LipoproteinsLipoproteins andand MembranesMembranes

P.P.LaggnerLaggner

LectureLecture atat

EMBOEMBO Practical CoursePractical Course on on

SolutionSolution Scattering from Biological MacromoleculesScattering from Biological Macromolecules

Hamburg September 2001Hamburg September 2001

Institute of Biophysics and X-ray Structure ResearchAustrian Academy of Sciences, Graz, Austria

ÖAW Research Centre Graz

Outstation atElettra / Trieste

Kepler 1596-1600Boltzmann 1870s

Schwarzenegger 1940-

Lipoprotein groupLipoprotein group::RuthRuth PrasslPrasslRobert SchwarzenbacherRobert Schwarzenbacher

HeinzHeinz Amenitsch Amenitsch Michal HammelMichal Hammel

MonikaMonika ZechnerZechnerSarahSarah TutzTutz

Veronika SattlerVeronika Sattler

JohnJohn ChapmanChapman (INSERM ,Paris)(INSERM ,Paris)

MembraneMembrane groupgroup::KarlKarl LohnerLohnerHeinzHeinz AmenitschAmenitsch

Manfred KriechbaumManfred KriechbaumMichaelMichael RappoltRappolt

Georg PabstGeorg PabstRichard Richard KoschuchKoschuch

Cilaine TeixeraCilaine TeixeraMarlene StroblMarlene Strobl

Monica Monica VidalVidal

MapMap ofof the Fieldthe Field

TechnaliaTechnalia

BiomedistanBiomedistan

MembraneMembrane

IslandIslandLipoproteinLipoprotein

ArchipellagoArchipellago

SeaSea ofof BiophysicsBiophysics

Cell BiologyCell Biology OceanOcean

CholesterolCholesterolCityCity

Pump Pump StationStation

BilayeronBilayeron

ReceptionReception

Lipoproteins are Lipoproteins are supramolecular supramolecular particlesparticles inin the the bloodstreambloodstream

Membranes are Membranes are the boundariesthe boundariesofof cellscells andandorganelles organelles

LIPOPROTEINSLIPOPROTEINS

VINTAGEVINTAGE

REVIEWREVIEW

LIPOPROTEIN METABOLISMLIPOPROTEIN METABOLISMNutrition

HDL2

LDL

Chylo-microns

Chylom.Remnants

VLDL

IDL

Extrahepatic

tissues

Macro-phage

Bile acids, Cholesterol

Atherosclerotic

plaques

Foamcell

HDL3

Intestine

EXOGENOUS ENDOGENOUS ATHEROSCLEROTIC

Liver

LIPOPROTEIN CLASSESLIPOPROTEIN CLASSES

Chylo-micronChylo-micron VLDLVLDL LDLLDL HDL2 HDL3HDL2 HDL3

Density[g/ml] 1.063 1.21

Size[nm] <1000 <80 18-25 8-13

1.00 1.019

LipoproteinFraction

LipoproteinFraction

PlasmaPlasma

Chem.Comp.

[%]

Apolipo-proteins

PRNPLFCTGCE

CBAIAIIE

CHEMICAL COMPOSITIONCHEMICAL COMPOSITIONChyloChylo--micronmicron VLDLVLDL LDLLDL HDLHDL

Size [nm]

Den

sity

[g/m

l]0.95

1.00

1.05

1.10

1.15

1.20

1.25

1.3010 20 30 40 50 10 0007060

HDL

LDLVLDL CHYLO-

MICRONS

LIPOPROTEIN PARTICLESLIPOPROTEIN PARTICLES

Electron microscopyElectron microscopy:: quasispherical particlesquasispherical particles

SAXS:SAXS: what is the internal structurewhat is the internal structure ??

HighHigh Density LipoproteinsDensity Lipoproteins(HDL)(HDL)

They are the smallest onesThey are the smallest ones ,, their main their main components are proteinscomponents are proteins andandphospholipids phospholipids

••TheThe SAXSSAXS curvescurvesof all HDLof all HDL are are closely similarclosely similar..

