Transcriptional regulation in the early phase of liver regeneration ; Transkripcinis ankstyvojo kepenų regeneracijos tarpsnio reguliavimas

Exj128

Sujets

• Biochemistry
• Gene expression
• Liver
• Transcription factor

Informations

• Publié par: vytautas_magnus_university
• Publié le: 01 janvier 2009
• Nombre de lectures: 22
• Langue: English
• Poids de l'ouvrage: 1 Mo

Extrait

VYTAUTASMAGNUSUNIVERSITY








Egl÷Juškevičiūt÷



TRANSCRIPTIONALREGULATIONINTHEEARLYPHASEOFLIVER
REGENERATION



Doctoraldissertation
Physicalsciences,biochemistry(04P)









Kaunas,2009VYTAUTODIDŽIOJOUNIVERSITETAS








Egl÷Juškevičiūt÷



TRANSKRIPCINISANKSTYVOJOKEPENŲREGENERACIJOS
TARPSNIOREGULIAVIMAS


Daktarodisertacija
Fiziniaimokslai,biochemija(04P)









Kaunas,2009

2The work described was carried out at Vytautas Magnus University and Thomas Jefferson
Universityin20022009.

Researchsupervisor:
Prof.habil.dr.VidaMildažien÷(VytautasMagnusUniversity)
Consultant:
Prof.dr. JanB.Hoek(ThomasJeffersonUniversity,Philadelphia)


3TABLEOFCONTENTS

ABBREVIATIONS ......................................................................................................................7
INTRODUCTION ........................................................................................................................9
1 LITERATUREREVIEW ...................................................................................................12
1.1 Generalfeaturesofliverregeneration.........................................................................12
1.2 Cellularmechanismsofliverregeneration .................................................................14
1.3 Thehepatocytecellcycle............................................................................................16
1.4 Hemodynamicchangesafterpartialhepatectomy ......................................................17
1.5 Earlyphaseofliverregeneration ................................................................................19
1.5.1 Matrixremodelingafterpartialhepatectomy .....................................................21
1.5.2 NotchandJaggedprotein....................................................................................21
1.5.3 ActivationofconstitutivelyexpressedtranscriptionfactorsafterPHx ..............22
1.5.4 ExpressionofimmediateearlygrowthresponsegenesafterPHx......................23
1.6 Signalingmechanismsinliverregeneration...............................................................24
1.6.1 Cytokinesignaling ..............................................................................................25
1.6.2 Theroleofgrowthfactorsinliverregeneration .................................................31
1.6.3 Growthfactorswithparacrineeffects.................................................................33
1.6.4 Thegrowthfactor–cytokineinteraction............................................................34
1.6.5 Signalingthroughadeninenucleotides ...............................................................36
1.6.6 Metabolicpathwaysandliverregeneration ........................................................37
1.7 Livermassandregenerationcapacity.........................................................................37
1.8 Maintainingliverfunctionsduringregeneration ........................................................39
1.9 Proliferationandapoptosisinhepatocytes:reactiveoxygenspecies .........................40
1.9.1 Concludingremarks............................................................................................43
2 MATERIALSANDMETHODS........................................................................................44
2.1 Materials .....................................................................................................................44
2.2 Animalprotocols.........................................................................................................44
2.2.1 Animalsanddiet .................................................................................................44
2.2.2 Surgicalprocedure ..............................................................................................44
2.3 RNAisolation .............................................................................................................45
42.4 Microarrayfabrication ................................................................................................45
2.5 Probepreparation........................................................................................................46
2.5.1 VectorRNAprobe ..............................................................................................46
2.5.2 RatlivertotalRNAsamples ...............................................................................49
2.6 Slidepreparationandscanning ...................................................................................50
2.7 Datanormalization......................................................................................................50
2.8 ANOVAresponsemodel ............................................................................................51
2.9 Clusteranalysis ...........................................................................................................51
2.10 Quantitativereversetranscriptionpolymerasechainreaction....................................52
2.11 Functionalannotation..................................................................................................54
2.12 Transcriptionalregulatorynetworkanalysis...............................................................54
2.13 Nuclearextractpreparation.........................................................................................54
2.14 Proteinquantification..................................................................................................55
2.15 Transcriptionfactoractivationassays.........................................................................56
2.15.1 TransAMassay ...................................................................................................56
2.15.2 TransFactoruniversalcolorimetricassay ...........................................................57
2.16 Chromatinimmunoprecipitationassay .......................................................................58
2.16.1 Chromatincrosslinking.......................................................................................58
2.16.2 Chromatinshearing.............................................................................................58
2.16.3 Immunoprecipitationreaction.............................................................................59
2.16.4 DNApurification ................................................................................................59
2.16.5 AnalysisofDNAfragments................................................................................60
3 RESULTS ...........................................................................................................................61
3.1 Identificationoftemporallyregulatedgenesduringliverregeneration......................61
3.2 Identificationofcoregulatedclustersofdifferentiallyexpressedgenes ...................71
3.3 ValidationofmicroarraydatawithqRTPCR............................................................72
3.4 Interferencewithpurinenucleotidesignalingaffectsc-fosexpression......................73
3.5 Liverregenerationfunctionrelevantgeneexpression................................................74
3.5.1 Transcriptionrelatedgenes.................................................................................75
3.5.2 Signaltransductionrelatedgenes........................................................................76
3.5.3 Cellproliferationandcellcyclerelatedgenes....................................................78
53.5.4 Stressandinflammatoryresponserelatedgenes ................................................79
3.6 TranscriptionalRegulatoryNetworkAnalysis ...........................................................80
3.6.1 CandidateTFsintheonsetofliverregeneration................................................80
3.6.2 Activationoftranscriptionfactors ......................................................................82
3.6.3 BindingofNFκBtoSod2,Mt1aandCebpb promoters....................................85
3.6.4 Functionalgenecategoriesregulatedbytranscriptionfactors............................86
4 DISCUSSION.....................................................................................................................90
CONCLUSIONS.........................................................................................................................93
SANTRAUKA............................................................................................................................94
ACKNOWLEDGEMENT ..........................................................................................................96
REFERENCES ...........................................................................................................................97
6ABBREVIATIONS
ANOVA analysisofvariance
AP activatorprotein
ARL augmentorofliverregeneration
aadUTP 5(3aminoallyl)2’deoxyuridine5’triphosphate
ATP adenosinetriphosphate
ATF activatingtranscriptionfactor
BSA bovineserumalbumine
BRN brain2classIIIPOUdomainprotein
BTG Bcelltranslocationgene
C/EBP CCAATenhancerbindingprotein
CRE cAMPresponseelement
CREB cAMPresponseelementbinding
CT cardiotrophin
Ct cycleatthreshold
DMSO dimethylsul