systems biology of the cell cycle - göteborgs universitet · e-mail: [email protected] icysb –...

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ICYSB, Göteborg Matteo Barberis

Swammerdam Institute for Life SciencesUNIVERSITY OF AMSTERDAM

Systems Biology of the Cell Cycle

Assistant ProfessorUniversity of Amsterdam, Swammerdam Institute for Life Sciences

E-mail: [email protected]

ICYSB – 6th International Course in Yeast Systems BiologyGöteborg, Sweden – 4 June 2013

Matteo Barberis



A “Baby” of UNICELLSYS

(by Stefan Hohmann at the Final UNICELLSYS Meeting, Innsbruck, 4-6 March 2013)

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Thank to …

Stefan Hohmann Marija Cvijovic



(Oltvai Z.N. & Barabási A.L., Science, 2002, 298: 763-764)

Life’s complexity pyramid

UNIVERSITY OF AMSTERDAMSwammerdam Institute for Life Sciences

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Cellular Systems Biology

• … the study of the integrated and interacting network of genes, proteins and metabolites that are responsible for the normal and abnormal cellular functions as well as the emergent properties that create life

• … aims at mechanistic understanding of cellular functions through interactions of constituent molecules in space and time



(from the Multidisciplinary Centre for Integrative Biology (MyCIB), The University of Nottingham, United Kingdom)

Multidisciplinarity


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(from the Multidisciplinary Centre for Integrative Biology (MyCIB), The University of Nottingham, United Kingdom)




The Cell Cycle

(Morgan D.O., The Cell Cycle: Principles of Control, 2007, pp. 210)


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Unravel molecular mechanisms underlying

Cell Cycle Control in budding yeast



(Lodish H., Berk A. et al., Molecular Biology of the Cell, Sixth Edition, 2008, pp. 973)

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4) Later in S phase, Clb3/4-Cdk1 activate replication origins finish S initiate the formation of mitotic spindle

5) As cells enter G2, Clb1/2-Cdk1 mitotic spindle formation trigger chromosome

segregation nuclear division

3) At the G1/S, Clb5/6-Cdk1 activate pre-replication complex

Cdk Activity during Cell Cycle Phases

(Lodish H., Berk A. et al., Molecular Cell Biology, Sixth Edition, 2008, pp. 973)

1) In G1 phase, Cln3-Cdk1 senses nutritional conditions drives cell growth

2) At the G1/S, Cln1/2-Cdk1 activate budding



DNA ReplicationDynamics

(computational analysis and testable predictions)

(Futcher B., Yeast, 1996, 12: 1635-1646;

Fitch I. et al., Mol. Biol. Cell, 1992, 3: 805-818)

Cdk/Cyclin and CkiRegulation

(integrating molecular biology, cell imaging and mathematical modeling)

SBI Seminar, Dublin Matteo Barberis 3.12.2012

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Oscillations of Cyclins

• The oscillatory behavior is widely known as “waves of cyclins”


(Lodish H., Berk A. et al., Molecular Cell Biology, Sixth Edition, 2008, pp. 973)



• Cdk1/Cyclins and Sic1 activities show alternate oscillations

Oscillations of Cdk1/Cyclins and Sic1

(Zachariae W. & Nasmyth K., Genes Dev., 1999, 13: 2039-2058)

Cdk1-Clb5,6

Cdk1-Clb3,4Cdk1-Clb1,2

Substrate/SubunitInhibitor of Cyclin-dependent kinase

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Mechanisms controlling S-Cdk Activation


Humboldt University BerlinInstitute for Biology

Max Planck Institutefor Molecular Genetics



Failure of Proper Timing – Cancer

• Decrease in p27Kip1 levels, due to its degradation, occurs in ~ 50 % of carcinomas (aggressive, high-grade tumors and poor prognosis, increased apoptosis)

• p27Kip1 cytoplasmic localizationcorrelates to enhanced cancer aggressiveness

(Blain S.W. and Massagué J., Nat. Med. 2002, 8: 1076-1078)

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Sic1 and p27Kip1: Two Sides of the Same Coin

(Barberis M. et al., Biochem. J., 2005, 387: 639-647)(Lacy E.R. et al., Nat. Struct. Mol. Biol., 2004, 11: 358-364)



(Kaizu K. et al., Mol. Syst. Biol. 2010, 6: 415)

Interaction Map of S. cerevisiae Cell Cycle

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Overview of the Cell Cycle Control System



