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(

Growth(medium( Biological(agent( Reac>on(stopping( reagent(

Clamp(A( Clamp(B(

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(