••SideSide maxima maxima indicate indicate quasiquasi--spherical shapespherical shape

••The scaling factorThe scaling factor xxis their reciprocal is their reciprocal sizesize

p ( r ) p ( r ) –– functionfunction

RadialRadial electron electron density distributiondensity distribution

HDLHDL CoreCore –– Shell Model Shell Model

•• Cholesteryl estersCholesteryl esters ((aboutabout 3.7 nm3.7 nm longlong)) are radially orientedare radially oriented inin the corethe core..

•• PhospholipidsPhospholipids ((aboutabout 2.2 nm) and2.2 nm) and cholesterol interdigitate from the cholesterol interdigitate from the surfacesurface.. TheThe polarpolar surface layer is aboutsurface layer is about 1.5 nm1.5 nm thickthick..

••Interdigitation is the size limiting principleInterdigitation is the size limiting principle of HDLof HDL

LOW DENSITY LIPOPROTEIN (LDL)LOW DENSITY LIPOPROTEIN (LDL)

••They contain aboutThey contain about 50% of50% of cholesterolcholesterol ((freefreeandand as fatty acid esteras fatty acid ester))

••Only one apoprotein copyOnly one apoprotein copy perper particleparticle:: apoapo--B, aB, a glycoprotein whichglycoprotein which has ahas a molecular molecular weightweight ofof aboutabout 550550 kD kD

CholesterylesterCholesterylester

ProteinProteinapoapo B100B100

PhospholipidPhospholipid

CholesterolCholesterol

LOW DENSITY LIPOPROTEIN (LDL)LOW DENSITY LIPOPROTEIN (LDL)

TriglycerideTriglyceride

Old Old ModelModel

LDLLDL showsshows aa pronouncedpronounced, reversible, reversible transition transition aroundaround 2020--35 °C 35 °C

fromfrom 3.73.7-- nmnm repeatrepeat totoisotropicisotropic

Concentrical twoConcentrical two--shell shell arrangementarrangement ofof cholesteryl cholesteryl estersesters

OldOld modelmodel

LDLLDL Core StructureCore Structure ::

LDLLDL--Core below the transitionCore below the transition: :

••inin which orientation are the cholesteryl which orientation are the cholesteryl esters arrangedesters arranged ??

••Fatty acidsFatty acids ofof thethe secondsecond layerlayer inin oror out ?out ?

AA task fortask for SANS !SANS !

SANSSANS:: selective deuterationselective deuteration ofofcholesteryl esterscholesteryl esters in LDLin LDL

Analysis ofAnalysis ofGuinier radiusGuinier radius

HH22O/DO/D22O O --

Contrast Contrast variationvariation

RRxx fromfrom aa comparison between deuteratedcomparison between deuterated andandundeuterated sampleundeuterated sample

oror,, betterbetter,, from the whole contrast from the whole contrast variation series variation series

Typical Guinier Typical Guinier plotsplots of SANS of SANS onon selectively selectively deuterateddeuterated LDL LDL

ImportantImportant::Concentration Concentration dependencedependence !!

Models A,B, and CModels A,B, and Cwould agree with would agree with the resultsthe results..

CholesterolCholesterol out /out /

Fatty acidsFatty acids in in

Which one is correctWhich one is correct ??

None completelyNone completely !!

The core transition temperature varies between The core transition temperature varies between individualsindividuals andand dependsdepends onon thethe CE/TG ratioCE/TG ratio

It showsIt shows aadiscontinuity around discontinuity around

CE / TGCE / TG ratiosratios of 7of 7

This is the core This is the core shellshell ratio in a ratio in a inin two two concentric concentric spheres with spheres with rrii//rroo of 2:1 of 2:1

The innermost coreThe innermost core(1/8 of(1/8 of the core the core

volumevolume)) retainsretains aafluidfluid,, oily state oily state even below the even below the

transitiontransition

The cholesteryl The cholesteryl esters phaseesters phase

separateseparate into theinto thesecondsecond layerlayer

T<TmCE/TG>7

Tmhigh

T<TmCE/TG<7

Tmlow

T>TmT G

C E

F C

P L apo B100

THE END :THE END :

NeitherNeither SAXSSAXS nornorSANSSANS can can differentiate between differentiate between the two modelsthe two models

ButBut:: modelmodel BB fits the fits the data fromdata from DSC and DSC and ESRESR

Can the core transition follow Can the core transition follow the physiological temperature the physiological temperature changeschanges inin the blood streamthe blood stream??

