1

Studies on Protein Dynamics with Fluorescence Studies on Protein Dynamics with Fluorescence Correlation SpectroscopyCorrelation Spectroscopy

Ton VisserTon Visser

BiophotonicsBiophotonics in Australia, Macquarie University, Sydney, 22in Australia, Macquarie University, Sydney, 22--25 February 200625 February 2006

Department of Structural Biologywww.bio.vu.nl/vakgroepen/structbiol/

MicroSpectroscopy CentreWageningen University

www.mscwu.nl

Micro is 1/millionNano is 1/billion

Providesbetter contrast

in opticalmicroscopy

MicrospectroscopyMicrospectroscopy is spectroscopy is spectroscopy performed on microscopic objectsperformed on microscopic objects

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Three strategic research objectives:

1. Visualisation of processes in the living cell:How, where, when and to which extent is information processedby ‘protein machines’ in a living cell after external stimuli?Systems biology approach

2. Functional genomics:Insight in higher-order organisation of the nucleus in living cells

plant transcription factorshuman nuclear receptors

3. Bionanotechnology:Development of new methods and techniques for biosensing and protein-protein interaction

FRET-FLIM‘single-molecule’ fluorescence (FCS, FCCS) microfluidics

The MicroSpectroscopy Centre also provides training courses

Organisation of four ‘FEBS Advanced Practical & Lecture Courses’FEBS: Federation of European Biochemical Societies

2000: ‘Microspectroscopy: Monitoring Molecular Behaviour in Living Cells’

2002: ‘Microspectroscopy: Monitoring Molecular Interactions and Reactions in Living Cells’

2004: ‘Microspectroscopy: ‘Visualisation of Biochemistry in Living Cells’

September 2006: ‘Microspectroscopy: ‘Imaging Biochemical Dynamics in Living Cells’

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OutlineOutline

Fluorescence Correlation Spectroscopy

Applications

Flavoenzyme p-Hydroxybenzoate Hydroxylase(PHBH): conformational dynamics during catalysis

Unfolding of apoflavodoxin

G-protein diffusion in Dictyostelium

Microfluidics

FCS: Fluorescence Correlation SpectroscopyFCS: Fluorescence Correlation Spectroscopy

Developed in early 1970s by Webb & Elson and co-workers

Practical improvements in early 1990s (Rigler & Eigen and co-workers): introduction of confocal microscopy:

small excitation volume high-efficiency collection (confocal) optics

stable lasersdetectors with high quantum efficiency and low dark noisestable and bright fluorophores distinguishable from background fluorescence

Applications include measurements ofDiffusion constantsAggregation statesPhotophysical characteristicsOligonucleotide hybridizationEnzymatic reactionsReceptor-ligand interactionsCellular processes

Review Article:S.T. Hess et al, Biochemistry, 2002, 41, 697-705

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An ultramicroscope from 1903

R. Zsigmondy and H.F.W. Siedentopf could observe light scattering of colloidal particles of sizes smaller than visible light

Source: Yan Chen. Analysis and applications of fluorescence fluctuation spectroscopy. Ph.D. thesis University of Illinois at Urbana-Champaign, 1999

T. Svedberg and K. Inouye counted colloidal gold-particles under an ultra-microscope witha frequency of 39 observations per minute. Results were published in Zeitschrift für physikalische Chemie 77, 145-191 (1911).