Cln3Cdk1-Cln3Cdk1-Cln3-Far1

Cdk1Cdk1-Cln3-Far1-p

Degradation

SBF/MBF

SBF/MBF-Whi5-p

Clb5,6

Cdk1-Clb5,6Cdk1-Clb5,6-Sic1Cdk1-Clb5,6-Sic1-6p

Sic1

Sic1-6p

Degradation

Cdk1-Cln1,2

Cln1,2

Far1

Far1-p

CLB5,6mRNA

CLN1,2mRNA

Cdk1

Cln3

Far1

CLB5,6mRNA

CLN1,2mRNA

Cdk1-Cln1,2

Cdk1-Clb5,6-Sic1

Cdk1-Clb5,6

CYTOPLASMCYTOPLASM

NUCLEUSNUCLEUS

BUDDINGBUDDINGDNASYNTHESIS

DNASYNTHESIS

24

44

43

42

50

40

1

24

3

51

28

3841

47 32

10

14

11

15

19

20

16

13

12

18

26

SBF/MBF-Whi5

Whi5-p

Degradation

Whi5

5

7

6

9

46

37

845Whi5

172234

36

39

48

2125

49

27

29

33

53

30

35

31

Whi5-p52

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(Barberis M. et al., PLoS Comput. Biol., 2007, 3: e64)

Kinetic Model

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Model Improvement

cytoplasm

nucleus

Compartmentalization

P0 PS

1

2

nucleus

cytoplasm

cyt

cyt

Vol

mcyt

ii

cyt

dt

dVolv

dt

dm

Cell size growth during the G1 phase

Volume Changes

r

jjij

i vndt

dm

1

nuc

cyt

Vol

Volcyttr

r

jjij

nuc mkvndt

dm

1

cyt

nucVolVol

nuctr

r

jjij

cytmkvn

dt

dm

1



Model Description by Mass Action Kinetics

Systems equations

r – number of reactionsSi – metabolite concentrationsvj – reaction ratesnij – stoichiometric coefficients

Network properties Individual reaction properties

r

jjij

i vndt

dS

1

p,pSvv

S1

S2S4

S3

v1 v2

v3

v4

v5 ijnN

d[S1]/ dt = v[1] v[2] = p1 – p2S1

d[S2]/ dt = v[3] v[4] = p3S1S4 – p4S2

d[S3]/ dt = v[5] = p5S2

d[S4]/ dt = v[3] + v[4] = – d[S2]/ dt

p1 p2

p3

p5

p4

0 1 2 3 4 50

0.5

1

S[t]S1

S2

S3 S4

Time

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Modeling Pipeline

Time courses for: Cln3, Far1, Clb5, Sic1Localization of: Clb5 and Sic1

(Rossi, R.L. et al., Cell Cycle, 2005, 4: 1798-1807)

(Alberghina, L. et al., J Cell Biol, 2004, 167: 433-443)0

0,5

1

1,5

2

2,5

3

3,5

4

0 50 100 150 200 250 300 350

Time (minutes)

Pro

tein

lev

els

Sic1

Clb5

Clb5/Sic1

ETHANOL 2%

0

0,5

1

1,5

2

2,5

3

3,5

4

0 20 40 60 80 100 120 140

Time (minutes)

Pro

tein

lev

els

Sic1

Clb5

Clb5/Sic1

GLUCOSE 2%



Modeling Pipeline

Kinetic constants (BIAcore analysis) for:binding of Sic1 to the Cdk-cyclin complex

(Barberis, M. et al., Biochem. J., 2005, 387: 639-647)

Cyclin A Cdk2

N-term

C-term

Sic1 (226-248)

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Monitoring of changes in mass concentration on the chip surface

How the Information is generated?



Study properties of small modules, e.g. Cln3 activity, transcription factors regulation, Cln1-2 and Clb5-6 activities, Sic1 localization, …

Cln3 activity

0 20 40 60 80 100 120 1400

0.00002

0.00004

0.00006

Time (minutes)

Con

cent

ration

(M

)

Cdk1Cln3nuc

Cdk1Cln3-Far1nuc

Cln3 activity

Simulated Time Courses for Protein and Protein Complex Concentrations

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Study properties of small modules, e.g. Cln3 activity, transcription factors regulation, Cln1-2 and Clb5-6 activities, Sic1 localization, …

Transcription

factors regulation

0 20 40 60 80 100 120 1400

0.002

0.004

0.006

0.008

0.01

0.012

Time (minutes)

Con

cent

ration

(M

)

