1: 1000
Process: 120 min.
Heat enzyme inactivation
200 – 10,000 Da
16

17. Obj.1- Results: properties
Chemical composition
Protein Moisture Lipid Ash
Atlantic mackerel (g 100 g-1 wet weight) 18.4 ± 0.3 64.0 ± 0.1 14.1 ± 0.3 2.3 ± 0.0
Hydrolysate (g 100 g-1 dry weight) 90.4 ± 0.2 0.9 ± 0.1 0.0 ± 0.0 6.4 ± 0.0
17

18. Obj.1- Results: properties
Chemical composition
Molecular weight
Pre-hydrolysis
Dehydrated NF retentate
18

19. Obj.1- Results: properties
Chemical composition
Molecular weight
Peptide characteristics
Enzymes Alpha actin
Cytochrome B Brain super conserved receptor
Zinc finger protein Histone H3
Other
19

20. Chemical composition
Molecular weight
Peptide characteristics
Total amino acid (g/100g protein)
High charged & hydrophobic amino acids
EAA 38.8 ± 0.2
NEAA 45.0 ± 0.3
AA 83.7 ± 0.2
BCAA 12.5 ± 0.2
CAA 48.4 ± 0.4
HAA 32.8 ± 0.2
Obj.1- Results: properties
20

21. Obj.1- Methodology: separation
techniques
EDUF
Charge & Size (<20 kDa, > 20 kDa)
21

22. Obj.1- Methodology: separation
techniques
EDUF
Charge & Size (<20 kDa, > 20 kDa)
Pressure-driven UF
Size (<1 kDa)
22

23. Obj.1- Methodology: separation
techniques
EDUF
Charge & Size (<20 kDa, > 20 kDa)
Pressure-driven UF
Size (<1 kDa)
SPE
Hydrophobicity
23

24. Obj.1- Results: migration rate
Isolation efficiency of EDUF
Effect of pH on migration pattern
Measured by BCA
Cationic
pH3 > pH6 > pH9
a
a
a
a
a
a
a
ab
ab
b
a a
b
b
b
0
250
500
0 30 60 120 180
Concentration
(µg
mL
-1
)
Time (min)
Cationic peptides
pH3 pH6 pH9
24

25. Obj.1- Results: migration rate
Isolation efficiency of EDUF
Effect of different pH on migration pattern
Measured by BCA
Cationic
pH3 > pH6 > pH9
Anionic
pH6 & pH9 > pH3
Total migration
pH3 > pH9 > pH6
a
a
a
b
b
a
a
a
a
ab
a
a
a
a
a
0
250
500
0 30 60 120 180
Concentration
(µg
mL
-1
)
Time (min)
Anionic peptides
pH3 pH6 pH9
25

26. Obj.1- Methodology: immunomodulation
activity
NO reduction and or stimulation effect
As index of iNOS activity
Based on Griess reaction
Read at 540nm
Positive control
Metformin and phenformin
% inflammation reported
J774A.1 macrophages
ATCC® TIB-67™
Overnight treatment of
LPS stimulated cells
Cytotoxicity of compounds
BCA assay
26

27. Obj.1- Results: anti- & pro-inflammatory
activity
Nitrite accumulation quantified as measure of NOS activity
pH3: pro-NO
*
*
*
0
100
LPS
2.5
ng
mL-1
P+3
1
ng
mL-1
P+3
1
ug
mL-1
P+3
10
ug
mL-1
P-3
1
ng
mL-1
P-3
1
ug
mL-1
P-3
10
ug
mL-1
F3
1
ng
mL-3
F3
1
ug
mL-1
F3
10
ug
mL-1
Phen
100
uM
%
inflammation
pH3
Cationic
peptides
Feed
recovery
Anionic
peptides
27

28. *
0
100
LPS
2.5
ng
mL-1
P+6
1
ng
mL-1
P+6
1
ug
mL-1
P+6
10
ug
mL-1
P-6
1
ng
mL-1
P-6
1
ug
mL-1
P-6
10
ug
mL-1
F6
1
ng
mL-1
F6
1
ug
mL-1
F6
10
ug
mL-1
Phen
100
uM
%
Inflammation
pH6
Obj.1- Results: anti- & pro-inflammatory
activity
Nitrite accumulation quantified as measure of NOS activity
pH3: pro-NO
pH6: insignificant Cationic
peptides
Feed
recovery
Anionic
peptides
28

