Systems Bio

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Results, Progress and Innovations from BMBF Funding

Systems Biology


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Imprint

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Forschungszentrum Jlich GmbH

Project Management Jlich (PtJ)

52425 Jlich

Orders

In writing to the publisher

Project Management Jlich (PtJ)

Auenstelle Berlin

PO Box 610247

10923 Berlin

Phone: +49-30-20199-457

Fax: +49-30-20199-470

E-Mail: [email protected]

Internet: www.fz-juelich/ptj/systembiologie

Supervision Contents

Dr. Sigrid Grolle, Dr. Gisela Miczka (PtJ), Jlich

Edited by

Dr. Stefanie Reinberger, Heidelberg

Translation

Language Services, Central Library, Forschungszentrum Jlich GmbH

Layout

FOCON GmbH, Aachen

Print

Bonifatius GmbH, Dr uck Buch Verlag, Paderborn

As of Jlich, Berlin 2008

Photo credits

Derichs Kommunikation GmbH, Jlich: Cover picture

Further photos and gures are provided from the respective authors (q.v.)


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Systems Biology


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PReFace2

Preface

Systems biology began well before the turn ofthe century in the USA and in Japan. Its empiricalmolecular genetics/genomics roots were moreAmerican, its physical chemistry/mathematicalbiology roots, including non-equilibriumthermodynamics and metabolic control analy-sis, more European. Hybridization arrays, QPCR,2-D electrophoresis, chromatography plus 2-D

mass spectrometry, and quantitative microscopyenable the quantication of changes in concen-trations of molecules and thus represent an ad-ditional basis for systems biology. Even if Europehas a historical lead in some of these, the majorinitiatives in systems biology started in the USAand Japan. There the time was ripe for systemsbiology whilst Europe was more sceptical withrespect to new research and development.

This barrier was overcome by the rst majorcoherent systems biology research programme inEurope, which started in 2004. The programmewas HepatoSys, funded by the Federal Ministry

of Education and Research (BMBF). In the follow-ing year, the UK BBSRC and EPSRC funded researchcentres and doctoral training centres for systemsbiology. The BMBF then funded four such researchcentres (FORSYS) in 2007. Setting up further fund-ing priorities (QuantPro, FORSYS Partner), BMBFcontinued to propel German systems biologyforward, also by supporting the training of youngscientists. At the same time BMBF announced atransnational research programme on microbialsystems biology (SysMO) with the Netherlands,the UK, Austria, Norway and Spain. In 2008 many

additional systems biology research programmesare now running in Europe, including Germany,where the new funding priority MedSys will fundapplied systems biology in medical research.

The functioning of living organisms does notonly depend on their individual components butalso on the interactions between these components,i.e. on dynamic networking. For good reasons,molecular biology focuses on individual macro-molecules. One paradox is therefore that systemsbiology needs to interface actively with molecularbiology, which itself shies away from studying

interactions and networking. Systems biologyalso needs to interface strongly with physiology,which itself frowns upon an analytical approachbased on individual components. Tuned to thesimplest possible systems and linear approxima-tions thereof, mathematics and physics considerbiology a mere set of special cases, too complexto resolve. Systems biology needs to integrateand add to these three paradoxical approaches.

Quite a number of research programmesthroughout the world call themselves systemsbiology, but do not integrate these threeapproaches. Some merely calculate theoretical

behaviour that may not actually function. Otherscollect data without interpreting how functionsarise from interactions. The BMBF researchprogrammes, and certainly HepatoSys, integratethe three approaches, and with appreciable success.The preparatory committees and the internationalsteering committees worked hard to bring aboutthis integration. The committees had to rejectexcellent research that lacked integration per-spectives, and they insisted on the integration ofdistinct proposals. Both types of action are unusualin evaluating and advising on scientic research.

The paradigm shift effected by systems biologyimplies that the success of systems biology pro-grammes should be judged by more stringentcriteria than the success of traditional research

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programmes. Of course, the research should beexcellent, as judged from the discoveries and ap-plications. In addition, the programmes should bedistinguished from the traditional research pro-grammes in molecular biology, mathematics andphysiology. Research that is excellent in terms ofmolecular biology but not in terms of mathematicsmay not be regarded as excellent systems biol-

ogy. On the other hand, the highest excellence insystems biology may conict with the paradoxicalstandards of the two neighbouring disciplines. Forthe steering committee, this makes life difcult,as the quality of research proposals/reports can-not be assessed from the number of publicationsin journals with high impact in molecular biology,or in conference proceedings in engineering.

Another paradox relates to the involvement ofindustry in systems biology programmes. The phar-maceutical industry understands why one shouldlook at disease and drug safety from the perspectiveof networks. However, since systems biology deals

with entire networks, the best expertise needs to beengaged; involving too many research groups forthe intellectual property to remain exclusive. Thepharmaceutical industry will only become involvedwhen research starts to become applicable. Thenthey plan dened bilateral projects with academicresearch groups. These kinds of projects are ex-pected for the funding priority MedSys, and maynow also become possible for Hepatosys. Systemsbiology is a matter for large-scale public funding inorder to support new application-relevant researchprogrammes in their early phases.

BMBF is to be complimented on the importantrole it has played in the emergence of systemsbiology. Japan and the USA may have been rstto engage in systems biology, but BMBF has nowput Europe into a leading position with the rstand by far the largest, truly integrated systemsbiology programmes. The integration of thevarious disciplines dealing with various aspects of

the human cell is of tremendous importance forhealth, disease and drug effectiveness. BMBF hasalso promoted the standardisation that is abso-lutely essential for the life sciences and for thesilicon human of the future. In addition, it hasenabled scientists to make scientic discoveriesthat could not otherwise have been made.

I invite you to study this brochure, and to assessand enjoy the progress made by systems biologyin Germany. Systems biology is working, also inEurope.

Hans V. Westerhoff

Hans V. Westerhoff is AstraZeneca Professor of Systems Biology and

Director of the Doctoral Training Centre for Integrative Systems

Biology from Molecules to Life (ISBML), Manchester, UK. Furthermore,

he is Professor of Molecular Cell Physiology at the Free University

Amsterdam and is Chairman of the HepatoSys Steering Committee.


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Contents

PrefaceHans V. Westerhoff 2

Contents 4

Progress and Innovation through Systems BiologyG. Miczka 6

HepatoSys Systems Biology of Liver Cells

HepatoSys - Systems Biology Studies of Liver Cells (Introduction)U. Heisner 10

Robustness of the Drug Detoxication Metabolism in Liver CellsM. Reuss, J. Bucher, U.M. Zanger 12

A Circuit Diagram for BiotransformationK. Mauch 14

High Tech for Liver CellsM. Athelogou, G. Schmidt, F. Owen 16

The Endocytosis Transport Network/SystemM. Zerial, J. McCaskill, A. Deutsch 18

Iron NetworkJ.G. Reich, M. Muckenthaler, M. Hentze 20

Central Data ManagementH.-P. Fischer, D. Bannasch 22

Liver Cells in Culture

J. G. Hengstler 24

Feedback for Liver RegenerationU. Klingmller, S.Dooley, J. Timmer 26

Liver Regeneration A Unique PhenomenonD. Drasdo, S.Hhme 28

HepatoNet - Modelling the Liver MetabolismH.-G. Holzhtter, K. Hbner, S. Hoffmann 30

FORSYS Centres of Systems Biology

FORSYS Research Units on Systems Biology (Introduction)B. Regierer 32


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Targeting Parkinsons DiseaseR. Baumeister, E. Schmidt 34

The Cells Suicide ProgrammeR. Eils 36

On the Track of Molecular Synergisms

W. Weckwerth 38

Microbes and Men A Complicated CoexistenceM. Naumann, R. Poltz 40

QuantPro Quantitative Analysis for the Description of Dynamic Processes in Living Systems

QuantPro Quantitative Analysis for the Description ofDynamic Processes in Living Systems, (Introduction)Y. Pfeiffenschneider 42

Biomarkers for Potato Breeding

P. Geigenberger 44

Transport Systems in the LiverU. Pehl 46

The Light Processing NetworkD. Osterhelt , M. Uefng 48

SysMO Systems Biology of Microorganisms

SysMO Systems Biology of Microorganisms (Introduction)M. Heidelberger 50

Lactic Acid Bacteria in ComparisonU. Kummer, B. Kreikemeyer 52

Stress in BacteriaV. Martins dos Santos 54

Clostridium acetobutylicum a Response to Dwindling Crude Oil ReservesP. Drre, A. Ehrenreich 56

ERASysBio 13 Countries Coordinate their Funding ActivitiesV. Simons 58

Data and Facts on Funding for Systems Biology in GermanyE. Stttgen 59


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In all societies, innovations form the basis forprogress and development. Innovations ensurecontinuous growth, prosperity and internationalcompetitiveness. The Federal Governments High-Tech Strategy for Germany is therefore specicallypromoting research elds with a high innovationpotential. This also includes the relatively young

discipline of systems biology.