SBF/MBFnuc

SBF/MBF-Whi5nuc

Transcription factors

regulation

Simulated Time Courses for Protein and Protein Complex Concentrations



Reproducing Experimental Data

Glucose Ethanol

Time (minutes)

Con

cent

ration

(M

)

0 20 40 60 80 100 120 1400

0.005

0.01

0.015

0.02

0

0.875

1.75

2.625

3.5

Sic1tot Clb5tot

0 50 100 150 200 250 300 3500

0.005

0.01

0.015

0.02

0

0.875

1.75

2.625

3.5

Time (minutes)

Con

cent

ration

(M

)

Sic1tot

Clb5tot

Experimental dynamics of Clb5

Experimental dynamics of Sic1

Simulated dynamics of Clb5

Simulated dynamics of Sic1

(Barberis M. et al., PLoS Comput. Biol., 2007, 3: e64)

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Model Validation



Model Validation

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Model Validation

0 20 40 60 80 100 120 1400

0.01

0.02

0.03

0.04

0.05

0.06

Time (minutes)

Con

cent

ration

(M

)

OE-CLN3

cln3

WT



0 20 40 60 80 100 120 1400

0.01

0.02

0.03

0.04

0.05

0.06

0 20 40 60 80 100 120 1400

0.001

0.002

0.003

0.004

0.005

0 20 40 60 80 100 120 1400

0.01

0.02

0.03

0.04

0.05

0.06

0 20 40 60 80 100 120 1400

0.0010.0020.0030.0040.0050.0060.007

Time (minutes)

Con

cent

ration

(M

)

Time (minutes)

Con

cent

ration

(M

)

Cdk1-Cln1,2cyt Cdk1-Clb5,6nuc

Time (minutes)Time (minutes)

OE-CLN3

cln3

WT

OE-FAR1

far1WT WT

OE-CLN3

cln3

WT

far1

OE-FAR1

Cdk1-Clb5,6nuc

Con

cent

ration

(M

)

Con

cent

ration

(M

)

A B

C DCdk1-Cln1,2cyt

Model Validation by altering the Gene Dosage

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SIMPLER ???

“ … modeling is a very concise

way of thinking about a system

and generating hypothesis … ”



(Barberis M.*, Beck C.* et al., Mol. BioSyst., 2011, 7: 2804-2812)

Stochastic Model

Sic1Clb5 nuc

SIC1 mRNA

Clb5 cyt

Model of Sic1 Expression and Degradation

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Mean Sic1

SD Sic1 CV Sic1

Q

Initial SIC1 mRNA

SIC1 mRNA transcri

ption

SIC1 mRNA degradatio

n

Transcriptional Noise at the G1/S Transition

Q =CV Sic1

CV SIC1 mRNACV Sic1 =

SD Sic1

mean Sic1

CV, Q = noise




Initial SIC1 mRNA = 2 Initial SIC1 mRNA = 10

Mean Sic1

SD Sic1 CV Sic1

Q

Low SIC1 mRNA ensures Robust G1/S TimingInitial SIC1 mRNA = 6

Initial SIC1 mRNA 2 6 10

Max mean Sic1 320 molecules 350 molecules 380 molecules

Max noise Q 250 275 320

Max Timing noise Q 390 min 450 min 500 min

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How many SIC1 mRNA Molecules?

(Barberis M. et al., Mol. BioSyst., 2011, 7: 2804-2812)

+

MS2 GFP (x3)

x12 loopsSIC1 ORF

3’ UTR

• Majority of cells show 0 or 1 SIC1 mRNAs

• Observed range between 0 and 10 mRNAs



• Cdk1/Cyclins and Sic1 activities show alternate oscillations

Oscillations of Cdk1/Cyclins and Sic1

(Zachariae W. & Nasmyth K., Genes Dev., 1999, 13: 2039-2058)

Cdk1-Clb5,6

Cdk1-Clb3,4Cdk1-Clb1,2

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Which Mechanisms control Cdk1/Clb Waves ?