29. Nitrite accumulation quantified as measure of NOS activity
pH3: pro-NO
pH6: insignificant
pH9: insignificant
*
0
100
LPS
2.5
ng
mL-1
P+9
1
ng
mL-1
P+9
1
ug
mL-1
P+9
10
ug
mL-1
P-9
1
ng
mL-1
P-9
1
ug
mL-1
P-9
10
ug
mL-1
F9
1
ng
mL-1
F9
1
ug
mL-1
F9
10
ug
mL-1
Phen
100
uM
%
Inflammation
Axis Title
pH9
Obj.1- Results: anti- & pro-inflammatory
activity
Cationic
peptides
Feed
recovery
Anionic
peptides
29

30. Nitrite accumulation quantified as measure of NOS activity
pH3: pro-NO
pH6: insignificant
pH9: insignificant
Hydrolysate: pro-NO
SPE-70%ACN: anti-NO
Obj.1- Results: anti- & pro-inflammatory
activity
Hydrolysate SPE-70%ACN
*
**
*
0
100
LPS
2.5
ng
mL-1
Phen
100
uM
1
ng
mL-1
1
ug
mL-1
10
ug
mL-1
1
ng
mL-1
1
µg
mL-1
10
µg
mL-1
%
Inflammation
30

31. Obj.1- Methodology: characterization
RP-UPLC
Total amino acid analysis (EDUF & UF fractions)
FPLC
Molecular weight distribution (EDUF fractions)
LC-MS/MS IM-Q-TOF
Sequence and precursor proteins (EDUF fractions)
1+9+2
1+9
1+9
31

32. Obj.1- Results: amino acid composition
EDUF total amino acids (g/100g protein)
Among charged fractions: highest
BCAA: pH6 & pH9 anionic fractions
CAA: pH6 anionic fraction
HAA: pH9 anionic fraction
pH3 pH6 pH9
Hydrolysate Feed recovery
Cationic
peptides
Anionic
peptides Feed recovery
Cationic
peptides
Anionic
peptides Feed recovery
Cationic
peptides
Anionic
peptides
EAA 38.8 ± 0.2 37.3 ± 3.0abc 40.4 ± 0.6abc 23.9 ± 0.4d 33.4 ± 4.2c 45.0 ± 1.8a 25.1 ± 1.6d 35.5 ± 0.5bc 42.7 ± 1.0ab 33.4 ± 0.9c
NEAA 45.0 ± 0.3 44.7 ± 2.4bc 27.1 ± 0.7f 35.3 ± 0.0e 39.0 ± 0.5de 19.2 ± 0.6g 48.5 ± 2.3b 41.8 ± 1.2cd 17.8 ± 0.1g 39.0 ± 1.0a
AA 83.7 ± 0.2 82.0 ± 5.5 67.5 ± 1.3 59.3 ± 0.4 72.4 ± 4.8 64.2 ± 1.2 73.6 ± 3.8 77.4 ± 1.6 60.5 ± 1.1 91.8 ± 1.9
BCAA 12.5 ± 0.2 12.4 ± 0.7ab 9.6 ± 0.0bc 9.5 ± 0.1bc 11.2 ± 2.4abc 8.7 ± 2.0bc 11.5 ± 0.6abc 11.3 ± 0.8abc 7.4 ± 0.2c 11.2 ± 0.5a
CAA 48.4 ± 0.4 39.0 ± 1.1ab 38.4 ± 0.8bc 30.1 ± 0.0d 34.3 ± 1.4cd 37.1 ± 0.8bc 38.0 ± 1.9bc 37.6 ± 1.1bc 36.7 ± 0.8bc 34.3 ± 0.7a
HAA 32.8 ± 0.2 32.4 ± 2.9ab 22.3 ± 0.3bc 23.1 ± 0.0d 29.0 ± 2.9cd 21.4 ± 1.7bc 27.5 ± 1.5bc 30.3 ± 0.3bc 18.9 ± 0.3bc 29.0 ± 1.2a
32

33. Pressure-driven UF total amino acids (g/100g protein)
Among fractions: highest
BCAA: retentate
CAA: retentate
HAA: retentate
Hydrolysate Permeate Retentate
EAA 38.8 ± 0.2 28.6 ± 0.2a 35.2 ± 2.4a
NEAA 45.0 ± 0.3 28.7 ± 1.0b 42.6 ± 1.1b
AA 83.7 ± 0.2 57.3 ± 0.8 77.7 ± 3.5
BCAA 12.5 ± 0.2 9.0 ± 0.2a 11.6 ± 1.2a
CAA 48.4 ± 0.4 25.0 ± 1.6b 36.8 ± 1.9b
HAA 32.8 ± 0.2 24.9 ± 0.4b 30.9 ± 1.2b
Obj.1- Results: amino acid composition
33

