1. “Studyingthe dynamicsof nucleotide excisionDNA repairusingsingle molecule approaches”
The seminarwasheldon the 6th
of May 2016 andwas hostedbyProfessorLouise Serpell andthe
talkwas givenbyDr Neil Kadfromthe School of Biosciencesatthe Universityof Kent.Duringthe talk
Dr Kad summarisedthe workhislabwasperformingandtheirmajorgoal that theyhope to achieve.
Dr Kad’s labstudiesnucleotideexcisionrepairfromaprokaryoticstandpoint,andtodo thisthey
utilise single molecule approachestotryand rebuildthe systeminsequence totryand understand
the complexityof nucleotide excisionrepairin prokaryoticorganisms,asDr Kadstatesthere are only
6 proteins involvedinthe prokaryoticsystemcomparedto30+ proteinsinthe eukaryoticnucleotide
excisionrepairsystem.HoweverDrKadstressesthatthisshouldn’tbe takentomeanthat the
prokaryoticsystemisa simple,linearsystemratherthatthe interactionsbetweenthe 6proteinsare
inrealityverycomplex andproduce acomplicatedsystemwhenstudiedinsolution.
Dr Kad explainsthathisprimaryfocusisto identifyhow DNA damage repair proteinsfindthe sitesof
DNA damage.His worklooksat DNA insolutionanditis explainedthatDNA molecules,whenin
solution,donotformlongstrandsinsteadtheytendtoform small bundleswhichprovideapoor
substrate whenlookingathowproteins interactwithDNA,particularlywhenlookingatindividual
proteins.Toovercome thisissue DrKad’steamuseda methodtocreate DNA “tightropes”thatwere
comprisedof single DNA molecules,thiswasdone usingglassbeadscoatedinpoly-glycineanda
DNA containingpolyethyleneglycol solution.The solutionwaspassedoverthe beadsand
occasionallyone endof aDNA molecule wouldbindtoone beadwhile the otherbindstoan
adjacentbead,thusprovidinga“tightrope”formedof asingle DNA moleculestretchedacrossthe
gap betweenthe beads.Oblique angle illuminationwasthenusedtofocuson individual molecules
and reduce the levelsof backgroundsignals. ThroughthisapproachDr Kadexplainedthathe was
able to identifythatthe UvrA proteininvolvedinnucleotide excisionrepairdoesn’tslide alongthe
DNA as expectedratheritbindstothe DNA,dissociatesandthenbindselsewhere inthe strand,
howeverwhenUvrBwasaddedto the solutionitwasfoundthatthe UvrAB complex wouldbindthe
DNA strand and thenslide alongittoscan for damage to the DNA.

2. "Dissectinghematopoieticstem- andprogenitorcell populationsusingmolecularandfunctionsingle
cell approaches”
Hematopoieticstemcellscanmaintainahighlineage rate forthe entire lifespanof the organism.
Thisis made possible bythe presenceof structuresdedicatedtolong-termmaintenance of the
multi-potentstemcell state,aswell asmechanismsforthe generationandsubsequentlineage
specificationof stemcell progenylackinglong-termself-renewal capacity.
Dr Nerlov’slab usesgeneticmethods,combinedwithgenome-wide geneexpression,to study the
complex nature of the hematopoieticstemcell population,the structures thatmaintainthem, and
the changeshematopoieticstemcells andstructures undergoduringaging. Theyfocusona range of
factors suchas transcriptionfactorsand signalingmolecules thatcontrol the commitmentof multi-
potenthematopoieticprogenitors,aswell asnormal cellularpathwaysthattheyspecify.DrNerlov’s
labhas produceda model suggestinghow mutationsaffectingthe normal transcriptionalcontrol of
myelopoiesisresultinacute myeloidleukemia,andaddresshow the stemcellsthatcause thisare
maintainedthroughouttheirlifecycle. Theydothiswiththe ultimategoal of understandingthe
molecularbasisbehindthe spatialorganisationof varioustypesof hematpoisesandtouse thisto
possiblydevise therapiesthatcanbe usedtotreat the effectsof these hematpoises.
A recentpaperpublishedby Dr Nerlov’slabidentifiedinterleukin-1asa keydriverinaccelerating
cell divisionandmyeloiddifferentiationthroughactivationof aPU.1-dependentgeneprogram.This
effectisessential forrapidmyeloidrecoveryfollowingbone marrow injury,though chronic
interleukin-1expressionrestrictsthe outputof hematopoieticstemcellsthatare crucial for blood
homeostasis.