<N>=1.55

1/<N>=0.64<N>=1.56

Residence timeparticle 1.5 s

Volume 1064 µm3

Particle concentration2.4x10-12 M

The first results of fluctuation spectroscopy and The first results of fluctuation spectroscopy and the analysis many years laterthe analysis many years later

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Fluorescence Fluctuation SpectroscopyFluorescence Fluctuation Spectroscopy

Fluorescence correlation spectroscopy Photon counting histogram analysisTime-dependent fluctuations Amplitude-dependent fluctuations

~1 µm

250 nm

FCS autocorrelates the relative fluorescencefluctuations over time

G (τ) = = 1 + <δI(t) * δI(t + τ)>

<I>2

<I(t) * I(t + τ)><I>2

Fluo

resc

ence

In

tens

ity

Time (s)

<I>

δI(t) = I(t) - <I>Quantum yield anddetector sensitivity

Describes observation volume

Function of concentration over time(physics of process)

∫= ),()()( trCrdrWkQtI

τ

6

Relaxation part Diffusion part (3D)

T)(TeT

NG(τ

relττ

−+−

⋅><

+=−

1111)

difz

xy

dif ττ

ωω

ττ

2

11

1

⎟⎟⎠

⎞⎜⎜⎝

⎛+⎟

⎟⎠

⎞⎜⎜⎝

⎛+

FCS model function to fit experimental dataFCS model function to fit experimental data

1E-3 0.01 0.1 1 10 1001.00

1.05

1.10

1.15

1.20

1.25

G(τ

)

τ (ms)

Rhodamine green Fit

Note that Dtran , in contrast to τdif , is independent from size of excitation volume

Obtained fit parameters:

Sp = (ωz/ωxy) = 7.5

τdif = 57 µs

T = 15%

τT = 3 µs

Volume ≈ 2πω2xyωz ≈ 0.76 fl

<N> ≈ 4.3 => [Rhod] = 10 nM

tran

xydif D

ω τ

4

2

=

htran r

kT Dπη6

=

1 + 1/<N>

τdif

How many fluorescence photons from a single molecule can be detected in a microscope?

Ground state: S0

Excited state: S1

absorbance(fs)

phosphorescence(µs-s)

fluorescence(ns)

Triplet state: T1

Photochemistry (O2)

Transit time of bright molecule: 1 msFluorescence lifetime: 1 nsMax # photons: 1 ms/1ns ~ 106 photonsPractice ~ 105 photons (triplet photobleaching!)Microscope detection efficiency 1%

1000 photons

3 µm

0.5 µmROS

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GFP expressed by baculovirus-infected Sf21 cells

1

1.1

1.2

0.001 0.1 10 1000

Auto

corr

elat

ion

Tau (ms)

Protonation/conformational kinetics1-50µs

Translational diffusion/aggregation0.1-1ms

Rotational diffusion/triplet kinetics10ns-5µs

FCS can monitor any dynamic process which causesfluorescence intensity fluctuations

What can be done with FCS?What can be done with FCS?

Outline PHBH applicationOutline PHBH application

The aim of this project is to visualize catalytic events and dynamics of single enzyme molecules in solution during turnover conditions with single-moleculefluorescence detection.

As a model enzyme the dimeric flavoenzymep-hydroxybenzoate hydroxylase (PHBH) is selected.

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Used approachesUsed approaches

Preliminary studies have shown that it is difficult to follow a single PHBH molecule via its weak flavin fluorescence.

Therefore:

PHBH was labelled at the accessible Cys116 residue by two different “donor” fluorophores: either Alexa488 or Alexa546 maleimide dyes.

Alexa488 has spectral overlap with FAD and can sense the flavinchromophore via Förster Resonance Energy Transfer (FRET), but only when flavin is oxidized.

The Alexa546 dye does not have such overlap.

Recap of FRETRecap of FRET

Donor must be fluorescent

There must be an overlap between Donor fluorescence spectrum and Acceptor absorption spectrum

The distance between Donor and Acceptor must be less than 2x R0

The angle between Donor and Acceptor transition moments should not be 90° ( then κ2 = 0)

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Spectral overlap of Alexa488 fluorescence with Spectral overlap of Alexa488 fluorescence with flavinflavin absorption (oxidized state)absorption (oxidized state)

For acceptor-donor pair FAD-Alexa488 R0=32 ÅFor acceptor-donor pair FADred-Alexa488 R0=60 Å