After the widespread introduction of themethods of molecular biology in medicine andbiology, systems biology is regarded as the secondkey technology for achieving progress in the lifesciences. At the same time, it forms the basis forexploiting new innovation potential in theknowledge-based bioindustry.

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In the past, the individual research disciplinesin the life sciences primarily focused on investigat-ing process ows down to the molecular details. Ina descriptive approach directed at achieving highquality and molecular details, a wealth of data wasgenerated concerning single cell components orfunctions. However, the interaction of these mol-ecular structures is highly dynamic and is control-led by cross-linkages with all cellular hierarchies.In order to understand such a biological systemas a whole, it is necessary to have a quantitativeunderstanding of the processes taking place init. This is the starting point for systems biology.

The aim of the systems biology research approachis to understand the behaviour, the dynamics of abiological system, for example a metabolic path-way, a cell organelle or - in the distant future - awhole cell or organism in its entirety. This requiresthe linkage of all molecular biology data from thelevel of the genome, through the transcriptomeand the proteome, up to and including the met-abolome, the analysis of interaction patterns andalso data integration with the aid of mathematicalmethods. A basic prerequisite for systems biologyapproaches is therefore the interdisciplinary

collaboration of researchers from the elds ofbiology, chemistry, medicine, computer science,mathematics, systems science and also engineering.The heart of the systems biology research approach

is an iterative process between laboratory experi-ments and mathematical modelling. The result ofthis process is an optimised mathematical modeldescribing the behaviour of a given biologicalsystem in a dened environment. This thus facili-tates predictions about the behaviour of the systemunder the inuence of internal and external factors.

Benets of systems biology

With its new concept, systems biology has thepotential to radically change the life sciences andto provide completely new ndings for biomedicalresearch and for biotechnology in industry andagriculture. Working with models and computersimulations offers the opportunity of proceed-ing in a targeted manner. Instead of looking forthe proverbial needle in a haystack, the most

probable processes can be calculated and ex-periments tailored accordingly. Systems biologythus offers the opportunity of raising knowledgeof dynamics and the interaction of vital func-tions to a completely new plane and of exploit-ing new potential for innovation in medicine,the pharmaceutical industry, the chemicalindustry and the biotechnology industry.The application of computer models may in future,for example, serve to nd new targets for treatingdiseases or forecasting possible side effects of newactive substances. Drug development will thus

become more effective and safer and, moreover,permit animal experiments to be restricted to aminimum. In the same way, biological applicationscan be specically optimised, for example increas-

PRogReSS and InnovatIon thRough SySteMS BIology6

Progress and Innovation through Systems Biology

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PRogReSS and InnovatIon thRough SySteMS BIology 7

ing the productivity of cell systems for certainsystems and also the development of novel synthesistechniques. First applications are already beginningto emerge in the ongoing research projects.

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Systems biology requires altered researchstructures in science and industry, new cooperationmodels and a new quality of interdisciplinary andinterindustrial collaboration in a national and inter-national framework. The Federal Ministry of Educa-tion and Research (BMBF) recognised this at an earlypoint and is reacting to these constraints. As partof the Federal Governments High-Tech Strategy,it is undertaking a selective expansion of systemsbiology support measures and the establishmentof relevant research and funding structures on a

national and international level. These measuresare being taken within the context of lines of actionplanned or implemented by the federal states, theHelmholtz Association, the Max Planck Society andother research and funding organisations in thiseld.

Back in 2001, BMBF initialised funding of thisinnovative research eld in Germany with its callfor proposals for Living Systems Systems Biology.The resulting pilot project Systems Biology of theLiver Cell - HepatoSys has now developed into anationwide network of expertise which also enjoys

international recognition.The support programme Research Units of

Systems Biology- FORSYS created the decisive basisfor systems biology in Germany. The establishmentof the four FORSYS centres in Freiburg, Heidelberg,Magdeburg and Potsdam in 2007 improved thesituation for systems biology, with respect to bothstructure and content. The FORSYS centres ensurethat the interdisciplinary collaboration essential forsystems biology is available under the same roof,and that there is a local concentration of researchexpertise that also provides training opportunities

for young scientists.The support measure announced in 2007Partners for Research Units of Systems Biology FORSYS Partners further strengthened systems

biology in Germany. This support measure consistsof two components. The FORSYS Cooperationsprovide support for a transfer of know-how be-tween the existing FORSYS centres and partnersfrom academia and industry and lay the founda-tion for the establishment of further competencenodes for systems biology in Germany. The FORSYS

Young Investigators Groups give young scientiststhe opportunity to conduct independent researchand thus to exploit their creative potential.

The support measure Medical Systems Biology MedSys announced early in 2008 focuses on theapplication potential of systems biology for medi-cine and drug development. Apart from academicresearch groups, it therefore primarily targets cor-porate research departments in the pharmaceuticaland biotechnology industries, which are concerned,among other things, with the development ofpatient-related tools for diagnosis and treatment orthe application of systems biology approaches forincreasing the efciency of clinical trials.

Systems biology also plays a not inconsiderablepart in the support measure on the topic of

BioEnergy 2021 Research on Utilising Biomassannounced at the beginning of 2008. The SystemsBiology for Bioenergy module will fund researchprojects that contribute towards optimising crops asenergy plants.

The discussion is currently focusing on furtherresearch measures including the expansion of themethodological and technological basis for systemsbiology, for which the foundation was laid by theresearch priority Quantitative Analysis to Describethe Dynamic Processes in Living Systems Quant-

Pro that started in 2004. In addition, attentionis also being paid to exploiting the potential ofsystems biology research for other elds ofapplication (e.g. for health in ageing).

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Apart from national commitments, the FederalResearch Ministry is also involved in the develop-ment of European support measures for systemsbiology. For example, in 2006 as part of the ERA-NetERASYSBIO, the transnational support measureSysMO (Systems Biology of Microorganisms) wasagreed jointly with six European partners. Funding

of the transnational collaborative projects began in2007 and due to the success of these projects will becontinued beyond 2010.

Bilateral support measures for Medical SystemsBiology were started with Slovenia in 2007. Other

cooperative projects are planned with Austria fromspring 2008.

Furthermore, another European support meas-ure for systems biology is under preparation as partof ERASYSBIO, which is receiving major supportfrom Germany and the UK. With these supportmeasures, BMBF is pursuing the goal of speci-cally strengthening national research and fundingpriorities in the eld of systems biology by interna-tional networking and of promoting cooperationbetween systems biology centres, which are at themoment mainly located in the Netherlands, the UK

and Germany.

The Federal Research Ministry is also involved inplanning a large-scale European project on theSystems Biology of the Metabolic Syndrome that isscheduled to be launched in 2009.

The strategy paper Systems Biology in the Euro-pean Research Area was published in April 2008by ERA-Net ERASYSBIO as a basis for the further

harmonisation of funding policy for systems biologyin Europe.

PRogReSS and InnovatIon thRough SySteMS BIology8

Schematic representation of the iterative cycle of experiment and

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With its early and comprehensive support forsystems biology, BMBF has helped Germany toestablish a leading position in this importantresearch eld that has great future potential. Thetotal BMBF funding of systems biology is 37 million

per year for national and international supportmeasures.

The aim of the BMBFs coordinated research andfunding measures is to support the establishmentof the research infrastructure required for systemsbiology together with major actors in Germany andEurope. It is taking up central research elds andtopics which are of major signicance for progressin the life sciences and for exploiting new inno-vation potential and contributes signicantly to thetraining and encouragement of young scientists.

In this way, the competitiveness of Germanywill be sustained in the eld of the life sciences andcooperation between academic and industrialresearch specically promoted.

Practical applications of the systems biologyresearch approach are already foreseeable in theelds of biotechnology and medicine. Since 2004,

the rst companies mainly small and medium-sized enterprises (SMEs) have already becomeinvolved in this research as pioneers in systemsbiology. The increasing maturity of systems biologyis manifested in the growing number of researchpartners from the pharmaceutical industry, thebiotech industry and other sectors who recogniseopportunities for the medium- and long-termdevelopment of new areas of business.

Gisela Miczka, Projekttrger Jlich (PtJ)

On the Internet: ww w.fz-juelich.de/ptj/systembiologie

Fascination Systems Biology

Dr. Vytaute Starkuviene-Ere has been head of the Young Inves-

tigators Group Screening of Cellular Net works since autumn

2007, which is part of the VIROQUANT centre in Heidelberg. She

spoke about her enthusiasm for systems biology and the excellent

opportunity to work in such a research centre.

When did you become interested in systems biology?