Humboldt University BerlinInstitute for Biology

Max Planck Institutefor Molecular Genetics




Cyclin-DependentKinase Inhibitor (CKI)

Sic1

InactiveComplexes

Cdk1 (Cdc28)

Cyclins

Cyclin-DependentKinase (CDK)

Clb5, Clb6Clb3, Clb4Clb1, Clb2

ActiveComplexes

ODE Model of Cdk1/Clb Activity

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Potential TranscriptionalRegulation of Clb Cyclins

CLB5,6

CLB3,4

CLB1,2

Clb6

Clb4Clb3

Clb5

Clb2Clb1

MBF

Fkh2

C

DB

TF

A



Sic1

Cdk1-Clb5,6

Cdk1-Clb1,2

Cdk1-Clb3,4

Clb5,6

Clb1,2

Clb3,4

6

1

8

10

C

A

B

9

7

D

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C1

C2

C3

Cdk1-Clb5,6-Sic1 Sic1

2

3Sic1

C1 C2 C3

5

4

Cdk1-Clb3,4-Sic1

15

16Sic1

C1 C2 C3

18

17

Cdk1-Clb1,2-Sic112

11

Sic1

C1 C2 C3

14

13

Ensemble Modeling of Sic1 Involvement

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Ensemble Modeling to study Sic1 Involvement



0 10 20 30 40 50 600

0.3

0.6

0.9

1.2

Time (minutes)

Clb3,4tot

Clb5,6tot

Clb1,2tot

Con

cent

ratio

n(a

.u.) • Global Sensitivity Analysis:

is the time delay independent on the parameter choice ?

The timing of Clb cyclinsappearance is Sic1-dependent

Sic1 potentially regulates the Appearance of Clb Cyclins

(Barberis M. et al., Biotechnol. Adv., 2012, 30: 108-130)

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G1 S G2 M

Cell cycle progression

Cyc

lin

leve

ls

Clb5,6 Clb3,4 Clb1,2

t5,6 t3,4 t1,2

(t3,4- t5,6)

(t1,2- t5,6)

(t1,2- t3,4)



0 2 4 6 8

0

5

10

15

20

0 2 4 6 8

0

5

10

15

20

t1,2 t5,6 (min) [Version 3]

t 1,2

t 5

,6(m

in)

[V

ers

ion

1]

t 1,2

t 3

,4(m

in)

[V

ers

ion

1]

t1,2 t3,4 (min) [Version 3]

The Time Delay is Sic1-dependent

tClb1,2 tClb5,6 tClb1,2 tClb3,4

r = 0.497

r = 0.0026

• The timing of appearance of Clb cyclins is due to the binding of Sic1 to all Cdk1/Clb complexes


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Structural Modeling of Sic1-Cdk1/Clb InteractionSic1-KID

Clb5

Cdk1

• Interfaces have steric features that allowthe formation of a stable ternary complex

(Barberis M. et al., Biochem. J., 2005, 387: 639-647;

Barberis M. et al., Biochem. Biophys. Res. Commun., 2005, 336: 1040-1048)

Leu 224

Val 225

(Barberis M., Adv. Exp. Med. Biol., 2012, 736: 135-167;

Barberis M., FEBS J., 2012, 279: 3386-3410)



Sic1 interacts with all Clb Cyclins


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(courtesy of Andreas Herrmann, Humboldt University Berlin)

Förster Resonance Energy Transfer (FRET)



Fluorescence Lifetime Imaging Microscopy (FLIM)

(courtesy of Andreas Herrmann, Humboldt University Berlin, Germany)

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mYFPmCer• m, A206K mutation

preventing any potential dimerization of fluorescent proteins



Transient Sic1-Clb Interactions in Living Cells

(Schreiber G.*, Barberis M.* et al., FASEB J., 2012, 26: 546-554)

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error bar = SEM (Standard Error of the Measurement)

Efficiency of Energy Transfer E (%)

D

DAE

1

• τDA, average lifetime of the donor measured in presence of the acceptor

• τD, average lifetime of the donor expressed alone

(Schreiber G.*, Barberis M.* et al., FASEB J., 2012, 26: 546-554)



0 10 20 30 40 50 600

0.3

0.6

0.9

1.2

Time (minutes)

Clb3,4tot

Clb5,6tot

Con

cent

ratio

n(a

.u.)

Does Sic1 regulate Timing and Oscillatory Behavior of Clb Cyclins?