AA task for synchrotron task for synchrotron radiationradiation SAXSSAXS

Temperature [°C]

10 20 30 40 50

Excess Heat Capacity

Tm

Liquid crystalline Isotropic oily16 - 32°C

DSC and SAXS

CORE LIPID TRANSITIONCORE LIPID TRANSITION

0.5

10°C

40°C

20x103

15

10

5

0Inte

nsity

(a.

u.)

0.40.30.20.10.0

s (1/nm)

TIMETIME--RESOLVED XRESOLVED X--RAY DIFFRACTIONRAY DIFFRACTIONKinetics of core transition

Melting: Laser T-jump10ms time resolution

time [ms]s [1/nm)

inte

nsity

[a.u

.]

Freezing of core lipids

Temperature drop: 40°C 10°C250 ms time resolution

0

5

10

0.3

0.15

0.45

time

[s]

s [1/nm]

inte

nsity

[a.u

.]

Melting isMelting is fast: onfast: on the millisecondthe millisecond timetime scalescale

Freezing isFreezing is onon the timescalethe timescale of sec, i.e. fastof sec, i.e. fastenoughenough toto follow thefollow the TT--changeschanges inin slowly slowly exchanging vesselsexchanging vessels

From structure analysisFrom structure analysis totosynthesissynthesis::

LDLLDL asas drugdrug carriercarrier

drug intercalation in core lipids

drug intercalation in surface monolayer Problems of incorporation:

drug intercalation results in a disordering of the core region and changes in apoB-100 conformation

Drug incorporation in LDL

C

O

CH2O

HO

OPO

CH2OO

-CHNH

CHOH

COC17H35

15H31

NHCH3

O N

5'-ceramide thymidine (CET)

O

CH3

3',5'-dioleyl thymidine(DOT)

CO

CH3(CH2)7CH:CH(CH2)7 O

OCO

CH3(CH2)7CH:CH(CH2)7

CH2O

NO

NH

O

H3

5'-monooleyl deoxythymidine(MOT)

CO

CH3(CH2)7CH:CH(CH2)7 OCH2O

NO

CNH

Characterisation of the LDLCharacterisation of the LDL -- DOT DOT drug complexes drug complexes with with SAXSSAXS

Experimental SAXS curves from LDL below the phase

transition

0 5 10 15 20 25

LD L con tro l 20 .4±0 .1 nm

LD L na tive 20.2±0.4 nm

LD L-D OT 21 .5± 0.4 nm

p(r

)

r [nm]

• The peak maximum at large distances for native LDL was rmax 20.2±0.4 nm, which corresponds to the electron density autocorrelation of the phospholipid headgroups and protein moiety.

• Broadening of maximum peak for LDL control without significant difference in rmax value indicate formation of LDL aggregates during incubation.

• Increase in rmax value (∆r=1.3±0.6 nm) and broadening of peak maximum for LDL-DOT indicate slightly increase in the maximum particle diameter and formation of LDL aggregates.

Real space electron-pair distance distribution

functions

0.0 0.5 1.0 1.5 2.0 2.5 3.0

LDL native LDL control LDL-DOT (5 DOT molecules per LDL)

log

I(h)

h [nm-1]

Characterisation of the LDLCharacterisation of the LDL--MOT MOT drug complexes drug complexes with with SAXSSAXS

Experimental SAXS curves from LDL below the phase

transition

• No significant differences have been observed in rmax value of peak maximum for native, reconstituted LDL as also for LDL-MOT complex with 50 molecules of drug per LDL particle.

• Incorporation of MOT have no significant effect on particle diameter and core lipid arrangement

Real space electron-pair distance distribution

functions

0.0 0.5 1.0 1.5 2.0 2.5 3.0

LDL native LDL control LDL-MOT (50 MOT molecules per LDL)

log

I(h)

h [nm-1]0 5 10 15 20 25

19.6 nm20.2 nm20.0 nm

LDL native LDL reconstituted LDL-MOT (50 MOT molecules per LDL)

p(r)

r [nm]

The StepThe Step to highto high resolutionresolution::

LDLLDL CrystallographyCrystallography

Images taken at EMBL Hamburg

LDLLDL crystalscrystals

ImagesImages takentaken atatElettraElettra: 180 ° rotation : 180 ° rotation inin stepssteps of 10°/of 10°/frameframe

CrystallographyCrystallographye.m., X-ray diffraction3D protein structure

ESR Spin LabellingESR Spin Labellinglipid mobilityorder parameter and polaritylipid-protein-interaction

SAXSSAXS

size and shapeinternal organization

DSCDSCprotein stabilitythermotropiccore-melting

core-transitionTm=24-31°C

Triglyceride

Cholesterylester

Free Cholesterol

Phospholipid

Protein (apo B100)

PHYSICAL METHODSPHYSICAL METHODS

MEMBRANES

Structure Dynamics

Thermodynamics

FunctionFunctionFunction

Non-Equilibrium

WHAT A MESS !