34. Oligopeptides of 2 – 6 amino acids <1000 Da
Free amino acids & water-soluble components <100 Da
Anionic fractions: 8 – 11%
Cationic fractions: 4 – 7%
Feed recovery: 5 – 6%
Obj.1- Results: molecular weight
distribution
Fractions % > 1000 Da % < 1000 Da % per compartment
pH3 Feed recovery 47 53 88
Cationic peptides 32 68 9
Anionic peptides 26 74 3
pH6 Feed recovery 47 53 63
Cationic peptides 30 70 10
Anionic peptides 43 57 27
pH9 Feed recovery 46 54 72
Cationic peptides 34 66 9
Anionic peptides 31 69 20
34

35. Obj.1- Results: peptide identification
EDUF: protein precursors mainly
Enzymes such as:
NADH dehydrogenase,
ATP synthase,
cytochrome oxidase & …
Tissue proteins such as:
α-actin, cardiac muscle & …
50 different precursor proteins
35

36. Obj.1- Conclusion
Hydrophobicity more vital for anti-inflammatory effect
Charge more vital for pro-inflammatory effect
pH3 more efficient in separating pro-inflammatory biopeptides
Negative charge more effectual on immuno-stimulating
attribute
Small size not important for immunoregulatory properties of
biopeptides
36

37. OBJECTIVE 2
Identification of gastrointestinal digestion stable antihypertensive
fish peptides from Atlantic mackerel (Scomber scombrus)
would be submitted for publication to the Journal Of Food Science And Human
Wellness
37

38. Objective 2
Separation of hypotensive peptides from hydrolysate using:
I- EDUF: MWCO 20 kDa (pH 3, 6 and 9)
II- Pressure driven UF: MWCO 1 kDa
III- SPE: hydrophilic and hydrophobic peptides
38

39. Obj.2- Methodology: hypotensive activity
Anti-ACE effect
Spectrophotometry method
Positive control: ACE inhibitor enalapril
% enzyme activity inhibition reported
Hippuryl-L-histidyl-L-leucine
(synthetic substrate)
ACE
+
H2O
+ 2,4,6-trichloro-s-triazine
(colorimetric reagent)
Hippuric Acid + His-Leu
Yellow color
read at 382nm
y = 10.615x0.3586
R² = 0.9171
0
20
40
60
80
100
120
140
0 200 400 600 800 1000
%
inhibition
Concentration (µM)
39

40. Obj.2- Results: anti-ACE activity
EDUF not suitable for production of hypotensive peptides
a
a
b
c
a
b
bc
c
a
a
a
a
a
b
b
b
40 20 10 5
a
a
b
c
a
ab
bc
c
a
b
c
d
a
b
c c
0
50
100
40 20 10 5
a
a
b
c
a
b
bc
c
a
a
b
b
a
ab
b
c
40 20 10 5
Total hydrolysate Feed recovery Positive peptides Negative peptides
pH3 pH9
pH6
Peptide concentration (µg)
%
Inhibition
40

41. Obj.2- Results: anti-ACE activity
EDUF not suitable for production of hypotensive peptides
UF could not improve anti-ACE effect of hydrolysate
UF
Peptide concentration (µg)
%
Inhibition
a
a
b
c
a
ab ab
b
a
a
b
b
0
50
100
40 20 10 5
Total hydrolysate
Retentate
Permeate
41

42. Obj.2- Results: anti-ACE activity
EDUF not suitable for production of hypotensive peptides
UF could not improve anti-ACE effect of hydrolysate
SPE enhanced bioactivity of hydrolysate
a
ab
bc
c
a
a
ab
b
a
ab
bc
c
a
ab
bc
c
0
50
100
40 20 10 5
Total hydrolysate
70% ACN
55% ACN
10% ACN
SPE
Fraction concentration (µg)
%
Inhibition
42