3. “Cryo superresolutionbothways:ElectronTomographyandCorrelative Microscopy”
Cryo-electronmicroscopyisanimagingtechniquewhichproducedthree-dimensional modelsof
biological samples,these samplesare usuallylive cells,butthe technique isnotlimitedtojustlive
cells.The cells are treatedwithacryogenwhich rapidly coolsthemsothat the waternaturally
presentin the cells cannotforma crystal that wouldpuncture the cell membrane,thisisoftenan
issue whenfreezingcellsasthe crystalline ice structuresare able topuncture cell membranescan
cause cell death.Thisrapidcoolingof the cellsmeansthatthey are preservedinanear-livestate.
The sample isthenrotatedrelative tothe electronbeam, usuallybysingle degree increments,and
the imagescan be consolidatesintoa3D model. Dr Schellenberger,whowasgivingthe presentation
at the time, presentedaprotocol forimagingcellsinsuperresolution,usinglight,fluorescence,
electronandcryo microscopy.
Dr Schellenbergerandherlab basedtheirtechniqueonCorrelativeLightandElectronMicroscopyto
attaina betterrange of precision,improvethe workflow andhandlingof samplesduringthe imaging
process.Herlab managedthisthroughthe additionof fluorescentmicrospherestocell samples
before theywere treatedwiththe cryogenicliquid.Thisfacilitatedcorrectionof the fluorescent
channelsthatbleedintoeachotherata laterstage afterimaging.The labtestedthisimproved
methodbyimagingadenovirusparticlesininfectedcells,theywere thenable tolocalisedviral
capsidswithan approximate precisionrange of 60nmwhichisa considerable improvementon
previousattemptswhichhave oftenstruggledtogetbelow 100nm.

4. “GliomaStemCells:takingaimonthe real target”
Dr Riva firstoutlinedthatglioblastomaisthe mostcommonmalignantbraintumourwitha
prognosisof only14 monthsin 50% of patientsandthat currenttherapiesare insufficientfor
extendingthis.DrRivastatesthat surgical therapiescanbe challengingespeciallyif the tumouris
locatedinan area of the brainthat isdifficulttooperate on,especiallysince the operationof the
brainstill remainsalarge mysteryandthe effectsof invasive operationsoncertainareasof the brain
can resultinsignificantside effectsof surgery.The tumourcellsare oftenresistanttoarange of
chemotherapiesavailable,thoughthe oral drugTemozolomide hasprovedeffectivewhenusedin
conjunctionwithradiotherapywhenadministeredinthe earlystagesof tumourgrowth,however,
laterstage cancer tumoursare generallyresistanttothistreatment.
Dr Riva statesthat the reasonwhyglioblastomaisbelievedtobe resistanttotherapyandfrequently
reoccursis because the tumourcontainscellsthatexhibitstem-celllikepropertieswhichhave tobe
totallydestroyedforthe cancerto be eradicated.Unfortunatelythisisnotoftenthe case when
treatingglioblastomasthroughsurgeryasitisimpossibletoremove everycellinatumourwith
currentsurgical techniquesandanumberof these stem-celllikecancercellsremainandare able to
replicate tore-generatethe original tumour.
Dr Riva and hislabhave studiedValporicacid – a drugnormallyusedinthe treatmentof epilepsy
and bipolardisorder–and have identifiedthatishaspotential asa therapeuticdrugtotreat
glioblastomas.Theyfoundthatvalporicacidinhibitshistone deacetylaseswhichremove the acetyl
groupsfrom histonestoincrease the DNA bindingaffinityof these histones. Inthe majorityof
glioblastomalinesandpatientsstudiedbyDrRiva andhis labthe applicationof Valporicacid
resultedinimprovedpatientsurvival aswell asasignificantreductioninmitoticdivisionsina
majorityof the glioblastomalinestreatedwithvalporicacid.