It was during my time as a postdoc at EMBL in Heidelberg. I had

established high-throughput assays for studies on trafcking

mechanisms in mammalian cells. In doing so, I began to under-

stand the signicance of being able to analyse many components

at the one time instead of concentrating on single proteins, which

is what I was familiar with from my earlier work. So thats why I

decided to orient myself in the direction of systems biology.

What is so fascinating about systems biology?

Systems biology throws light on a specic issue from all sides both

from the level of the gene and the protein right up to the effects in

the system and vice versa. It accounts for a wide variety of factors in

this process, and can be expanded to areas where classical biologywas not admitted in the past, such as biophysics, bioorganic

chemistry or even quantum physics. This gives us an overall picture

of whats going on it doesnt just show us how a single protein

behaves, but rather how the whole system or at least a part of it

behaves. Thats absolutely fascinating. Systems biology has the

potential to help us understand every system, every organism in

its entirety and complexity at some point in time. At the moment,

weve only just begun, but the process of getting to this stage alone

is very captivating.

What does it mean for you to head a Young Investigators Group

in the VIROQUANT centre?

As soon as they have penetrated the host cell, viruses become

involved in a number of cellular processes and therefore inuence

the entire system of the cell. VIROQUANT investigates these pro-

cesses and this information is what attracts me. In the BIOQUANT

building in Heidelberg, I work side-by-side with virologists and cell

biologists, as well as with mathematicians, bioinformaticians, and

modellers. We have lively discussions and our cooperations are ex-

tremely fruitful. This is the only way that systems biology can really

function well. I believe that my research benets from this to a largedegree and I hope that I too can contribute a great deal.


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hePatoSyS10

HepatoSys

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With its call for proposals for Living Systems Systems Biology in 2001, the Federal Ministry ofEducation and Research (BMBF) gave the go-aheadfor the funding of systems biology in Germany.HepatoSys a national network of expertise for re-search into liver cells was initiated in 2004. Today,HepatoSys is the largest systems biology consortium

working on interdisciplinary principles anywherein the world.

The liver is the central metabolic organ in verte-brates and is in many respects a very special organ.Each day, it synthesises, converts or degrades morethan 10,000 substances and thus contributes to theutilisation of food and purifying the body of meta-bolic products, drugs, alcohol and other harmfulsubstances. As the largest gland in the body, in 24hours it produces almost a litre of bile and thus as-sists the bodys digestive system. The organ serves tostore glucose and vitamins and also produces vital

proteins such as the blood clotting factors. The liveris furthermore characterised by its unique abilityto regenerate itself almost completely after dam-age by injury or toxic agents. Hepatocytes expressmore genes than most other types of tissue in themammalian organism. They therefore have a verywide range of enzymes and metabolic networks.

The HepatoSys network of expertise investigatesregeneration, differentiation, endocytosis, detoxi-cation and the iron metabolism in liver cells. To

this end, experimental research teams from biol-ogy, chemistry, pharmacology and medicine workhand in hand with representatives of theoreticalphysics, mathematics and with computer scientistsand engineers. The long-term objective is to createa model for predicting vital processes in the liver,which would represent an enormous increase inknowledge for medicine and pharmacology. Drugscan be developed more efciently and economi-cally with the aid of such tools. In silico modelsopen up new possibilities of individualising treat-ments and signicantly reducing the numberof animal experiments in drug development.

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During the rst funding phase of the Hepa-toSys consortium from 2004 to 2006, attentionwas initially focused on creating a functioninginfrastructure. Today, more than 40 teams workin HepatoSys in the four regional networks ofdetoxication, iron metabolism, endocytosis andregeneration in addition to the two platformsof cell biology and modelling using comparable

cells according to jointly agreed lab protocols.

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hePatoSyS 11

The important results, laboratory regulationsand background information are made accessible toall members of the network by means of central dataand knowledge management. The second fundingperiod started at the beginning of 2007. Until 2009,all activities are focused on result-oriented research.

The project is monitored by an internationalpanel of high-calibre experts who provide valu-able stimulus for further developments. Hepato-

Sys is steered by a project committee consistingof the coordinators and representatives of thenetworks and platforms. The project committeetakes up the recommendations of the steeringcommittee and is responsible for implementingthe milestone planning. The project committeecoordinates the interdisciplinary collaboration,organises the dates and keeps itself informed ofthe scientic progress made in the consortium. Inorder to handle the wide range of organisationaltasks, the project committee has a central projectmanagement unit. In addition, each network and

platform has its own local project management.

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There is a very high level of knowledge trans-fer in the HepatoSys consortium due to the inter-disciplinary collaboration and the numerousexternal cooperation. A steadily increasing numberof companies are joining the association. This devel-

opment is encouraged by the fact that the regionalnetworks are located at important biotechnologysites throughout Germany. For example, the regen-eration network has its main sites in Freiburg andHeidelberg in the immediate vicinity of the world-famous hospitals in Heidelberg and Freiburg andalso close to BioValley e.V., an association bringingtogether industrial companies and research insti-tutions in Germany, France and Switzerland. Thedetoxication network maintains close contactswith the process engineering industry in theStuttgart region. The iron regulation network col-

laborates closely with the Charit university hospi-tal in Berlin and the Heidelberg university clinic.The network of expertise is also very well known

outside Germany. In 2006, HepatoSys attractedinternational attention as the organiser of the rstSystems Biology of Mammalian Cells (SBMC)conference. At the beginning of 2008, the HealthProgramme of the European Union grantedfunding for a HepatoSys project devoted to studyingcancer of the liver. This project, which will be start-ing in October 2008, means that the HepatoSysnetwork of expertise is extending its activities onthe European level.

Dr. Ute Heisner

Systems Biology of Liver Cells - HepatoSys

Central Project Management

Institute for Physics, University Freiburg

Phone: +49(0)761-203 5803www.systembiologie.de

www.hepatosys.de

[email protected]

ar:


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Robustness of the Drug

Detoxication Metabolism in Liver CellsCompensation for genetic polymorphisms and environmental inuences inri xbiis i ir

Humans and animals are subjected to perma-nent exposure to different kinds of xenobioticsubstances, including plant toxins, medicinal drugs,and environmental poisons. In vertebrates, the liverhas the task of making these substances water-solu-ble and thus preparing them for excretion. In hepa-tocytes, this process consists of a complex sequence

of reaction steps (biotransformations) that are cata-lysed by an extensive enzyme system, in particularby the cytochrome P450 monooxygenases (CYP450).

Caused by a number of variations in geneticmake-up, so-called genetic polymorphisms, and asa result of illness or environmental factors such asthe intake of food and drugs, these enzymes exhibita pronounced interindividual variability in theirexpression and functionality, in other words in howmuch of the effective enzyme is available in the cell.

So that the metabolism of xenobiotic substancesprogresses smoothly, it must be robust compared

to the individual factors of inuence. Robustnessis a central concept in the systems-theory analysisof networks. It describes how well a system cancompensate for disturbances that have eitherbeen caused internally, for example as a resultof mutations, or externally, for example throughenvironmental inuences. Experimental andtheoretical groups within the HepatoSys Compe-tence Network are working on our project, whichaims to mathematically model and simulate thedetoxication metabolism of drug substances. Aspart of this project, we are studying how the largevariability of the CYP450 system is compensated.

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For the characterisation of the catalytic proper-ties of the CYP450 system, we chose dextromethor-phan, a cough suppressant, and propafenone, ananti-arrhythmic agent as model substances. In orderto identify the CYP450 variants involved in convert-

ing these substances, we rst conducted activitymeasurements of recombinant enzymes. On thisbasis, we established the topological structure ofthe metabolic system: it is a multi-reactive system inwhich individual isoenzymes have an overlappingsubstrate specicity.

Both of the model substrates were degraded by anumber of enzyme variants. This redundancy is

extremely important for the robustness of thesystem. It increases the number of possible degrada-tion pathways and reduces the risk that the loss ofan individual CYP could endanger the functionalperformance of the degradation. This becomes clearthrough a comparison with a reduced model inwhich each reaction step is only realised by the mostactive master isoenzyme. In this case, the loss of anindividual enzyme would have a more drastic effecton the degradation of xenobiotic substances.

In order to identify the parameters of the mathe-matical model, we conducted experiments on

microsomal fractions, membrane-limited vesiclesfrom human liver tissue, in which CYP450 agentswere anchored. We determined the formation ratesof the degradation products for different starting

Model structure of the drug detoxication metabolism of the sub-

strates dextromethorphan (DTM) and propafenone (PPF) for the phase I

metabolites and CYP450 enzymes involved in the complete isoenzyme

model (left) and in the reduced model (right).

In the liver, cytochrome P450 monooxygenases are responsible for thedegradation of xenobiotic substances. Here, the percentage distribu-

tion of different CYP450 agents is shown.