Clb1,2tot

SIC1

0 10 20 30 40 50 600

0.1

0.2

0.3

Time (minutes)

Clb1,2tot Clb3,4tot

Clb5,6totCon

cent

ratio

n(a

.u.)

sic1


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• Wild type strain shows Clb periodicity, which is delayed but still present in the SIC1-0P strain

Clb2

Clb3 Clb5 Sic1

0 30 60 90 100 110 120 130 140 150 160 170 180

SIC1

Time (min)

180

160

140

120

100

90

60

30

0

58/46

46/53

14/81

34/67

65/29

74/9

97/4

99/3

100/0

1C/Bud

SIC1

Sic1 may be Responsible for Clb Timing during Cell Cycle Progression

Clb2

Clb3

Clb5

sic1Δ

1C/Bud

42/74

49/73

50/58

58/47

63/38

67/25

70/14

76/14

80/13

sic1Δ• In the sic1Δ strain, timing of Clb appearance is lost, although cells proceed into S phase

G1 S G2 M




Hcm1 is a Transcriptional Activator of S Phase

(Pramila T. et al., Genes Dev., 2006, 20: 2266-2278)

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The Sir2 Histone Deacetylase

• Sir2 is a NAD-dependent histone deacetylaseinvolved in silencing telomeres and mating locus, maintaining genome integrity and blocking inappropriate gene expression

• sir2Δ shortens life span, whereas SIR2 extra copy extend lifespan

(Guarente L., Genes Dev., 2000, 14: 1021-1026)

Calorie excess Calorie restriction



Sir2 interacts and influences Hcm1 Localization

(Rodriguez-Colman M.J. et al., J. Biol. Chem., 2010, 285: 37092-37101)

Hcm1-HA

Is Sir2 involved in the regulation of Fkh1/2 for the timing of mitotic events?

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(Rodriguez-Colman M.J. et al., J. Biol. Chem., 2010, 285: 37092-37101)

Sir2 interacts with Fkh1 and Fkh2

(Linke C., …, Barberis M.* and

Krobitsch S.*, under revision)



Sir2 interacts with Fkh Transcription Factors


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(Linke C ., …, Barberis M.* and Krobitsch S.*, under revision)

Sir2 inhibits Cell Growth in a Different Extent

SDII SDIVpBTM + pACT

pBTM-Fkh1 + pACT

pBTM-Fkh1 + pACT-Sir2

pBTM-Fkh2 + pACT

pBTM + pACT-Sir2

pBTM-Fkh2 + pACT-Sir2

pBTM-Hcm1 + pACT

pBTM-Hcm1 + pACT-Sir2

Sir2 inhibition: Fkh1 > Fkh2 > Hcm1



Sir2 binds to CLB2 Promoter

Sir2 binds to CLB2 promoter in G1 and M phases

0

1

2

3

4

5

6

ACT1 CLB2

Exponential phase

G1 phase

S phase

G2/M phase

Fold

chan

ge


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Sir2-VC+ VN-Fkh1Ndd1-VC+ VN-Fkh2




Transmitted light

Venus

FACS

Transmitted light

Venus

FACS

50100 4020 30

60 70 80 90 100 110

Sir2_VC +VN_Fkh1

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Sir2 binds to CLB2 Promoter via Fkh1/Fkh2

Fold

chan

ge

0

1

2

3

4

5

ACT1 CLB2

Exponential phase

Stationary phase

2 mM H2O2

0

1

2

3

4

5

ACT1 CLB2

Fold

chan

ge

wild type

fkh1

fkh2

(Linke C., …, Barberis M.* and

Krobitsch S.*, under revision)



Sir2 Nuclear Localization requires Fkh1/Fkh2


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Activation and Repression in Clb Regulation

Clb2 Sir2Ndd1

Fkh1

Fkh2Physical Interaction

Transcriptional activation

Transcriptional repression

Sir2 Hcm1

Fkh1

Fkh2




Conclusion

Timing of Cdk1/Clb activation is achieved by coupling multiple mechanisms:

(i) Sic1-dependent Clb inhibition

(ii) Sir2-dependent inhibition via Fkh1/Fkh2

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StructuralBiochemical

Computational

Microscopy

PPIs Modeling

Modeling

DNAPIs

SYSTEMS

BIOLOGY

Assays

(Barberis M., FEBS J., 2012, 279: 3386-3410)







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From Ph.D. Student to Assistant Professor

Prof. Edda Klipp Prof. Hans WesterhoffProf. Lilia Alberghina



Claudia Beck (Bachelor)

Aouefa Amoussouvi (Ph.D.)Christian Linke (Ph.D., Post-doc)

Alberto González-Novo (Post-doc)

Gabriele Schreiber (Post-doc)

Christine Klaus (Technician)Silvia Scolari (Ph.D.)

Adriana Supady (Master)

Miquel Á. Adrover (Ph.D.)

Thomas Spiesser (Master, Ph.D.)Christian Diener (Ph.D.)

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Thank You!

Questions welcome.

systems biology of the cell cycle - göteborgs universitet · e-mail: [email protected] icysb –...

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