Starting from the components:

Lipid self-assembly

LIPID POLYMORPHISM

A typical T/c -phase diagram

A little bit of theory...

(Biologists may doze now for a while, and dream of Monica)

Uncorrelated bilayers / Vesicles

Continuous scattering

The solution:

Electron density profile

Multilayer stack

Liposome

Bilayer Stacks / Liposomes

Bragg - reflections

Multilayer stack

Liposome

Swelling series

Small- and wide-angle scattering

This can also be done with natural membranes, e.g. erythrocyte ghosts

Unilamellar vesicles and multilamellar liposomes can coexist

Catching up speed, slowly.

Temperature scanning

main

transition

Lβ

Lα

maintransition

pre-transition

Lβ’

Pβ’

Temperature

Pressure

Equilibrium StructuresSlow T-scan 500 sec / frame (sealed tube 2kVA)

small angle wide angle

Equilibrium ?

NO

Synchrotron T-scan

medium speed

Phase epitaxy

Physiological relevance(Biologists wake up)

LHC complex shifts L/H equilibrium in reconstituted membranes towards bilayer

Excess lipid is stored as hex-phase

Getting faster – chasing intermediates

T/p jumps

Head of Project: Peter Laggner1) Fibrlagg@mbox.tu-graz.ac.atLocal Contact: Heinz Amenitsch1)* Amenitsch@Elettra.Trieste.It

igrid Bernstorff 2) Bernstorff@Elettra.Trieste.ItScientists: Pavo Dubcek2) Pavo.Dubcek@Elettra.Trieste.It

Michael Rappolt1)* Michael.Rappolt@Elettra.Trieste.ItTechnician: Cristian Morello1) Cristian.Morello@Elettra.trieste.it

1) Institute for Biophysics and X-ray Structure Research (IBR) 2) Sincrotrone Trieste Austrian Academy of Sciences Strada Statale 14, km 163.5 Steyrergasse 17, 8010 Graz, Austria. 34012 Basovizza (TS), Italy.Tel 0043-316-812 004 Tel 0039-040-375 81 Fax 0039-040-938 0902 Fax 0043-316-812 367 *) Working site: IBR c/o Sincrotrone Trieste

DYNAMICSDYNAMICSIRIR--LASER TLASER T--JUMPJUMP

IR-Laser:λ = 1.54 µmEmax = 4 Jτ = 2 ms∆Tmax = 20°C

Intermediate also in lam/hex transition

ShortShort--lived Intermediatelived Intermediate

Lαααα *Lα Lα

Non-Equilibrium StructureOrdered Intermediate Phase

0 5 10 15

6.2

6.3

6.4

6.5

d (n

m)

t (s)

30 40 50 60 70

6.40

6.45

6.50

T (°C)

lateral expansion

com

pres

sion

T-jump:1-2 ms

∆T = 5 - 15 °C

Relaxation:~ 30 - 40 s

Lαααα Lαααα*

Re-diffusion of water

T-jump probes the elastic properties of liposomes

Milos Steinhart(IMC Prague)

Heinz Amenitsch (IBR)Sigrid Bernstorff (Sincrotrone Trieste)

High-pressure SAXS:Lipids and Cholesterol

Monica Vidal (IBR)Cilaine Teixera (IBR)Manfred Kriechbaum (IBR)

0 5 10 15 20 25

0,004

0,006

0,008

0,010

0,012

0,014

0,016

0,018

0,020

0,022

|∆

d|/d

eq

xC (mol %)

Effects of Cholesterol on Bilayer ElasticityT-jump induced change in lamellar repeat as a function of cholesterol content in POPC

HardeningSoftening

Reference: M.Steinhart, M.Kriechbaum, K.Pressl, H.Amenitsch, P.Laggner and S.Bernstorff, Rev.Sci.Instrum. 70, 1540-1545 (1999).