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hePatoSyS 13

Prof. Matthias Reuss is Director of the

Institute of Biochemical Engineering at

the University of Stuttgart. His research

interests include systems biology and its

possible applications for biotechnology

and medicine.

Phone: +49(0)711-68 564 573

[email protected]

Dipl.-Ing. Joachim Bucher studied process

engineering at the University of Stuttgart

and is currently completing his Ph.D. at

the Institute for Biochemical Engineer-

ing, University of Stuttgart, in the eld of

systems biology.

Phone: +49(0)711-68 566 324

[email protected]

Prof. Ulrich Zanger is head of the research

eld of Molecular and Cell Biology at the

Dr. Margarete Fischer-Bosch Institute of

Clinical Pharmacology in Stuttgart.

For the last 20 years, he has been working

on drug metabolism and genetic poly-morphisms.

Phone: +49(0)711-81 013 704

[email protected]

ars:

concentrations of the model substrates - individu-ally or in combination. On the basis of these data,model parameters such as the maximum enzymaticdegradation rate were estimated with the aid ofwhat is known as an evolutionary algorithm.

The results show that the parameters - compa-rable with the interindividual differences in CYPconcentrations and activities - may vary stronglyin the isoenzyme model. Despite high variable pa-rameters, a constant good adaptation was achieved,which can be interpreted as the maintenance of the

functionality of the drug detoxication metabolism.

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Using model simulations of substrate degra-dation, we investigated how well protected theCYP450 system is against inter-individual vari-ability. In order to do so, we selected the data of 150individual isoenzyme concentrations in the liver cellfrom a comprehensive liver bank and integratedthem into the reaction kinetics in the model simula-tions.

The following is true of the robustness of themetabolism of drug substances: the smaller thedeviations of the half-lives of the substrate degrada-tion, the more robust the system. It was shown thatthe complete isoenzyme model exhibited a lowervariance of half-lives than the reduced model,which lacked isoenzymes that appeared to be lessimportant but were critical for redundancy. Theredundancy of the isoenzymes therefore representsa decisive factor for the compensation of individualdifferences.

a ri f pssib ppiis

That the CYP450 system represents an extremelydiverse and robust chemical defence system is not

new. The advantage of systems biology analyses liesin the fact that many different conditions can besimulated with the models created. In the future,this could help us to gain a better understandingof the circumstances under which the robustnessof the system is no longer sufcient to perform anadequate detoxication, or to simulate the degra-

dation of new substances under different conditionsin early phases of drug development.

Furthermore, we want to link other systemsbiology model assemblies, which contain the generegulation of the CYP450 enzymes, or relevantaspects of the central carbon metabolism of the cell,and thus achieve a comprehensive modelling andsimulation of the degradation processes of drugsubstances in the liver cell.

The inter-individual differences in the CYP isoenzyme concentration is

reected in the half-life for substrate degradation. The example shows

the simulation proles for two different livers.


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hePatoSyS14

A Circuit Diagram for Biotransformation

Dynamic ux analysis of the central metabolism in human hepatocytes

The enzyme system of the central metabolismplays an important role in the functioning of anorganism. It supplies energy equivalents that makeit possible for vital processes to occur and it alsoallows the biotransformation of endogenous andforeign substances so that these can be eliminatedfrom the body. The mathematical description of

this enzyme system represents a decisive basicprinciple for predicting the dosage of medications.The objective of our research project is therefore tocreate a type of circuit diagram that quantitativelyrecords the transformation of the componentsinvolved in the central metabolism. Coordinatedby Insilico Biotechnology AG, Stuttgart, a teamof chemists, biologists, engineers, and computerscientists analyse the activities of the centralmetabolism and integrate the data into a computermodel before they simulate the behaviour of themetabolism with the aid of supercomputers.

Fwi mbi pws

We have already succeeded in integratinga few hundred enzyme-catalysed reactions andmore than 400 metabolites, in other words thedegradation products and intermediate degrada-tion products of biochemical metabolic processes,

into the model system. In quantifying intracellularmetabolite concentrations, it is essential that themetabolism of cells be stopped immediately aftersampling. This is the only way of ensuring that our

results reect the actual situation at a selectedpoint in time. For this purpose, researchers at theInstitute of Biochemical Engineering in the Uni-versity of Stuttgart apply an ingenious procedurewhich involves briey treating hepatocytes thathave been quickly separated from the mediumwith water at a temperature of over 90 degrees

Celsius. This inactivates enzymes ensuring that themetabolites are not degraded further. With theheat treatment, we simultaneously achieve a celldisruption that releases intracellular metabolites.

Following this, colleagues in the Dr. MargareteFischer-Bosch Institute of Clinical Pharmacology,Stuttgart, determine the amount of metabolitesin the samples. In order to do so, they combine gaschromatography and high performance liquidchromatography with mass spectrometry. Thehighly sensitive measuring procedure does not justguarantee a precise determination of the metabo-

lites; it also allows the detection of isotopes, whichare labelled compounds that only differ fromunlabelled compounds by their mass numbers.In this manner, it is also possible to investigateconversions in parallel metabolic pathways.

Metabolic network of human hepatocytes (circles symbolise metabo-

lites, enzyme-catalysed reactions are represented by rectangles). From

the modelling and simulation environment, precise mathematical

equations for the components are formulated, evaluated and

compared with experimental data.

The determination of intracellular metabolite concentrations requires

precise analysis. A combination of high-pressure liquid chromatogra-

phy and mass spectrometry is, for instance, used for this purpose.


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hePatoSyS 15

For example, we use glucose labelled with13C carbon atoms and trace the distribution dy-namics of the heavy isotope in the glucose deg-radation products. Computer simulations on thebasis of these studies then allow us to calculatemetabolic uxes in parallel pathways and reac-tion cycles. Using this method, we succeeded for

the rst time in determining the production ratesof NADPH - an important cosubstrate for medi-cation degradation in the liver - in hepatocytesusing the pentose phosphate pathway. We nowknow that NADPH is not a limiting factor for theapplication and disposal of therapeutic agents.

Mbi mis fr

miisri f mii

During the rst project phase in the period 2004to 2006, the focus was on the analysis of station-

ary metabolic uxes in the central metabolismof hepatocytes. On this basis, we will direct ourattention in the second phase from 2007 to 2009to the metabolic dynamics after the administra-tion of cholesterol-lowering drugs. The aim is todescribe the effect of these therapeutic agentsmechanistically on a metabolic level and to predictthe inuence of the medication dosage. We wantto know how the enzymes involved control theproduction of cholesterol and how side effects canbe avoided. Furthermore, we are interested in theeffect the substances have in people with differentgenetic backgrounds so that we will be able to take

steps towards creating individualised treatments.In this way, we are making an economically rel-evant contribution to reducing the amount of timeand the high costs involved in studies that aim todetermine the appropriate medication dosage.

In cooperation with other partners in theNetwork Detoxication from the Stuttgart groupinvolved in HepatoSys, we are working on a modelsystem which can be used to predict the optimumdosage of medication using a computer. The rststep requires experiments for recording the dynam-ics of the central metabolism after the administra-

tion of the active ingredient and then imaging themusing the computer. Since the kinetic parametersfor the majority of the enzyme reactions involvedare unknown, we use sophisticated estimation

procedures known as evolution strategies in orderto determine the unknown parameters. The resultspreviously obtained with this procedure are en-couraging. With one of the rst dynamic models,we thus succeeded in achieving good agreementbetween the simulated and measured metaboliteconcentrations. This model is available to HepatoSysproject partners so that further models can be incor-porated.

Computer simulation of the time course of selected metabolites after

excitation of the system by a sudden reduction in the external nutrient

concentration.

Klaus Mauch is co-founder and CEO of

Insilico Biotechnology AG

Phone: +49(0)711-674 2164

[email protected]

www.insilico-biotechnology.com

Projectpartners involved: Insilico Biotechnology AG (Klaus Mauch),Dr. Margarete Fischer-Bosch Institute of Clinical Pharmacology (Ute

Hofmann), Institute of Biochemical Engineering in the University of

Stuttgart (Klaus Maier, Anja Niebel, Gabriele Vacun, Matthias Reuss).

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hePatoSyS16

High Tech for Liver Cells

am im sis f sis

As part of the HepatoSys initiative, the Endocy-tosis Network (EndoSys) focuses upon analysis ofendocytosis and its inuence on signal transductionusing systems biology. The members of the con-sortium, for instance, investigate the formation ofvesicles (cell compartments enclosed by the cyto-plasmic membrane with which membrane proteins

or nutrients are ingested into the cell), and howthe vesicles are transported within the cell. Theseinvestigations produce large quantities of veryheterogeneous image data, such as two- or three-dimensional microscopic images of hepatocytesand their components, as well as simulated imagedata of different biological processes. Relevantparameters for investigation are simultaneously de-termined using both experiments and simulations.