The FRET efficiency (E)FRET efficiency (E) depends on the inverse sixth power over the relative distance between Donor and Acceptor:

1

1 + RR0

6E =

R

100%

50%

0%

E

R0

R0 = 3.2 nm for Alexa488Alexa488 (donor) and FADFAD (acceptor)

AcceptorDonor

No FRET:Donor is excited and fluoresces with

maximal intensity

R >> 1.5 R0

AcceptorR < 1.5 R0

Donor

FRET:Donor is excited and fluoresces with

less intensity

Recap of FRETRecap of FRET

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Structure of labeled PHBH

Distance R between Alexa488 and FAD in one subunit: 39Å => E = 23%Distance R between Alexa488 and FAD in other subunit: 57Å => E = 3%

Catalytic cycle of Catalytic cycle of pp--hydroxybenzoatehydroxybenzoate hydroxylasehydroxylase

Oxidized state of flavin,Spectral overlap with

donorFRET (Alexa488),

Minimum detected emission

of the Alexa488 fluorescence.

Reduced states of flavin, No overlap with donor,No FRET (Alexa488),Maximum detected

emissionof the Alexa488 fluorescence.

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FCS curves before and during reactionFCS curves before and during reaction

Rhodamine110 Rhodamine110 -- calibrationcalibration

PHBHPHBH--AlexaAlexa

Structure parameterStructure parameter

The data were fitted with “Diffusion” model. The data were fitted with “Diffusion” model.

PHBHPHBH--AlexaAlexa+ + pOHBpOHB

PHBHPHBH--AlexaAlexa+ NADPH+ NADPH

PHBHPHBH--AlexaAlexa + NADPH+ NADPH+ +

pOHBpOHB(reaction)(reaction)

The data were fitted with The data were fitted with “Diffusion“Diffusion--Relaxation” model.Relaxation” model.Diffusion time from previousDiffusion time from previous

step was fixed. step was fixed.

PHBHPHBH--AlexaAlexa + + pOHBpOHB+ +

NADPHNADPH(reaction)(reaction)

FCS: experimental and fitting strategiesFCS: experimental and fitting strategies

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FCS: model functions to fit experimental dataFCS: model functions to fit experimental data

( )

DD

Tbefore

τspτ

ττ

A)(AA

N(τG

211

11

exp111)

+⎟⎟⎠

⎞⎜⎜⎝

⎛+

⋅−

−+−⋅+=

ττ

( )

( )

DD

R

Treaction

τspτ

ττB)(

BBA)(

AAN

(τG

211

11exp1

1exp111)

+⎟⎟⎠

⎞⎜⎜⎝

⎛+

⋅−

−+−

⋅−

−+−⋅+=

ττ

ττ

“Diffusion” model

“Diffusion-Relaxation” model

Global analysis of FCS dataGlobal analysis of FCS data

Structure of the FCS Data Processor software

Global analysis of fluorescence autocorrelation curves

V. Skakun et al. Eur. Biophys. J. 34, 323-334 (2005)

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“Diffusion-Relaxation” model

The relaxation time for the reaction: τrel = 23 µs

Concept:Eox ERED 1/τrel = kred + kox

FCS curves before and during reactionFCS curves before and during reaction

“Diffusion” model

τT = 2.6 µs (triplet lifetime)

Dtrans = 37 µm2/s (diffusion constant ~ 80 kDa)

kred

kox

Catalytic cycle of Catalytic cycle of pp--hydroxybenzoatehydroxybenzoate hydroxylasehydroxylase

Oxidized state of flavin,Spectral overlap with

donorFRET (Alexa488),

Minimum detected emission

of the Alexa488 fluorescence.

Reduced states of flavin, No overlap with donor,No FRET (Alexa488),Maximum detected

emissionof the Alexa488 fluorescence.

