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hi my name is Brandi Jones I'm a

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postdoctoral fellow with the NAI Center

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for a ribosomal of origins and evolution

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at Georgia Tech and today I want to talk

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to you about a special group of

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extremists Al's they were kind of

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skipped during the morning session but

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they're rotifers they are eukaryotes

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they're microscopic animals and they

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live in very diverse habitats in my work

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focuses on the molecular biology of how

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they're able to survive in those

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habitats and we know a lot is known

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about prokaryotes the very little is

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known about eukaryotes extreme of house

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so that's what my work practices are

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just some examples of where you can find

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rotifers and they live in alkaline lakes

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acidic blakes you can find them in hyper

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saline habitats as well as i know we've

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heard a lot about dr ali and target hell

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you can find them there as well they

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also are able to adapt to their habit

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the habitats in which they live where if

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you get extreme desiccation they can go

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from a very active living form to a

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desiccated dormant form that upon

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rehydration years later will become

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active adult and able to let live

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metabolize and just to give you an idea

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the adult can go from active swimming to

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that desiccated form within 30 minutes

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and upon rehydration you get the active

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swimming adult form within the same time

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period and so my question is how do the

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ribosomes are they able to survive and

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become active when that within that

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short of time period the model organism

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for my study is bright be honest Maha

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vacas is a rotor for a microscopic

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aquatic organism is able to withstand

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extreme heat desiccation UV irradiation

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and this here is a female carrying a

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sexual eggs they undergo both asexual

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and sexual life cycles usually it's a

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sexual where they develop the asexual

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eggs but if you get some type of

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environmental key or signal usually it's

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an environmental stress they it

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tourism to go into a sexual life cycle

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where they produce these resting eggs

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that can remain dormant for hundreds of

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years they can become completely

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desiccated you can expose them to

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extreme heat UV radiation but upon

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rehydration they're able to continue

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developing until normal adults and this

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is just an experiment I did with the

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active rotifers adult rotifers from

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three different species and then those

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desiccated forms that was just telling

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you about if you incubate and this is a

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survival ship curve and it's on the

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y-axis is the proportion surviving on

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the exes the temperature exposed to so

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the adults when exposed to temperatures

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higher than 40 degrees they die but

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those are dormant cysts you can expose

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them to temperature is greater than 100

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degrees and then upon rehydration they

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go on metabolizing so they're very um

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they can withstand extreme temperatures

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so my goal is to gather insight on how

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the ribosomes are able to withstand

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those environmental stresses how once

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you remove those organisms from the

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environmental stress the ribosomes are

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able to continue translating and form

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proteins so what I know from other

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eukaryotes is that usually when you have

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some type of environmental stress

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translation of housekeeping genes in

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normal genes they it stops the ribosome

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will fall apart you'll get the small

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subunit falls apart from the large

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subunit and the small subunit associates

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with proteins known to initiate

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translation as well as mrnas that are

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not even translated and they move into

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these stress granules and here's just an

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example from a human cell line here

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we're looking at and we're just we type

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of proteins fluoresce with fluorescent

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markers this is from a paper from 2009

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you have the large subunit and green the

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small subunit in red and then you have a

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initiation factor shown here in blue

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apply

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of stress with arsenite you get

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co-localization of the large the small

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subunit as well as the initiation factor

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but the large subunit doesn't colocalize

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with them and this right here is what we

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call a stress granule you see it here

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and shown over here is the same thing

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these three proteins or three initiation

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factors upon I'm stress you get

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co-localization of all three proteins

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and stress granule production so my

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hypothesis is that these stress granules

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form and the rotor first or in periods

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of environmental stress and they protect

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the ribosome during those periods and

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then upon removal of the environmental

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stress you get reassociation of the

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ribosome and the organism are able to go

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on metabolizing so what I know from

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previous studies done with both use the

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mammalian cells is that unstressed cells

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do not have stress granules they're not

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present he upon heat stress you get

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stress granule production as well as

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starvation hyperosmolarity and then if

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you incubate them in the presence of an

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antibiotic called cycloheximide it

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inhibits stress final formation but the

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antibiotic promote stress for Niall

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formation so I decided to test all these

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conditions with my rotifers and the

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different environmental stresses i use

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whereas modoch stress you normally grow

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the Rutherfords in fifth artificial

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seawater containing 15 ppt salt I buried

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it up to 45 p bt i deprived them of

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nutrients for several days and then I

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heat shock them and I did all of those

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things and then I followed the movement

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of three proteins known to be associated

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with stress granules the three proteins

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are TI a one is that an rna-binding

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protein is involved in mrna splicing and

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then to eukaryotic initiation factors

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eif4e and ya s-3b all three of these

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proteins are known to co-localize the

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stress granules upon stress

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environmental

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stress in yeast and among yourselves so

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just so you can follow the rest of my

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experiment to follow the proteins i use

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anna antibodies and they were

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fluorescently tagged so here TI a one

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will always appear red in my pictures

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eif 3b will appear magenta eif4e will be

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green and then the DNA and some pictures

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I sing with that be in an appears blue

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and this is just an example of the

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microscopy pictures you'll see here's a

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bright field image showing just the

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rotifer here's a confocal image you see

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the nuclei and some of the red and

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magenta and some of the green and those

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are the three proteins and wherever

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those proteins co-localize you'll see a

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yellow or orange color shown here you're

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here and then I have them labeled so you

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can see the organs and which they

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colocalize so in my first experiment I

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deprived them of nutrients for 48 hours

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and just so you can see each column here

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is the individual protein that I

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followed so y is 3 be as you continue

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with starvation you see aggregation of

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eif 3b as well as eif4e and CIA one they