As the technology partner of the HepatoSysnetwork, Deniens AG Munich has the task ofgenerating automated image analysis so as: to en-

able these heterogeneous datasets to be combined;to generate parameters from the experimentalimage les; and, conversely, to produce simulatedimages from the experimental measurements.

Rii

Originating from the idea of mapping elemen-tary mechanisms of human perception simplyand naturally into an image analysis process, Gerd

Binnig and his team developed Deniens CognitionNetwork Technology. It is based upon the concept ofinformation processing through cognition net-works. This image analysis process involves gen-

erating semantic networks that describe objects.In simple terms, a cell, for example, is representedtogether with its properties such as size or shape,and these attributes are linked hierarchically (thecell is large, elongated and granulated). Thesehierarchies are then in turn linked and they collec-tively form a network - the cognition network.

Considering the example of a liver cell, im-aged with the aid of a confocal microscope, thenetwork has the following structure: the lowestlevel contains pixels as objects, which are com-bined on the next higher hierarchical level to formlarger units such as cell nuclei, endosomes andcytoplasm; these are further combined into ob-jects representing individual hepatocytes, whichare nally combined into groups of liver cells. Thisprocedure can be further continued, for example,by combining clusters of cells into organs andorgans into organisms. If the necessary data is not

present in a single image, it is possible to relatethe contents of several images. Metadata such asmeasurement information can also be utilised.

Frm im prmr

simi bk i

Cognition Network Technology can be used toanalyse both image data (e.g. three-dimensionalimages of hepatocytes) and results from modellingand simulations (e.g. the modelling of endocytoticprocesses).

In the case of microscopic images, information is

recorded such as the following:

the segmentation and classication ofiii ps, i, sis,

sms psm;

i sripi f ps ir s;

qii sripi f mris bw bjs i,

s s is f sms frm

i.

In simulations like those involving vesiclesduring endocytosis, point coordinates are availablewhich describe the image e.g. its extent andorientation in space. Similarly, data from

Image analysis of three-dimensional (confocal) pictures of a hepato-

cyte based upon Deniens Network Technology (red: cell nucleus;

blue: marker; green: cytoplasm; yellow: cell boundary for the

membrane representation; image data: Steven Dooley).


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hePatoSyS 17

experiments and statistical analyses can be used tocompile simulated images.

Since information from real and simulatedimages and also experimentally-obtained data can

be processed at one common level, these differenttypes of information can be interconverted e.g. in-formation from a confocal image can be convertedinto experimental data in order to produce a simu-lation. Experiment, modeling and simulation canbe linked in this way and the parameters resultingfrom the analysis can be used to optimise experi-ments and models.

lr ms f

ris ps f s

In order to investigate the endocytotic proc-

esses in liver cells, Endocytosis project partnershave developed assays that have to be performed inextensive screening programmes, thereby gener-ating large quantities of data sets. The algorithms

developed by Deniens are used to analyse thesedata sets, to record them quantitatively and toextract information from them. The algorithms,which were rst developed for small volumes ofdata, can then be adapted to the requirements oflarge volumes of data. Benets of this approachusing Cognitive Network Technology include: good

transferability and high precision in applying theimage analysis approach to a large number of im-age les; full-automation; user-friendly softwarethat is easy to operate; and an implementationthat is exible and adapts easily to changing usagepatterns. The algorithms that Cognition NetworkTechnology employ for image and table analysis willin future also be used to investigate other cell types.

Image analysis of three-dimensional (confocal) pictures of hepatocytes

(white: individual endosomes; magenta: cell nuclei; green: cytoplasm;

image data: Marino Zerial).

Image analysis of three-dimensional simulated image data. The

gure shows a time sequence from the development of vesicles

during a simulation. Each individual vesicle is represented by a dif-

ferent colour. In the Deniens image analysis platform, images are

automatically generated from the point coordinates of the simulated

vesicles and then analysed via Cognition Network Language (CNL)

rules (Simulation: J. S. McCaskill).

Dr. Maria Athelogou, is s enior research

scientist with Deniens AG and is

concerned with project management

and image analysis.

Phone: +49(0)89-231 180 14

[email protected]

www.deniens.com

Dr. Gnter Schmidt is senior research

scientist at Denens AG and addresses

issues of software development and im-

age data analysis.

Phone: +49(0)89-231 180 15

[email protected]

Owen Feehan concentrates on software

development and image data analysis.

Phone: +49(0)89-231 180 97

[email protected]

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hePatoSyS18

The Endocytosis Transport System

Mr swis r mri rspr isi

Endocytosis is a central cellular process in whichmembrane components and dissolved substancesare taken up by the cell surface. In this process, thecell membrane folds around the object thus form-ing vesicles which transfer their cargo to a set ofintracellular membrane compartments that consti-tute the endosomal transport system. Depending

on the purpose, the endocytosed cargo, it is eitherrecycled or degraded within the cell. Endocytosiscontrols processes such as nutrient uptake, proteintransport within the cell and the signalling responseto growth factors and hormones. Diseases such asAlzheimers, asthma or viral and bacterial infectionshave been associated with defects in this transportsystem, which makes endocytosis important andinteresting from a biomedical perspective.

To date, the mechanisms underlying endocytosisremain largely unexplored. At present, there is nopossibility of predicting the course of endocytosis

under different physiological and pathologicalconditions. Within the general frame of Hepato-Sys, the aim of the EndoSys network is to analyseendocytosis and its inuence on cellular signallingnetworks by a systems biology approach, focus-ing on liver cells. The ultimate goal is to developboth specic mathematical models and a generalsimulation platform. This will serve for a quantita-tive prediction of endocytotic processes and signaltransduction in hepatocytes under predenedphysiological or pathological conditions.

Simi pfrms fr sis

In systems biology, the analysis of endocytosisin liver cells presents us with entirely new chal-lenges. Current studies demonstrate that molecularreactions as well as changes in transport and shapein cellular compartments such as endosomes areclosely coupled. The necessary integration includeschemical activities on several spatial levels - startingwith individual molecules, via supramolecular proc-esses, up to the dynamics of compartments, such asprotein sorting by vesicle budding and nally theentire cell.

In order to improve our understanding of theseprocesses, within the EndoSys network at the RuhrUniversity in Bochum we are developing a novelhierarchical simulation platform. Complex objects

for example, collections of proteins at the molecularlevel and surface elements of vesicle membranes atthe next higher level are regarded as a hierarchyof container systems. The individual containers areloaded with molecular structures of the next lowerlevel. The simulation therefore bridges the gapbetween molecular processes such as the

interaction of proteins on the vesicle membraneand the dynamic processes on the level of completeendosomes, for instance for the processes ofmembrane deformation, membrane fusion andprotein exchange. For the rst time, a systematicand molecular-based simulation platform has beenestablished combining the chemical kinetics andphysical self-organisation of structures for a spatial-ly and temporally resolved investigation of cellularprocesses. This method will be of major importancefor future computer-based studies of endocytosis inliver cells in systems biology but the simulations can

also be systematically adapted to many differentproblems.

nw risi priip

To gain a full understanding over and abovethis, we must also unravel interested in the precisemolecular mechanisms underlying endocytoticmaterial transport. To this end, at Dresden Univer-sity of Technology we have translated the molecularswitches that regulate the transport between earlyand late endosomes into a system of partial differ-ential equations. These can be used to represent, for

example, the concentration of typical key regula-tory proteins Rab5 for early endosomes, Rab7 forlate endosomes as a function of time and positionon the vesicle membrane.

Primary mouse hepatocytes with endosomes under the microscope.

Early endosomes are stained green, late endosomes red and the cell

nuclei glow blue.


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hePatoSyS 19

Rab5 and Rab7 are molecular switches that caneach recruit a specic ensemble of partner effec-

tor proteins which undertake different tasks in thesorting, recycling and degradation of transportedmaterial up to and including vesicle movement anddeformation. Fluorescence microscopy investiga-tions at the Max Planck Institute of Molecular CellBiology and Genetics in Dresden have shown thatabsorbed material of endocytosed cargo destinedfor degradation is rst concentrated in Rab5 endo-somes and then collected and transferred to a Rab7endosome. In this process, Rab5 appears to playtwo conicting roles. On the one hand, the proteincontrols the accumulation of cargo by fusing severalRab5 vesicles. This requires a sufciently high Rab5

density on the vesicle membrane which is regulatedvia a positive feedback mechanism.