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FCS curves before and during reactionFCS curves before and during reaction

Note that amplitude decreasesduring reaction:

# of molecules increases

Dimer – monomer equilibriumis influenced

Percentage increase in number of Alexa488Percentage increase in number of Alexa488--PHBH PHBH molecules during reaction obtained from FCS analysismolecules during reaction obtained from FCS analysis

Kd ~ 5 nM for dimer-monomer equilibrium

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We are watching only a few enzyme molecules transiting during a fraction of a second through a tiny volume; many are briefly watched; diffusion is one source of fluctuation, another one theshuttling of enzyme between oxidized and reduced forms.

Autocorrelation traces of PHBH-Alexa yield diffusion constants corresponding to a dimeric flavoprotein of 80 kDa molecular mass.The enzymatic reaction of PHBH-Alexa488 yields an additional relaxation time of 23 µs corresponding with a protein fluctuating between ‘oxidized’ and ‘reduced’ conformations.This relaxation is absent in experiments with PHBH-Alexa546 (data not shown).The dimer-monomer equilibrium is affected during reaction: conformational dynamics during reaction influence strength of subunit-subunit interface.

ConclusionsConclusions

A. Westphal et al., (2006) J.Biol.Chem. in press

Outline Outline apoflavodoxinapoflavodoxin unfoldingunfolding

The aim of this project is to characterise the unfolded state of apoflavodoxin with fluorescence correlation spectroscopy. More knowledge about the unfolded state of a protein is strongly required, as it forms the starting point for protein folding.

General reference: S.E. Radford (2000) TIBS 25, 611-618.

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Schematic of a protein folding energy Schematic of a protein folding energy landscape landscape

C.P. Schultz (2000) Nature Struct. Biol. 7, 7-10

• 179 residues (~20 kDa)

• Tryptophans

• Cysteine (position 69)

• Alexa 488

Apoflavodoxin labelled with Alexa 488 maleimide

•Labelling does not affect protein stability

Thermal unfolding mid-points:Apoflavodoxin: 47.02 ± 0.06 0CApoflavodoxin-A488: 45.88 ± 0.24 0C

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1

1.2

1.4

1.6

1.8

2

2.2

0.001 0.01 0.1 1 10 100

2 M GuHCl: Alexa 488

Apofld

1

1.2

1.4

1.6

1.8

2

2.2

0.001 0.01 0.1 1 10 100

0 M GuHCl: Alexa 488

Apofld

Diffusion of labelled apoflavodoxin in increasing denaturant concentrations

Time (ms)

G (τ

)

1

1.2

1.4

1.6

1.8

2

2.2

0.001 0.01 0.1 1 10 100

4 M GuHCl: Alexa 488

Apofld

0.9

1.1

1.3

1.5

1.7

1.9

0.0 1.0 2.0 3.0 4.0[GuHCl]

Nor

mal

ized

diff

usio

n tim

e

Apoflavodoxin diffusion becomes slower as it unfolds

Apoflavodoxin-A488

Free dye - Alexa 488

• Normalized diffusion time corrected for refractive index changes

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Apoflavodoxin unfolding followed with FCS

2 State-fit95% CI

Rh = 28 ÅRh = 38 Å

0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.50.9

1.0

1.1

1.2

1.3

1.4

[GuHCl]

Rela

tive

diffu

sion

tim

e

• Normalized diffusion time corrected for refractive index and viscosity changesUnfolding mid-point Cm = 1.55 M

(similar value obtained with steady state fluorescence)

10 100 1000

10

100

Number of residues

R h (Å

)

Denatured apoflavodoxin has random coil – like dimensions

Apoflavodoxin-A488

Hydrodynamic radii of denatured proteins of various chain lengths (Wilkins et al., (1999) Biochemistry 38 16424)

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FCS work in progress

Effect of macromolecular crowding on protein folding (to mimic cellular situations)

(Ruchira Engel et al., manuscript in preparation)

Further characterization of denatured apoflavodoxin

spFRET (single-pair FRET)Ultrafast time-resolved Trp fluorescence in ensembles

(homo-FRET => molten globule)NMR (structure of unfolded state)

Aims / questionsDiffusion of G-protein β and γ subunits in Dictyostelium

• Is it affected due to initiation of chemotaxis?