The HP-X-ray Cell

Cell body: Stainless steel (5 x 3.5 x 2 cm)

Max. pressure: 2-3 kbar (5-90°C)

Windows: Be or diamond C (2r = 3 mm, d=1.5 and 0.75 mm, respectively)

Aperture: entrance: 1.5 mm, exit: 2 mm (2r); 60° angle cone (covers SAXS and WAXS).

Sample: optical path: 1.5-3 mm.

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.06.2

6.4

6.6

6.8

7.0

7.2

7.4

7.6 POPC 20% + 1% mol cholesterol, T=20°C

d [n

m]

P [Kbar]

time

[s]

scattering vector

335 bar

1782 bar

300 bar

0

220

P-scans of POPC (20%) with different cholesterol concentrations (0/1/5/10/20 mol%) at different

temperatures (5/10/15/20°C)

Pm

Pm

110 frames, 2s/frame

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.06.2

6.4

6.6

6.8

7.0

7.2

7.4

7.6 POPC 20% T=20°C

d [n

m]

P [Kbar]

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.06.2

6.4

6.6

6.8

7.0

7.2

7.4

7.6POPC 20% + 5% mol cholesterol, T=20°C

d [n

m]

P [Kbar]

0.0 0. 2 0.4 0.6 0.8 1.0 1.2 1.4 1. 6 1.8 2. 06.2

6.4

6.6

6.8

7.0

7.2

7.4

7.6 P OPC 20% + 1% mol cho lestero l, T=20°C

d [n

m]

P [Kbar]

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.06.2

6.4

6.6

6.8

7.0

7.2

7.4

7.6POPC 20% + 10% cholesterol, T=20°C

d [n

m]

Pressure [Kbar]

Hysteresis for POPC 20% and different molar concentrations of cholesterol at 20°C d-spacing as a function of pressure during a p-scan (red: compression and blue: decompression)

0 % 1 %

5 % 10 %

Pressure-Hysteresis

Transition pressure Pm of 20% POPC at different molar cholesterol concentrations (0-20%) asa function of temperature obtained from pressure-scans followed by SAXS.

Clausius-Clapeyron slopes

dP/dT

0 5 10 15 2045

50

55

60

65

70

dP/d

T

% cholesterol0 2 4 6 8 10

-40

-30

-20

-10

0

10

20

30

40

20°C 15°C 10°C 5°C

hyst

eres

is/ra

te

% cholesterol

Rate-dependence of hysteresis

Both, Clausius-Clayperon slopes (dP/dT) and hysteresis/rate show a maximum at low concentrations of cholesterol (1-5 mol%). This agrees with the notion that low amounts of

cholesterol induce a softening/plastification and higher concentration cause hardening.

Pressure scanning of PC / Cholesterol mixtures

0 5 10 15 20 25

0.050

0.055

0.060

0.065

0.070

0.075

0.080

dP/d

T

xc (mol %)

0 2 4 6 8 10 12 14 16 18 200.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

delta

Pm

xc (mol %)

Transition pressure Pressure hysteresis T-jump induced change of d-spacing

Pabst G., Rappolt M., Amenitsch H., Bernstoff S., and Laggner P. (2000). Langmuir 16, 8994-9001

Comparison of p-Scan and T-jump Results

20% POPC, 0.33M LiCl, 15°C 25% POPC, 0.33M LiCl, 15°C

Lαααα*Lαααα*L ααααL αααα

L ααααL αααα

Lαααα*Lαααα*

L αααα /Lαααα* - Equilibrium Phase Separation by Li+-Ions

Pressure stabilizes L αααα

Surface Diffraction

φ

θΟ

θΙ

Scattered beam

sample

Incident beam

In-Plane structure

Depth structure

Surface Diffraction Cell

M y la r

H 2 O

L ip id s

S i

X - r a y s

25 µµµµm~50 µµµµm

500 µµµµm

Type I:

Surface Diffraction

Lββββ „Gel-Phase“

Lαααα „Liquid crystalline-Phase“

6 nm

Fig.: Surface diffraction pattern of SOPE heated from 20 - 40 - 20 ºC with a rate of 1ºC/min showing the diffraction peaks from the2nd to the 6th order at fixed incidence angle ω (1.2º). The upper resolution limit was just given by the dimension of the vacuum tube and the detector length. The phase transition Lβ - Lα- Lβ (@ 35 ºC) is clearly visible.