With the aid of simulations, we sought an an-swer to the question of which organisation principleenables Rab5 to best full its task in accumulatingmaterial before the protein is displaced from thesurface of the vesicles. The model analysis suppliedan astonishing answer. Rab5 does not defend itselfagainst its supposed opponent Rab7 but rather actu-ally activates the Rab7 protein. As a consequenceof vesicle fusion, the density of Rab5 increases withtime, which initially promotes the accumulation.

However, at the same time more Rab7 is also recruit-ed on the membrane until it displaces its predeces-sor Rab5 through a negative feed-back mechanism.

By a combination of modelling, model analysis,simulation, living cell microscopy and observationof individual endosomes in image sequences wesucceeded in unravelling the organisation principlethat enables the directed and effective transport ofmaterial via the endocytotic pathway. We term thisprinciple the cut-out switch, and it may also play a

part in other biological contexts.Our simulations and the detailed investigation

of the mechanisms and organisation principles ofendocytosis will make a contribution towards abetter understanding of this phenomenon. On thisbasis, it is possible to identify new targets for treat-ing such diseases as Alzheimers, asthma, bacterialor virus infections such as tuberculosis, HIV andu and even cancer, in which endocytosis plays adecisive part.

Simulation (mprDPD from BioMIP) of budding vesicles in liver cells.

The model shows the self-organisation of different proteins and mem-

brane lipids which induce cascades of protein recruitment processes.

The coat protein complexes (green) show the formation of distinct

domains and the budding of new vesicles.

Dr. Marino Zerial is a director at the

Max Planck Institute of Molecular Cell

Biology and Genetics in Dresden and is

coordinator of the Endocytosis network

of HepatoSys.

Phone: +49(0)351-210 2636

[email protected]

www.mpi-cbg.de

Prof. Dr. McCaskill is head of the Biomolec-

ular Information Processing research

group at the Ruhr University in Bochum.

His research concerns the interplay of

genetic information and self-organisation

in synthetic and biological systems.

Phone: +49 (0) 231-9742 6420

[email protected]

Dr. Andreas Deutsch is head of the depart-

ment of Innovative Methods for Comput-

ing at the Centre for Information Services

and High Performance Computing,

Dresden University of Technology. He is

interested in the organisation principles

of biological systems.Phone: + 49(0)351-463 31943

[email protected]

http://rcswww.zih.tu-dresden.de/~imc

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hePatoSyS20

Iron Network

Ssms ss f ir mbism i ir

Ionic iron is an essential trace element butalso a dangerous poison. Ionic iron mediates theelectron transfer during cellular respiration, asrequired for energy supply to the body. Moreover,it is indispensable for detoxication of foreignsubstances by the liver. Above all, however, ironis an important component of hemoglobin,

the red blood pigment, without which oxygencould not be supplied to the bodys organs.

Iron depletion - as a result of illness, of unbal-anced diet, or during growth phases and pregnancy,as well as after repeated blood donation - is there-fore a serious health problem which aficts about500 million people world-wide. However, excessof iron is also problematic, for example in patientswho due to certain other diseases depend on regu-lar blood transfusion therapy, or even in the case ofa certain genetic disease, an iron overload disorder,which causes excessive accumulation of iron in the

liver, and can lead to liver cirrhosis, liver cancer,and ultimately even to death. Nowadays, iron canbe ushed out by application of certain drugs butthen again an iron deciency must be avoided.

t ir s r r f ir mbism

Iron metabolism is therefore of central sig-nicance. It is controlled by a complex regulatorysystem that steers absorption, distribution andexcretion of the trace element. The intestinal tract,the liver, the spleen, several kinds of macrophagesand also the muscular system play a key role in this

system. A special coordination task is performedby the small intestine as the organ that absorbsiron, and the liver as the control centre. The liverhas sensors for the iron requirement of the entireorganism and sends an appropriate dose of thepeptide hormone hepcidin as a signal to the smallintestine and macrophages, which ne-tunesabsorption and distribution of iron in accordancewith the overall requirements of the organism.

The IronLiver collaborative project com-bines a theoretical working group (Max DelbrckCentre, Berlin) with two experimental workinggroups (EMBL and University Clinic Heidelberg).

Their objective is to study the regulatory processesof the iron metabolism in more detail. We aredeveloping a computer model of iron regulationwhich reects the interactions of the liver withother organs of the body in the form of a dynamicnetwork integrating absorption, transport, inter-conversion and excretion of iron-related proteins.

The iron storage metabolism is controlled by a complex system. The

gure shows the owchart of body iron. The thickness of the arrow

symbolises the conversion rate (small arrow: 12 mg iron per day; thick

arrow: 2030 mg iron per day; Hb: haemoglobin, red blood pigment;transferrin: iron-transport protein; ferritin: iron storage protein)

lr h, Prsi f Isi f

Ssms Bi i S

t w r f prii, pri prs-

is mii m pssib b ssms bi

rprss ri i mii wi

imp m sps f r is.


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hePatoSyS 21

t ms s m im

We use mice for the physiological studies - thebasis for developing the model. Using geneticmodications, we selectively inhibit or switch offcertain components in the iron metabolic system.We then analyse the iron content in the various

organs involved in the network in these animals,for example in the liver and the intestines, as wellas in the blood. In this way, we elucidate the regu-latory roles of the components of the iron system.This will allow us to modify, for example, the fer-roportin gene, which codes for the iron transportprotein at the entry port from the intestine intothe blood, such that it can no longer react to thehepcidin signals from the liver. This will result,of course, in iron excess of the body. It is of greatinterest to discover how the iron metabolismsystem and the complete organism of the mouse

react to this drastic perturbation of the system.The physiological data acquired from the ex-periments with genetically modied animals are in-corporated into a ux model. Basis of comparison isa model of iron content and distribution rate of thehealthy mouse which we have already developed.

In parallel to our work on genetically engi-neered animals, we also prepare an analogousmodel for humans. The basis here is the ux modelof the mouse into which we feed literature dataon human iron metabolism. The aim is to qualita-tively and quantitatively simulate the physiologicalhuman iron turnover, as well as its pathological

deviations - on the basis of interactions between thelevels of cellular and organismic system hierarchy.

This type of overall model serves as basis fora detailed study of iron-related human diseases.It is also hoped that it can be used for computer-controlled therapy planning in conditions of eitheriron deciency or iron overload. With the aid ofcomputer simulations, it could become possible towash out or replete iron as required thus avoid-ing excessive as well as insufcient dosage of iron.

Prof. Dr. Jens Georg Reich is head of the

working group for bioinformatics at

the Max Delbrck Centre for Molecular

Medicine in Berlin-Buch and is a member

of the German National Ethics Council.

His research interests are the molecular-

genetic and systems-biology principles of

the cholesterol and iron metabolism.

Phone: +49(0)30-940 628 33

[email protected]

Prof. Dr. Martina Muckenthaler is head of

the Department of Molecular Medicine at

University Clinic Heidelberg. She studies

the role of iron for health.

Phone: +49(0)6221-56 69 23

[email protected]

Prof. Dr. Matthias Hentze is head of

the working group Cytoplasmic gene

regulation and molecular medicine at

EMBL in Heidelberg. He focuses on the

molecular biology of the iron storagemetabolism.

Phone: +49(0)6221-38 75 01

[email protected]

Liver tissue of a healthy mouse (wild type, left) and of a knock-out

mouse (right), where theHfe-gene that is involved in the production of

the iron sensor Hepcidin is switched off. If the gene is absent or dam-

aged, an iron storage disease appears. The iron uptake of the duode-

num is out of control and a deposit is build in the liver (brown colour).

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hePatoSyS22

Central Data Management

The scientic communications platform of the HepatoSys consortium

The main aim of the HepatoSys network is touse the methods of systems biology to achieve ascomprehensive an understanding as possible of thecellular processes in hepatocytes. This requires closeinterdisciplinary collaborations between scientistsfrom widely differing disciplines. More than fortygroups from universities, clinics and other research

institutions and industry throughout Germanyconduct research within the framework of a largealliance. In order to allow the various teams tocollaborate efciently, a central infrastructure is re-quired to collect essential research data and allow itto be exchanged between the groups. This functionis fullled by the central data management system.

In the planning phase of HepatoSys, it hadalready become clear that the volumes of data gen-erated in the research network are considerable,particularly as a result of the application of high-throughput processes. The central data manage-ment system therefore had to be designed in such away that it could scale with large volumes of data.

Furthermore, the central storage of the datain a relational database was essential, as was thesystematic structuring and integration of differenttypes of data on the level of the gene, RNA andprotein, and also tools in order to biologically

interpret the various types of data in the contextof the liver cell. The concept of the central datamanagement system was developed by the mem-bers of the HepytoSys consortium and then put

into practice by Genedata, a company providingcomputational systems for life sciences research.The system is administered by the coordinators forcentral data management within the HepatoSysconsortium at the Max Planck Institute for Dynam-ics of Complex Technical Systems in Magdeburg.

tr mps fr mm

A signicant function of the central datamanagement system is to create a joint com-munications platform for the partners in theresearch network through which they canexchange data, ndings and information onmodels. To this end, the application was installedon a central server at the Max Planck Institutein Magdeburg. All HepatoSys groups havepassword-protected access via the Internet.The system is composed of three modules:

t xprim bk ws xprimndings obtained within the consortium to

b sr r.

t mp bk is s srifrmi s, mRnas,

pris sii pws r

isii.