•Does it play a role in polarization of cell?

Strategy•Study diffusion of GFP labeled proteins using fluorescence correlation spectroscopy (FCS)

G-protein diffusion in Dictyostelium

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Dictyosteliumlife cycle

Chemotaxis of Dictyostelium cells to cAMP (adenosine 3′,5′- cyclic monophosphate)

http://dictybase.org

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OUT

IN

membrane

receptor

G-protein

Chemotaxis Mechanism

pseudopod formation

PH domain-proteins

cGMP

GCPLC

PI3K

cAMP

Gβ

GγGα

GTP

GTP

Gβ

GγGαGDP

GDP

Myosin II mediated contraction

Actinpolymerisation

Actindepolymerisation

cAMP

Part I: Chemotaxing cells are polarised Does diffusion play a role?

PH domain translocation

Gβγ localization

1% c

AM

P gr

adie

nt

22

0

0.2

0.4

0.6

0.8

1

-2 -1 0 1 2

Cytosol: 3D diffusion

10 µm

GFP-Gβ and GFP-Gγ diffusion in Dictyostelium cells

10 µm

GFP-Gγ

10 µm

GFP-Gβ

65 kDa

34 kDa

G(τ

)

Time (ms) log scale

0

0.2

0.4

0.6

0.8

1

-2 -1 0 1 2

Membrane + Cytosol: 3D + 2D diffusion

GFP-Gβ.GFP-Gγ

100 kDa

Diffusion of Gβγ subunits in cytoplasm becomes faster upon cell polarization

• Diffusion of free GFP in polarized cells 1.6 times faster

Ruchira et al. J. Biol. Chem. 2004 (279) 10013-10019

GγGβpolarized

GFP-Gβ:

GFP-Gγ:

13 µm2/s

13 µm2/s

0

2

4

6

8

10

12

14

D (µ

m2 /s

)

GFP-GγGFP-Gβ

vegetative

9 µm2/s10 µm2/s

GFP-Gβ:GFP-Gγ:

GγGβ

0

2

4

6

8

10

12

14

(65 kDa)

(34 kDa)

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Cells incubated in buffer Cells incubated in buffer + 1µM Latrunculin A

Detailed look at GFP-Gβ.GFP-Gγ diffusion in chemotaxing cell cytoplasm

Stimulation with 10 µM cAMP

20 µm 20 µm

•Diffusion in latrunculin A treated vegetative cells is ~1.2 times faster than in untreated cells

GFP-Gβ.GFP-Gγ diffusion is faster in front part of chemotaxing cell

9.6 µm2/s (39)Mid

9.5 µm2/s (41)Back

11.9 µm2/s (50)Front

front mid back

D (µ

m2 /s

)

back

frontmid

02468

101214

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FRONT

Faster diffusion in front: a role in signal amplification?

• Recent study using FRET: more G-protein activated in front than back of cellXu et al., Mol .Biol. Cell 2005 (16) 676-688

BACK

Rapid diffusion in front

Faster recycling of Gβγ to the front

Spatial polarization of cytoskeleton

cAMP signal

Dissociation of Gβγ from Gα

Conclusions

• Gβ and Gγ are present in a complex with each other

• Diffusion of GFP-Gβγ subunits in cytoplasm becomes faster in polarized cells

• Diffusion in membrane of vegetative and polarized cells similar

• In chemotaxing cells faster diffusion in front than in the back: a role in signal amplification?