Problem: Absorption and angular correction!!Poiseuille shear cell for neutrons: Hamilton et.al., Physica B 221, (1996) 309-319

Interface correction factor and Cross-section correction factor

Film deposition & microscopyFilm deposition & microscopy

Spray coating „air brush“

Air brush: IWATA HP-A as microdispensersystem, max volume 1ml

• constant N2 pressure 0,4bar

Microscopy picture (160x magnification)of 10mg/ml POPC / isopropanol solution,deposited on Si-wafer

Film deposition & microscopyFilm deposition & microscopy

Spin coating

Microscopy picture (160x magnification)of 10mg/ml POPC / isopropanol solution,deposited on Si-wafer

Transpipette: TRANSFERPETTOR, max volume 50µlDrilling machine: BOSCH CSB550

POPC/ 10mg/ml POPC/ 10mg/ml (from (from isopropanolisopropanol) full hydration ) full hydration

spray coating (air brush)spray coating (air brush)

Image plate Omega – scan : x-axis = s-scaley-axis = omegaz-axis = intensity

DPPC: dry, at partial and fully hydration:DPPC: dry, at partial and fully hydration:

Sample No 8: 10 mg/ml DPPC/isopropanol on Si-wafer (spin-coated)

dry partial hydrated fully hydrated

LiCl Concentration d (Å) Multilayer Peak d (Å) Isotropic Ring

0M 63.1

0.10M 64.6

0.33M 64.8 57.8

0.55M 65.09 56.0

0M, after removal of LiCl 63.9

Lithium / Lipid - Interaction In-Situ

0.1M 0.33M

0.5M 0M

POPC at full hydrationPOPC at full hydration

Sample No 23 – spin-coated

Image plate Omega – scan : x-axis = s-scaley-axis = omega

z-axis = intensity

High Pressure Surface Diffraction

Entrance Nipple

IncidentX-ray beam

Diamond Windows

Pressure Media

Silicon Waver

Sketch of the Surface Diffraction High Pressure Cell

10 mm

Specularreflectedbeam

•X-ray energy: 16 keV

•Diamond windows: 0.75 mm thick

•Beam size: 0.5 x 0.2 mm2

•max incidence angle: 4°

•exit aperture: 60° total

•sample surface: 3 x 3 mm

•p : 0-3 kbar

•temperature range: 0-80 °C

Fig.: set-up

High Pressure Surface Diffraction

Fig.: Diffraction pattern of DMPC in the Lα Phase at 30 °C during the ω-scan. FWHM of ω-scan: < 0.03°.

Fig.: Diffraction pattern at 30 °C:p-scan (1 -

1736 bar) Lα/Pβ’/Lβ’

DMPC (Lββββ ’/Pββββ ’/Lα α α α @ 15/25 °C)

High Pressure Surface Diffraction

Fig.: Diffraction pattern of POPE at 25 °C/1 bar

Tim

e (s

)

s (1/nm)

POPE (Lββββ/Lαααα transition at 22 °C)

POPE: p-scan (1000 - 1 bar, Lβ/Lα transitionat 280 bar)

WHERE DO WE GO FROM HERE ?

NanocompositesNanocomposites at Interfacesat Interfaces

Solid-state Soft (bio)materials

Bio-inspired design of functional devices

Specific Recognition

Semiconductor

NANOBIONICS : MEMBRANE ON CHIP

Sensitive Amplification

Electronics

SolidSupport

Mechano- Electronic Handling

Protein

Specificity Triggering

Lipid

Sensitivity

COST D-22: WG Membranes at Surfaces

Institute of Biophysics and X-ray Structure Research Austrian Academy of Sciences http://www.oeaw.ac.at/ibr/

IBR / Elettra Heinz AmenitschSigrid Bernstorff Pavo DubcekMichael Rappolt Georg Pabst

IBR / Graz: Manfred Kriechbaum, Ruth Prassl,Ingrid Winter, Karl Lohner,Richard Koschuch, Monica Vidal,