I m bk, i sii ms f simi f mis f

mbi sii pws i

ir s r sr, ik wi

iii mps x

bw hpSs prrs.Wi xprim mpbks r bs sfwr r

ib frm g, m bk

b p frm sr.

d mm fr

ssms bi rsr prss

Systems biology research requires the deni-tion and application of new standards, such asstandard operating procedures (SOPs) forin vivo

experiments, the unied processing and nor-malisation of data, and the introduction of jointdata formats within the research consortium. Thecentral data management system is therefore

Design of the central data management system: the data within

(1) the experiment block, (2) the component and reaction block,

and (3) the model block are linked to each other and centrally

managed and retrieved.


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hePatoSyS 23

based on established IT Community Standards,such as Systems Biology Markup Language (SBML)for the exchange of mathematical models thatrepresent molecular biology cellular processes.

The central data management system also hasanalysis software for processing systems biologydata and for interpreting data. Of critical impor-tance here is the automated, computer-assisted dataquality and consistency control. Only after qualitycontrol and the subsequent steps for normalisingand standardising the data, can the different types

of biological data be compared to each other. Forinstance, this allows an informative comparison ofthe expression of the gene that codes for a particu-lar enzyme with the intrinsic enzyme activity.

The systems biology investigation into livercells also requires tools for the analysis and inter-pretation of data within a biological context. Thisdemands a technology-independent data analy-sis, which is also performed by the central datamanagement system. Specialised cross-omicsanalyses were developed for this purpose, which

help us to analyse and gain a better understand-ing of signal transduction pathways and theunderlying regulation processes in hepatocytes.

d mm i fr

The HepatoSys consortium has taken on apioneering role in developing and establishingdata management software for systems biologyresearch processes in cooperation with Genedata.The central data management system currentlysupports systems biology investigations into liver

cells. The functionalities of the system, however, willalso be used to address other challenges in systemsbiology in the future.

Database in use: a screenshot of the user interface shows the gene

expression of hepatocytes stimulated with rifampicin from three

patients (HH26, HH27, HH44). (1) The raw data were loaded, processed

and the quality of the data was evaluated. (2) The data were then

analysed further with the functions of the data management system.

The analyses show that the expression of the gene CYP3A4 is much

higher after treatment with rifampicin, while the other genes,

for example from PXR(NR1I2) and from HNF4a, remain largely

unaltered. (The diagrams were provided courtesy of Thomas Reichart,

ITB, University of Stuttgart).

Dr. Hans Peter Fischer has been head

of Genedata Phylosopher, biology data

management and data analysis,

since 1999.

[email protected]

Dr. Detlev Bannasch is responsible for

the central data management within the

HepatoSys network. He focuses on

designing and monitoring the develop-

ment and administration of the database,

user support and cooperations with exter-

nal data providers and database providers

for HepatoSys.

Phone: +49(0)391-6110 216

[email protected]

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hePatoSyS 25

The use of appropriate standards such asrecombinant proteins allows us to calibrate ourinvestigations. This does not just make it possibleto determine the proportion of phosphorylatedfactors but also the number of molecules.

Knowledge and control of interfering param-eters, which include cell density for example, arealso important for quantitative and reproduciblework with this type of system. We discovered thatconuent, i.e. densely growing, hepatocytes reactin a different manner to stimulation with cytokinesthan less densely growing subconuent cells. Forexample, the phosphorylation of the proteins ERK1

and ERK2 is weaker and briefer in conuent cellsafter the HGF growth factor has been added. This ef-fect can be attributed to the stronger expression ofphosphates in the conuent cultures. In order to beable to generate reproducible results, it is thereforeessential that we establish an exactly dened stan-dard protocol for experiments with our cell culturesystem, which would then hold for all of the work-ing groups involved in the network.

g rm wi i i sii

With the aid of suitable cytokines, such as HGFor the epidermal growth factor EGF, proliferationcan be induced in the cell cultures. The percentageof hepatocytes that proliferate in the culture is

comparable with the amount of proliferating cellsin a mouse liver regenerating after damage bycarbon tetrachloride (CCl

4). The processes of signal

transduction also progress in a similar manner inboth culture and living animals. We conrmed thisusing experiments, for example, those with isolatedperfused livers - in other words whole organs that

were kept in culture. If such an organ is stimulatedwith the HGF or EGF growth factors, the inducedphosphorylation kinetics of factors such as ERK1/2or the Akt kinase, another important nodal point inthe system, are similar to those in cultivated hepa-tocytes. This conrms that research with primarymouse hepatocytes leads to ndings that reectthe situation in living organisms well. The system istherefore suitable for use as an experimental basisfor the modelling of signal transduction processesin hepatocyte proliferation. It is applied by thescientists involved in the HepatoSys consortium

in accordance with mutually agreed standardisedworking conditions and culture protocols.We expect that these studies will lead to a basic

understanding of the regulation of signal transduc-tion networks as well as an understanding of thecomplex and dynamic processes that lead to the de-cision on whether hepatocytes proliferate or remaindormant. This knowledge provides the foundationfor therapies that will improve liver regeneration inthe future.

Proliferation of hepatocytesin vivo andin vitro. Cells with brown nuclei

(BrdU insertion) have gone through the S phase of the cell cycle and

are therefore about to divide. Left: Cultivated hepatocytes 48 h after

cytokine stimulation. Right: The culture cells exhibit a proliferation

that is comparable with the situation in a mouse liver 48 hours after

treatment with the liver toxin CCl4.

Prof. Jan G. Hengstler is head of the re-

search group Susceptibility to Toxicants

at the Leibniz Research Centre for Work-

ing Environment and Human Factors

at the University of Dortmund. He is the

coordinator of the cell biology platform

in the HepatoSys Network of Excellence.

Phone: +49(0)231-1084 348

[email protected]

www.ifado.de

ar:


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hePatoSyS26

Feedback for Liver Regeneration

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The liver boasts the astonishing property of be-ing able to regenerate itself after injury and dam-ages caused by toxic agents so that it is once morein full working order. This process is coordinated bythe complex interaction between different growthfactors. For the regeneration, differentiated but dor-mant hepatocytes gain the ability to multiply. Once

the liver mass has been restored, the proliferation isdisabled in a controlled manner.

A master regulator of this process is the neuro-transmitter TGF. It is responsible for stopping theproliferation of the liver cells and thus preventingexcessive growth. An excess of TGF can lead toinammatory processes and scarring in the liverright up to brosis, a pathological proliferation ofthe connective tissue, and can thus encourage theformation of hepatocellular tumours.

Interestingly, TGF is already secreted in an earlyphase of liver regeneration. This sounds paradoxicaland gives rise to the question of what mechanismsare responsible for the fact that hepatocytes canmultiply despite TGF when this neurotransmitterlater ensures that the proliferation ends - without

the danger of a brotic response. We investigatethis phenomenon on the basis of data generatedexperimentally and with the aid of a mathematicalmodelling of the TGF signalling cascade.

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For systems biology investigations, we requirequantitative time-resolved data of the highestquality. An important prerequisite for this is work-ing with a standard cell system. In cooperationwith other HepatoSys groups, we have developed

standard operating procedures (SOPs) with whichwe can obtain primary hepatocytes from male BL6mice aged between 6 and 12 weeks as a matter ofroutine in a two-stage collagenase perfusion. They

are subsequently cultivated on collagen-coatedplates and starved, in other words, we removegrowth factors so that all of the signalling pathwayscan adopt a ground state. On the rst or second dayafter preparation, the hepatocytes are stimulatedwith TGF. They are subsequently lysed at differentpoints in time in order to obtain protein extracts

or RNA. We use this standardised sample materialto investigate the control mechanisms of the TGFsignalling pathway.

In order to generate appropriate data for math-ematical modelling and to quantitatively inves-tigate the TGF-mediated signal transduction inhepatocytes, we also developed strategies to reducethe errors associated with identifying proteins. Oneexample is that we study the specimens of a time se-quence experiment in random order. This preventsdeviations, which can occur simply as a result ofdirect proximity in immunoblotting.

With the aid of GelInspector computer software,the data obtained can be automatically processedand combined. By using the software, we are in aposition to produce very extensive data sets for therst time with a high temporal resolution, whichrepresents signicant progress for dynamicmodelling.