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each begin to aggregate amongst

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themselves but then i'm showing you a

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merged image and you can see that

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they're colocalize into the same

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location and just in case you can't see

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what i see i use a software that labels

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white and where those three proteins

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co-localize so you see as you increase

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the time period of starvation you're

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going to increase of colocalization of

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the three proteins within the major

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organ systems which is indicative of

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stress granule formation and here I've

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just zoomed in and you see the yellow

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and orange here I've also done some

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statistical analysis using a matters

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the coefficient when you get a zero it

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means there's no colocalization of the

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proteins and a one means perfect

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colocalization and you can see it goes

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from point 344 at 24 hours and it peaks

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at point seven on at 48 hours does that

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mean five left or left okay and then my

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next experiment is osmotic shop where I

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buried it from the 15 ppt asw to the 35

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on ppt so the 15 ppt is the control and

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as you see as we increase the osmolarity

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you get an increase in aggregation of

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each of the individual proteins it's not

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as dramatic as we saw before in the

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nutrient deprivation but you do see

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co-localization and it goes from point

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three seven in the control 2.79 so

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that's indicative of colocalization is

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stressful any old formation I under heat

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stress I buried the time that we heat

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shock them from zero to 20 minutes and

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you see the same pattern you see an

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increase in aggregation of each of the

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individual proteins and an increase in

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colocalization and the major organ organ

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systems which is like the other is

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indicative of coagulation and stress

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granule formation so here I wanted to

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show you that the stress final formation

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is dynamic you get stress granules form

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but when you remove the stress those

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stressful angles will go away so here at

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unstressed rotifers you don't see any of

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those allegations and co-localization

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you see it with 30 minutes of heat

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stress and then I let them recover for

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three hours so I put them back at their

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normal temperature to grow and that was

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room temperature and you see a decrease

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in the amount of stress my angles formed

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so these stress granules are dynamic

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they form and then they dissipate and

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here

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antibody puromycin that's known to

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promote stress for information and other

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organisms we see that when you incubate

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them with the pur- inand the heat stress

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you get on the dramatic amount of stress

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granules forming and then the other

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antibiotic cyclohexyl might that's known

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to prevent stress triangle formation I'm

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just the same here you don't see any

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colocalization of those proteins and

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here it's a deviation from my other

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pictures the green here is actually the

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large ribosomal subunit I labeled it I

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labeled puromycin with the green

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fluorescent well here is 50 which is

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green and then I incubated it with the

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rotor first after they have been heat

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stress though it's going to bind to the

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large ribosomal subunit which is known

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in other organisms not to move into

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stress granules and usually on the

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periphery and you see aggregation with

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heat stress so these are the other two

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proteins that you saw before you see

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that there are co-localized like they

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did before the red and magenta but the

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green which is the large ribosomal

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subunit is not co-localize arrest

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renewals so just in conclusion what I've

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seen in rotor first is consistent with

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what we see in both yeast in mammalian

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cells that an unstressed cells you do

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not get stressful annual formation but

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in other environmental stresses that we

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test it we got stress granule formation

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just as you see in yeast and mammalian

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cells the antibiotic purim I

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cycloheximide prevented stress mariel

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formation and pura myosin promoted it

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and the components of the stress

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granules and rotor first were consistent

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with what we see in other organisms so

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just what I can conclude from this study

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is that the stress granules are most

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probably of an ancient origin because

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you see them early in the evolution of

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animals rotifers as well as in mammals

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and I want to thank on the labs I work

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with the snow lab

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at the school of biology at Georgia Tech

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the Williams lab in school chemistry at

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Georgia Tech and the Dunham lab at Emory

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as well as NASA and my Center on the

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Center for ribosomal origins and

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evolution thank you do we have any

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questions for brandy hi sorry if I miss

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this but is there anything special about

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the stress granules of the rotifers that

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makes them better able to withstand

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straw so that would be what I want to

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determine later on we don't know now is

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the first time they've ever been seen in

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rotifers but I would like to see if the

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stress granules swarm quicker and

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rotifers if you if they're more dynamic

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i'm not sure but that's something I

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definitely want to look into I could you

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go back to your slide with the the heat

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stress over time early earlier yeah that

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one the very bottom with the T 1 a 1

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where it's not showing any sort of and

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any red dump yes what's going on there I

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mean why is it just disappeared do you

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think it has a lot of it I don't know if

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you can see it yeah it has a lot of it

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okay because all right maybe I've just

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read blind for the bottom there but I

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doesn't see anything yeah it's there I

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do think I agree that there is more here

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than there but it's still a lot I'm

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present but I think there's a limit to

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the stress granules form they're

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supposed to be dynamic and I really

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didn't give the animals and opportunity

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to it just like they would normally do I

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just take them and move them from their

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normal growth temperature to this heat

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shock and normally they would have time

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to acclimate to it so I think probably

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20 minutes was pushing the limits for

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them and so they start to kind of guy

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let's do you use pure mind sending what

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was the other cycloheximide do you know

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how that works how that antibiotic and

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so cyclohexyl might prevent

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elongation and Pyramus and prevent

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initiation of I'm translation and the

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prevent initiation it promotes a signal

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a signaling cascade that promotes these

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stress modules formation and the cycle

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hexa might because you're stopping it at

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a different time point it doesn't start

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that cascade by segment with cascading

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the same one would it be worth looking

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at other antibiotics to give you clues

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about Eric um they have been like with

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the east of mammalian cells there are

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there a number of different antibiotics

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that they have looked at and that just

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grabbed two from the list but if you are

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the antibiotics that prevent an

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initiation of translation all promote

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stress granule formation because you end

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up getting on phosphorylation of one of