43. Obj.2- Results: anti-ACE activity
EDUF not suitable for production of hypotensive peptides
UF could not improve anti-ACE effect of hydrolysate
SPE enhanced bioactivity of hydrolysate
IC50 (µg protein)
Hydrolysate 8.8 ± 0.1def
UF Permeate (< 1 kDa) 9.0 ± 0.4def
Retentate (> 1 kDa) 8.2 ± 3.7ef
SPE 10% ACN 14.3 ± 5.2cde
55% ACN 5.7 ± 2.6f
70% ACN 5.0 ± 2.9f
EDUF pH3 Neutral peptides 21.5 ± 3.2bcd
Positive peptides (< 20 kDa) 23.2 ± 0.4bc
Negative peptides (< 20 kDa) 43.7 ± 0.4a
pH6 Neutral peptides 27.3 ± 6.1bc
Positive peptides (< 20 kDa) 23.3 ± 5.9bc
Negative peptides (< 20 kDa) 40.8 ± 4.3a
pH9 Neutral peptides 8.9 ± 0.2def
Positive peptides (< 20 kDa) 11.5 ± 0.5cdef
Negative peptides (< 20 kDa) 9.4 ± 0.0def
43

44. Obj.2- Methodology: digestion stability
Static simulated GI digestion model
Continuous &
Non-continuous:
Pepsin, trypsin & pancreatin
Control:
sample + inactivated enzymes
% ACE inhibition activity test
Oral:
pH 6.8, 2 min.
Gastric:
pepsin, pH 2, 60 min.
Duodenal:
pancreatin, trypsin, bile extract
pH 7.5, 30 min.
Intestinal:
acetone powder,
pH 8.2, 30 min.
44

45. Obj.2- Results: prediction of
bioavailability
Most potent fraction: SPE-70%ACN was tested
Continuous digestion ACE inhibition activity (%)
Condition Sample Control
After oral phase 88.4 ± 1.1a 88.3 ± 0.8a
After gastric phase 87.9 ± 1.3a 88.1 ± 1.2a
After duodenal phase 56.9 ± 2.1a 57.9 ± 5.3a
After intestinal phase 52.5 ± 2.2a 59.7 ± 4.8a
45

46. Obj.2- Results: prediction of
bioavailability
Most potent fraction: SPE-70%ACN was tested
Continuous digestion
Non-continuous digestion
ACE inhibition activity (%)
Condition Sample Control
After oral phase 88.4 ± 1.1a 88.3 ± 0.8a
After gastric phase 87.9 ± 1.3a 88.1 ± 1.2a
After duodenal phase 56.9 ± 2.1a 57.9 ± 5.3a
After intestinal phase 52.5 ± 2.2a 59.7 ± 4.8a
Enzyme ACE inhibition activity (%)
Pepsin 88.1 ± 3.6a
Trypsin 88.2 ± 2.3a
Pancreatin 86.8 ± 0.7a
46

47. Obj.2- Methodology: characterization
RP-UPLC
Total amino acid analysis (SPE fractions)
LC-MS/MS IM-Q-TOF
Sequence and precursor proteins (hydrophobic fraction)
3
1
47

48. SPE total amino acids (g/100g protein)
Among fractions: highest
BCAA: 55% & 70%ACN fractions
CAA: 10%ACN fraction
HAA: 55% & 70%ACN fractions
Hydrolysate 10%ACN 55%ACN 70%ACN
EAA 38.8 ± 0.2 38.1 ± 0.1b 48.0 ± 1.8a 49.3 ± 0.7a
NEAA 45.0 ± 0.3 41.7 ± 0.6ab 42.4 ± 1.5a 37.7 ± 0.8a
AA 83.7 ± 0.2 79.8 ± 0.7 90.4 ± 3.3 86.9 ± 0.1
BCAA 12.5 ± 0.2 12.4 ± 0.8b 21.7 ± 1.1a 23.2 ± 0.3a
CAA 48.4 ± 0.4 35.8 ± 0.4a 30.7 ± 1.8b 26.8 ± 0.7b
HAA 32.8 ± 0.2 32.8 ± 0.4b 47.9 ± 1.2a 49.2 ± 0.4a
Obj.2- Results: amino acid composition
48

49. Obj.2- Results: peptide identification
SPE-70%ACN
Small MW
19 precursor proteins
49

50. SPE-70%ACN
Leucine rich motifs
GRAVY values ≤ +4.5
Hydrophobic
KGVLGDLSSR,
HVVTPTLAPP FPF
Obj.2- Results: peptide identification
50