The generation of quantitative,

time-resolved data with the

LumiImager. The chemilumines-

cence detected with the aid of

the LumiImagers CCD camera

is linear over a large area and

therefore particularly accurate.

Primary hepatocytes that were cultivated after isolation for 16 hours

in collagen-coated cell culture dishes. In order to make the cell nuclei

and plasma membrane visible, they were coloured with Hoechst 33342

(blue) and Dil (red) and studied with the aid of confocal microscopy.


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hePatoSyS 27

t sr fr fbk misms

The proteins Smad2 and Smad3 are important

information carriers in the TGF signalling cas-cade. If TGF binds to its receptor on the surfaceof the cell, these proteins are phosphorylated andthey undergo trimerisation with another proteincalled Smad4, and then migrate to the cell nucleus.A multitude of target genes are activated there,which in turn inuence the dynamic behaviourof the signalling cascade. A number of feedbackmechanisms for controlling this type of informa-tion transfer can be found in the literature. It cantherefore be assumed that this type of mechanismalso exists for ne-tuning the TGF cascade. Thisis the reason why we stimulate hepatocytes with

TGF and use microarray analyses to investigatethe transcription of the target genes. Genes frompotential control proteins, which are more stronglyexpressed after the TGF stimulation, are potentialcandidates for the ne-tuning of the signallingcascade. The rst results suggest that a proteintakes over control here, which is something thatwas not apparent in earlier investigations usingthe traditional methods of molecular biology.

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Our data-based mathematical model allows usto investigate processes of the TGF signal transduc-tion in silico and to plan promising experiments.

In this way, we have an opportunity of clarifyingfor the rst time the effect that reducing or increas-ing individual components has on the dynamicbehaviour of the entire system. In the long-term,the model will be used to identify approaches thatcan be used to selectively control the termination ofhepatocyte regeneration and to prevent damage.

Dynamic modelling of the TGF signal transduction. (A) Schematic

of the TGFbeta signal transduction. (B) Time-resolved analysis of the

Smad2 phosphorylation in primary hepatocytes using immunoblot-

ting. (C) Mathematical modelling of experimental data.

PD Dr. Ursula Klingmller is head of Systems

Biology of Cellular Signal Transduction at

the German Cancer Research Center in

Heidelberg. Her work centres on the

combination of quantitative data and

mathematical modelling for the analysis

of the dynamic behaviour of signalling

pathways.

Phone: +49(0)6221-42 4481

[email protected]

Prof. Dr. Steven Dooley, as head of the section

of Molecular Alcohol Research in Gastro-

enterology at the Medical Clinic of the Uni-

versity of Heidelberg, focuses on explaining

the molecular mechanisms of chronic liver

disease. Priority areas are the signal trans-

duction of the master brosis cytokine TGFb

and the development of treatments.

Phone: +49(0)621-383 3768

[email protected]

Prof. Dr. Jens Timmer is spokesman for

the HepatoSys project committee. As

head of Data Analysis and Modeling of

Dynamic Processes in the Life Sciences at

the Institute of Physics in the University

of Freiburg, he develops mathematical

methods for the analysis and modellingof biomedical data.

Phone: +49(0)761-203 5829

[email protected]

ars:


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hePatoSyS28

Liver Regeneration A Unique Phenomenon

t rri f mpx r rir i spi-mpr m

The liver is a very special organ. It is capable ofalmost complete regeneration after damage evenif more than 50 percent of its total mass is affected.This is of central signicance since the liver is avery important metabolic organ amongst otherfunctions, for the detoxication of the blood. Ithas a complex anatomy. The lobes of the liver are

composed of a large number of lobules with amaximum size of one to two millimetres, whichmainly consist of liver cells known as hepatocytes.The special structure of the lobule ensures thatduring its passage through the liver the bloodcomes into optimum contact with the liver cells.The portal vein collects the blood and transport itto the liver lobules where it ows through smallvessels called sinusoids along one to two cellsthick layers of hepatocytes to the central vein.

In liver cirrhosis, caused by drugs, alcohol orvirus infections, the complex architecture of the

liver lobules is destroyed and the liver can no longerfunction properly. This also affects the regenerativecapacity of this organ. In the case of advanced cir-rhosis the only treatment is liver transplantation.Since the transplantation of an organ can involve awide range of complications there is great interestin developing alternative, drug-based treatments.

For this reason, it is important to analyse moreprecisely the processes that take place during theregeneration of the liver on the level of the indi-vidual cells. The aim of our project is to reconstructthese processes on the basis of animal experimentsand then mathematically model them in order to

better understand the underlying mechanisms.

Frm iss si rri m

In our detailed investigation of the regenera-tion processes, we inject mice with the hepatotoxincarbon tetrachloride (CCl

4) by which necrosis

of the hepatocytes in the vicinity of the centralvein in induced. We then reconstruct the regen-eration process from tissue sections, which weevaluate statistically in several image processingsteps, and use the data to model the process.

For image processing, we rst perform uores-cence staining of the tissue samples according tocharacteristic cell properties in order to distinguishbetween hepatocytes and the endothelial cells

which line the sinusoids. Also those cells whichare proliferating can be detected with the aid ofsuitable staining techniques. In several imageprocessing steps, we then determine the positionand size of the cells and blood vessels. We meas-ure parameters such as the radius and density ofthe sinusoids, the size and direction of the hepa-

tocytes, and also the distance of the hepatocytesfrom each other and from the central vein.

From this information, we calculate a statisti-cal liver lobule for the computer simulation. Inthis simulation, each hepatocyte is represented asan isotropic, homogeneous, elastic object, whichinteracts with adjacent hepatocytes or sinusoids.This interaction is modelled by forces composed ofthe forces arising during the deformation, com-pression and adhesion of the cells. Mathematicalequations describe the movement of each cell.We apply the same concept to the modelling ofthe sinudoid deformations and displacements.

Cell division is simulated in two phases

within the model. The diameter of the cell rstincreases until its volume is doubled. At a constantvolume, the cell is then deformed until twoseparate cell bodies are formed from one cell.

From tissue section to computer simulation (A) Liver cells around a cen-

tral vein under a confocal microscope. (B) Visualisation of the sinusoids.

(C) Section of a liver lobule in the computer simulation with hepatocytes

(pink) and sinusoidal structures (red). (D) Sinusoidal network.


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In the model, we vary different additional

parameters in order to investigate their possibleinuence on the regeneration process. These pa-rameters include the spatio-temporal cell divisionpattern, the strength of the cell-cell and cell-sinu-soid adhesion, the rigidity of the sinusoids, as wellas mechanisms that control the orientation of thedaughter cells after cell division, for example, alongthe sinusoid structures, towards the destroyed areaof the liver lobule, towards the central vein, or intoa random direction. All the parameters are quanti-tatively estimated, either directly from the experi-ments or from the published literature.

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The calculated models of liver regeneration aresubsequently experimentally validated so that thecomputer model can be improved in the next step.In this way, we have already been able to rule out anorientation of cell division towards the central vein.

On the other hand, it was possible to experimen-tally observe another mechanism predicted bythe simulations. It is based on the assumptionthat the sinusoidal cells secrete cytokines, i.e.molecules, which can inuence the orientation

of the liver cells. In the simulation, the daughtercells re-arrange after cell division in such a waythat their connection line orients parallel tothe adjacent sinusoid. In this orientation, they

migrate towards the central vein until the de-stroyed region is completely regenerated.

We are currently working on integrating mo-lecular models of the metabolism into each modelcell. It thus becomes possible to directly simulatethe inuence of regeneration on the liver metabo-lism. In the next steps, we intend to investigate

the role of growth factors in regeneration and alsoto search for ways of improving the regenerativecapacity of the liver by means of molecular agents.It is furthermore planned to extend the simulationmodel to include the regeneration of the wholeliver after surgical removal of a part of it. In future,it will also be possible to use the model to investi-gate the development of liver tumours from thedegenerated cell up to the macroscopic tumour.

Various models of liver regeneration. Experimental results conrm

that complete regeneration requires a reorientation of the daughter

cells towards the sinusoids. (A) Complete regeneration scenario after

damage with CCl4 (day 0). If no reorientation takes place (B) or if cell

migration is too slow (C), the simulated regeneration is incomplete

after 10 days.

Dr. habil. Dirk Drasdo is head of the Multicel-

lular Systems Research Group at the IZBI in

Leipzig and research director at the French

National Institute for Research in Computer

Science and Control (INRIA) at Le Chesnay

near Paris. His main research interests are in

the mathematical modelling of processes of

tissue organisation.

http://ms.izbi.uni-leipzig.de/drasdo.html

Phone: +33(0)139-635 036

[email protected]

Stefan Hhme is working on his PhD at the

University of Leipzig in the Multicellular

Systems research group. His main focus

is on the visualisat

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