Ruchira Engel et al., submitted

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MicrofluidicsMicrofluidics: square quartz fibre: square quartz fibre

100 µm2 internal diameter

Capillary mounted on the ConfoCor™ I microscope stage and connected to FPLC pump

Flowing molecules measured with FCSFlowing molecules measured with FCS

B.H. Kunst et al., Anal. Chem. 74, 5350-5358 (2002)

1E-3 0.01 0.1 1

1.0

1.5

2.0

Flow of Rhodamine green at different flow settings

norm

aliz

ed G

(τ)

τ (ms)

flow 0.00 ml.h-1

0.34 0.68 1.0 1.4 1.7

0 10 20 300.00

0.05

0.10

Flow of Rhodamine green through 100 µm2 capillary5 nM Rgreen, ND=0.3, 514 nmpumpflow = 0.34 - 1.7 ml.h-1

Flow

vel

ocity

(m.s

-1)

Flow (µl.min-1) (by weighing)

40.75 40.80 40.850.000

0.025

0.050

Poiseuille flow profile of Rhodamine green in a 100 µm2 capillaryPump speed: 1 ml.h-1.

Flow

(m.s

-1)

Position y-axis (mm)

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Screening and sorting in Screening and sorting in microfluidicsmicrofluidics chipchip

Fluorescent particles are detected in a microchannelusing FCS instruments.

The detection of single fluorescent particles takes place in a 0.5-3 µm confocal detection element.

A liquid flow and an electric field is used to focus particles in the middle of the microchannel to enhance detection probability.

Fluorescent particles are sorted out of the main channel by switching of electric fields.

Chips can be sterilisedChips can be sterilisedChips can be disposed off to prevent crossChips can be disposed off to prevent cross--contamination contamination Reduced consumption of reagentsReduced consumption of reagents(channels ~ 5 (channels ~ 5 µµm wide and deep)m wide and deep)Fast parallel analysis possibleFast parallel analysis possibleNovel analysis methods Novel analysis methods LabLab--onon--aa--chipchip

Why screening and sorting on a chip?Why screening and sorting on a chip?

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TTL pulses from photon detector

Data storage andcharacterization

NoBurst ?

Yes

Wait x ms

Send TTL pulseto sorter

1

5

2 3

4

Detection of particles through photon burstsDetection of particles through photon bursts

B.H. Kunst, et al. Rev. Sci. Instrum. 75, 2892-2898 (2004)

Detection, focussing and sorting of Detection, focussing and sorting of microspheresmicrospheres

Fluorescent microspheres (0.9 µm) in suppressed EOF environmentIntensified CCD imaging using argon laser light

Fluorescent microspheres (0.9 µm)in suppressed EOF environment

CCD imaging of fluorescence usingmercury lamp illumination

Fluorescent microspheres (0.9 µm)in suppressed EOF environment

CCD imaging of fluorescence usingmercury lamp illumination

Detection Focussing Sorting

B.H. Kunst, et al. Rev. Sci. Instrum. 75, 2892-2898 (2004)

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Future is bright for FCSFuture is bright for FCS

FCCS for selective protein-protein interaction in cell(better VFP-couples now available); 2-photon

Concentration measurements of protein complexes in cells (transiently)

PCH, FIDA, FIMDA, FILDA: comparison of in vitro and in vivo

Image correlation spectroscopy

Microfluidics => nanofluidics

AcknowledgementsAcknowledgements

PHBH

Adrie WestphalAndrey MatorinArie van HoekMark HinkJan Willem BorstWillem van Berkel

Apoflavodoxin

Ruchira EngelAdrie WestphalDaphne HubertsSanne NabuursNina VisserArie van HoekHerbert van AmerongenCarlo van Mierlo

Dictyostelium

Ruchira EngelJaco Knol (RUG)Mieke Blaauw (RUG)Peter van Haastert (RUG)

MicrofluidicsMicrofluidics

Niek Kunst

Funding

NWO ALWSTWEU

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Jan Willem BorstMark Hink

RuchiraEngel

AcknowledgementsAcknowledgements

Adrie Westphal

Carlovan

Mierlo

AndreyMatorin

Willemvan

Berkel

Niek Kunst