51. Obj.2- Conclusion
Most hydrophobic fraction having highest anti-ACE activity
Charge and size not as important for anti-hypertensive effects
Most potent anti-ACE fraction being GI digestion stable
Leucine rich motifs presumably vital for hypotensive effects
51

52. OBJECTIVE 3
Bioactivity of Atlantic mackerel peptides on obesity and insulin
resistance, an in-vivo study
would be submitted for publication to the Journal Of Food And Function
52

53. Separation of anti-obesity and anti-insulin resistance peptides from
hydrolysate using:
I- EDUF: MWCO 20 kDa (pH 3)
setting different from
objectives 1 & 2
Objective 3
53

54. Obj.3- Methodology: anti-MetS activity
In-vivo effects in diet-induced obese and diabetic rats
Effects compared against hypercaloric & or chow diet
Samples:
Hydrolysate & EDUF pH3 cationic peptide fraction
8-weeks experimental diet, 208 mg/kg BW
Modulation of obesity, dyslipidemia & diabetes markers
Food intake, total & organ weight gain, TG & TC, adiposity,
insulin & glucose levels
54

55. Obj.3- Results: in-vivo analysis
Insignificant effect on
Weight gain: total, organ & tissues
Weights (g) Chow HFHS
HFHS +
Hydrolysate
HFHS + EDUF
fraction
Body weight
Initial 23.8 23.6 24.0 24.2
Final 25.6 31.6 32.9 32.9
Weight gain 2.1 8.4 9.5 9.2
Organ
Brain 0.44 0.43 0.44 0.44
Heart 0.13 0.13 0.13 0.13
Liver 1.06 1.01 1.05 1.03
Pancreas 0.26 0.26 0.26 0.27
Kidney 0.30 0.32 0.33 0.32
Adipose tissue
Mesenteric 0.18 0.43 0.48 0.49
Epididymal 0.41 1.70 1.86 1.91
Inguinal 0.27 0.68 0.77 0.75
Retroperitoneal 0.19 0.67 0.74 0.78
Brown 0.06 0.09 0.10 0.10
Muscle
Skeletal
gastrocnemius
0.25 0.25 0.25 0.25
Leg muscle 0.01 0.02 0.02 0.02
Caecum
Full 0.53 0.20 0.20 0.19
Empty 0.14 0.06 0.07 0.07
Stomach-cecum 35.7 32.4 32.7 33.0
Cecum-anus 7.0 6.3 6.7 6.6
55

56. Obj.3- Results: in-vivo analysis
Insignificant effect on
Weight gain: total, organ & tissues
Body composition
Food intake
56

57. Obj.3- Results: in-vivo analysis
Insignificant effect on
Weight gain: total & organ
Body composition
Food intake
Glycemia & hyperinsulinemia
Dyslipidemia &
hepatic steatosis
57

58. Obj.3- Conclusion
Fish type dependent obesity and insulin resistance benefiting
effects
Immunomodulatory and hypotensive attributes not correlated
with anti-obesity and anti-IR activities of biopeptides
Possibly higher dosage required for tested bioactivities
58

59. General conclusion
Peptide migration rate rather pH dependent
Importance of separation technique in isolation efficiency of
biopeptides
In-vitro experiments:
59

60. General conclusion
Peptide migration rate rather pH dependent
Importance of separation technique in isolation efficiency of
biopeptides
In-vitro experiments:
Hydrolysate: pro-inflammatory & hypotensive
EDUF fractions: pro-inflammatory
SPE fractions: anti-inflammatory & hypotensive
UF fractions: insignificant effects
Non-cytotoxic biological activities
60

61. General conclusion
Peptide migration rate rather pH dependent
Importance of separation technique in isolation efficiency of
biopeptides
In-vitro experiments:
In-vivo experiments:
Insignificant on diet-induced obesity &
Metabolic syndrome associated risk factors
61

62. Future directions
Study of SAR of selected fractions
Further examination of hypotensive peptides in well controlled
animal & or cell studies on MetS risk factors
Purification of active peptide & in-depth characterization
Process optimization for efficient production of biomolecules
Incorporation of therapeutic biopeptide into commercial
products for general & clinical use
62

63. Acknowledgment
Supervisors:
Dr. Lucie Beaulieu
Dr. Laurent Bazinet
Supervisory committee members:
Dr. André Marette
Dr. Ismail Fliss
Funded by Natural Sciences & Engineering Council of Canada
(NSERC)
63

64. THANK YOU
64