Ryerson(SSEP( Microgravity(has(lots(of(effects(on(biological( - PDF document

Ryerson(SSEP( • Microgravity(has(lots(of(effects(on(biological( systems( • Vertebrates(lose(bone(density,(muscle(mass(( • Immune(func>on(is(compromised( • Bacteria(have(altered(virulence( • What(else(is(happening?( Gene(changes(in( Salmonella ( associated(with(space(flight( • ~167(genes(are(altered(in(expression( • Increased(biofilm(forma>on(genes( • Changes(in(iron(u>liza>on(genes( • Changes(in(stress(response(genes( • All(of(these(genes(are(known(to(affect(ability( to(induce(disease( Wilson(et(al,(PNAS,(2007(

Salmonella( Typhimurium ( produces( polysaccharide(in(space( Wilson(et(al,(PNAS,(2007( Salmonella( Typhimurium ( virulence( increased(aTer(in(space(flight( • Major(cause(of(foodVpoisoning( • Useful(model(of(bacterial(pathogenesis( • Causes(lethal(typhoid(fever(in(mice( Wilson(et(al,(PNAS,(2007(

Other(bacteria(studied(in(space( • Pseudomonas(aeruginosa (–(increased(biofilm( forma>on( • Three(other(pathogens(( Listeria ,(MRSA,( Enterococcus )(were(studied(and(reduced( virulence(observed(in(space( • Studies(are(few(and(far(between( – Lots(of(opportuni>es(to(learn(new(science!( Reac>on(vessel( !

Reac>on(vessel( Clamp(A( Clamp(B( ! Biological(agent( Reac>on(stopping( Growth(medium( reagent( Types(of(experiments( • Effect(of(microgravity(on(some(metabolite( – Degrada>on(of(toxin( – Rate(of(a(reac>on( • Change(in(a(gene/protein(level((one,(many(or( all?)( • Growth(rate?( • Cellular(composi>on?( • etc(

Biological(degrada>on( • During(growth(–(biomolecules((DNA,(RNA,( protein,(lipid,(carbohydrate,(etc)(are(rou>nely( synthesized(and(degraded( • In(order(to(measure(the(level(of(a(par>cular( biomolecule(at(a(specific(>me(–(it(is(important( to(prevent(degrada>on(during(storage/ processing( Experimental(design( • Wait(period((from(assembly(to(start)( • Start(reac>on(by(mixing(biological(agent(with( medium((remove(clamp(A)( • Incuba>on(period((experiment(running)( • Stop(reac>on(by(mixing(organisms(with( reac>on(stop(reagent((remove(clamp(B)( • Wait(period((experiment(stop(to(delivery)(

Experimental(design( • Wait(period((from(assembly(to(start)( • Start(reac>on(by(mixing(biological(agent(with( medium((remove(clamp(A)( • Incuba>on(period((experiment(running)( • Stop(reac>on(by(mixing(organisms(with( reac>on(stop(reagent((remove(clamp(B)( • Wait(period((experiment(stop(to(delivery)( Wait(period( • Biologicals(have(to(be(stabilized(in(some(way( – Growing(cells(will(die(during(shipment,(wai>ng( period( • Spore(forming(microbes( • FreezeVdried(microbes(

Experimental(design( • Wait(period((from(assembly(to(start)( • Start(reac>on(by(mixing(biological(agent(with( medium((remove(clamp(A)( • Incuba>on(period((experiment(running)( • Stop(reac>on(by(mixing(organisms(with( reac>on(stop(reagent((remove(clamp(B)( • Wait(period((experiment(stop(to(delivery)( Growth(experiment(

Time(course(experiment( Stopping(reac>ons( • Flash(freezing( – Fast,(cheap,(reac>ons(generally(stopped(immediately( • Solvents/detergents( – Dissolve(membranes/proteins,(stabilize(small( molecules( • Using(crossVlinking(fixa>ves( – Formaldehyde( – Paraformaldehyde( – Glutaraldehyde( • These(preserve(structures(for(microscopy;(don’t(necessarily( preserve(other(biological(molecules(

Considera>ons( • What(is(the(analyte(being(considered?( • What(type(of(analysis(will(be(done(back(on( earth?( • What(are(the(constraints(of(the(experimental( system?( Choice(of(stopping(reagent( • Different(molecules(can(be(stabilized(by( different(reagents( • Proteins(–(protease(inhibitors( • RNA(–(aqueous(RNA(stabiliza>on(reagents( • DNA(–(aqueous(DNA(stabiliza>on(reagents( – Trizol(–(allows(isola>on(of(all(three(major( molecules( • Other(reagents(possible(depending(on(Qs( asked(

Ques>ons?( • Dr.(Joe(McPhee( • jbmcphee@ryerson.ca( ( (( ( (@JoeBMcPhee(