Download 1 Dissertation Project – 2nd Cycle BACKGROUND

DissertationProject–2ndCycle
Student´sName:
Studentemailaddress: No.
Supervisor(s):ClaudiaNunesdosSantosandReginaMenezes
Supervisor(s)emailaddress:[email protected]
Lab/Institution:MolecularNutrition&HealthLaboratory/iBET/ITQBNova
TITLE:Unravelingphenolicswithpotentialtherapeuticapplicationforneurodegeneration
BACKGROUND
Plantssynthesizeastaggeringvarietyofsecondarymetaboliteswhosebiodiversityprovidesapristine
poolofhigh-valuecompoundswithpotentialapplicationtohumanhealth.Oneofthelargestclassesof
suchphytochemicalsarethephenoliccompounds,whicharepresentinhighcontents,greatdiversity
and unique profiles in berry fruits. Indeed, the health benefits of these plant metabolites as well as
their functional properties towards the prevention of many disorders, including neurodegenerative
diseasesandtheassociatedchronicinflammatoryprocesses,havebeenextensivelydescribed.Thus,it
becomes imperative the identification of new bioactivities and the characterization of the molecular
mechanismsunderlyingcellularprotection.Afterthebioprospectionofaworldwideberrygermplasm
usingayeastSMARTplatform,theextractfromRubusgenevieriwasidentifiedasapotentprotectant
againstpathologicalprocessesassociatedtoAmyotrophiclateralsclerosis(ALS)whereasRubusidaeus
var Prestige displayed protective effects for Huntington’s disease (HD) and inflammation. Extract
fractionation and re-testing identified the fractions and potential compounds conferring protective
activities.Itremainstoelucidatethemolecularmechanismsbehindtheprotectivepotential ofthese
compounds.
OBJECTIVES
The main aim of this project is to investigate the molecular mechanisms underlying protection
mediatedbypolyphenolsusingyeastandmammaliancellularmodelsofthesediseases.
Thespecificobjectivesare:
- To identify the phenolics conferring protective activities in the yeast models of ALS, HD and
inflammation;
- Tocharacterizetheunderlinedmolecularmechanismsofprotection;
- Tovalidatetheresultsinmammaliancellularmodelsofneurodegenerationandinflammation.
PROJECTDESCRIPTION
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PROJECTDESCRIPTION
ThepresentprojectisalignedwiththeaimofEuropeanprojectFP7-KBBEBacHBerryandwillinvolve
collaborativeworkwiththeconsortiumpartners.
Task 1: Identification of protective phenolics from Rubus genevieri and Rubus idaeus var Prestige
extracts
Potential compounds relieving proteotoxicity of FUS (FUsed in Sarcoma), whose aggregation is a
pathological hallmark of ALS, will be identified using a yeast model of ALS encoding human FUS.
Protectiveactivitieswillbeinferredbygrowthcurveanalysisandextrapolationofparameterssuchas
lagphase,doublingtimeandfinalbiomassofthecultures.Asimilarprocedurewillbeusedtoidentify
compoundswithprotectiveactivitiestowardspathologicalprocessesofHD,whichincludeaggregation
andtoxicityofpoly-glutamin(polyQ)expandedhuntingtin.Theyeastmodelfortheseanalysisexpress
the polyQ103-HTT-GFP fusion. In both cases, the models recapitulate the molecular aspects of the
diseases.Identificationofphenolicspotentiallyattenuatinginflammatoryprocesseswillbeperformed
by the measurement of beta-galactosidase activity, using a reporter strain encoding the lacZ gene
underthecontrolofapromotercontainingCrz1recognitioncis-elements.Crz1istheyeastorthologue
of NFAT, which controls pro-inflammatory responses in mammals, and shares with this transcription
factorconservedmechanismsofactivation.
Task2:InvestigationofthemolecularmechanismsunderlyingprotectionbyRubusphenolics
Onceidentifiedthecompoundsdisplayingprotectiveactivities,thenextstepwillbetheinvestigation
ofthemolecularmechanismsassociatedtoeachspecificresponse.TheALSmodelwillbesubjectedto
adose-responseanalysistoestablishtheprotectiveconcentrationrangeofR.genevieriphenolics.An
additionalstrain,encodingaGFP-FUSchimera,willbeusedtoevaluatehowtheyaffectproteinlevels,
clearance, subcellular localization and aggregation by means of immunoblotting, fluorescent
microscopy and flow cytometry. Similar approaches will be performed to evaluate the activity of R.
idaeus var Prestige phenolics towards polyQ103-HTT pathological processes using the GFP fusion
describedinTask1.Also,adose-responseanalysiswillbedonetoestablishtheconcentrationrangein
whichphenolicsfromthesamematrixcontrolCrz1activationbyenzymaticactivityassays.Thesedata
willbestrengthenedbyqRT-PCRtomonitortranscriptionalactivationofendogenousCrz1targetgenes
such as PMR1and PMC1.Astrainencoding CRZ1-GFPdriven by thenativeCRZ1promoter will allow
followingCrz1nucleartranslocationuponstimulationandtheinfluenceofidentifiedRubusphenolics
inthisprocess.
Task3:Validationofkeyresultsinmammaliancellmodelsofneurodegenerationandinflammation
Final validation of previously identified compounds will be done in mammalian cell models. In
particular neuroprotection will be evaluated in a human neuroblastoma cell injured with an oxidant
agentbyflowcytometry.Moleculartargetsoftheidentifiedcompounds(Task2)willbeapproachedin
thehumancellmodeleitherbyimmunologicalorqRT-PCRtechniques.AN9murinemicrogliacellline
will beused asa neuroinflammation cellmodel.Thepotential of identified phenolicsfor attenuating
neuroinflammatory process in ATP-stimulated cells will be evaluated as regard calcineurin activity,
NFAT phosphorylation status and subcellular compartmentalization using enzymatic assays,
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immunoblottingandfluorescencemicroscopy,respectively.
TIMELINE(usefilltoolforthecells)
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Thesis
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