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[Music]

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[Applause]

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so Bree the speaker set up very nicely

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how the proton gradient is related to

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ATP synthesis so but I also have a

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little bit of an introduction on that

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which I'll go through faster now thanks

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to his his presentation but just to get

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us all on the same page I had these

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first two slides

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so basically metabolism consists of two

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parts the part where you build up

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complex organic molecules from simpler

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ones

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that's called anabolism and that

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requires energy which is obtained by

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converting ATP to ADP and oxidizing NADH

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to NAD+ so that's shown in this arm of

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the schematic diagram here and the other

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part of metabolism is the breakdown of

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the complex molecules which releases

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energy and that's called catabolism and

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that released energy is then stored in

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the formation of ATP and NADH and that's

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shown in this arm of the cycle on the

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schematic the abbreviations are shown

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over here so how is this ATP synthesize

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ATP then is a very key molecule for all

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metabolism and and so are these

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so-called cofactors such as nad so in

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there are various ways of synthesizing

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ATP but in most meta depending on the

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metabolism of the organism so in most

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organisms except for the fermenters ATP

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synthesis depends on the formation of a

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pH gradient that's called a chemiosmotic

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potential across the cell membrane and

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coupled to the generation of this pH

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gradient is the generation of NADH from

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nad and this this entire process is

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achieved by transferring electrons down

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an electron transport chain that

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involves enzymes I'll go through the

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schematic in a minute but let me just

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finish reading these bullet points the

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movement of protons down the

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chemiosmotic potential drives the

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synthesis of ATP by this ATP synthase

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molecule that was presented in the

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previous presentation as well the

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enzymes that are involved

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establishing the chemiosmotic potential

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contain these iron sulfur clusters in

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the subscript and simply means that this

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is the the stoichiometry of the iron and

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sulfur so they contain these iron sulfur

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clusters at their active sites and it's

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been therefore proposed for a long time

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in the literature that iron sulfur

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minerals might have played the roles of

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these enzymes and protocells so what are

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we talking about over here here's the

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membrane the phospholipid membrane shown

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here in the background this represents

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the interior of the bacterial cytoplasm

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and that's the outside or the the

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periplasm and in the case of this

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picture is taken for photosynthetic

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metabolism the electron transport chain

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so you have photosystem ii where the

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photolysis of water produces sorry not

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photolysis of the water but where water

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is broken down to form oxygen and two

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protons so this generates protons out

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here and the electrons in the process

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are picked up and transferred across to

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the cytochrome complex here by a quinone

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plastoquinone and the plastoquinone and

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the cytochrome complex spit out more

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protons out here for the transfer the

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electron using cluster sign on to first

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photosystem one for the system one

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transfers it using ferredoxin to nab 10

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ADP reductase and at NADP reductase a

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proton is combined with nad to convert

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it to NADH and so a proton is consumed

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over here so protons are produced on

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this side and consumed on this side and

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this generates a net enrichment of

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protons on this side of the membrane a

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net depletion of protons on this side of

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the membrane so this creates this kemiya

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osmotic potential and the fact that

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these electrons are being transferred

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from one enzyme to another this is the

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electron transport chain and here are

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the iron sulfide clusters that are in

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the active sites of all these different

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of all these different electron

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transport enzymes once it here's another

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picture now continuing on once you've

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generated the select this chemiosmotic

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potential of more protons on one side

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than on the other side if they flow back

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through the ATP synthase where ADP

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combines with phosphorus and is

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converted into ATP so the question was

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posed in the previous talk and we will

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talk about it here as well as to how did

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this chemiosmotic potential first arise

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in order for ATP synthesis to occur so

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the hypotheses that we have built in our

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in our model in our experiment here are

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based on a couple I mean the the

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experimental system we've built here are

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based on a couple of hypotheses the

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first hypothesis is the earliest

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protocell metabolism was fermentative

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this was proposed by Las Carnot and

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Miller in the 90s and the idea is that

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those organisms didn't have this proton

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gradient to drive ATP synthesis but at

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some point there was some evolutionary

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transition to metabolisms that did have

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the electron transport chain and

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chemiosmotic potential in order to drive

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ATP synthesis so the hypothesis we have

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in our work here is that photocatalytic

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minerals may have played the roles of

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enzymes in this evolutionary transition

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including iron sulfur minerals which are

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such as pyrite which are photocatalytic

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so we're going to build an experimental

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system to test this hypothesis whether

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photocatalytic minerals can actually

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drive this nad to NADH reduction

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reaction as well as create and

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transmembrane chemiosmotic proton

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gradient so we've devised a model

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protocell consisting of a lipid membrane

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the lipid vesicles bilayer we're going

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to react we're going to use a

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photochemical mineral for the catalytic

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mineral and shine UV light on it which

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generates a hole and an electron since

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it is a photocatalytic mineral on the

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outside of this membrane we have a

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reductant we are choosing any common

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reductant that might have been present

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in the environment such as an amino acid

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we just picked serine it can be any

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amino acid or any simpler organic

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compound the serine takes up the whole

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and combines with water to produce GOx

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like a sadhana and it also produces

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protons in the process meanwhile the

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electron is picked up by a molecule

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called a poly aromatic hydrocarbon which

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was included in the membrane during the

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synthesis of this protocell vesicle so

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the poly aromatic hydrocarbons have been

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shown previously to be electron shuttles

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and their structure is actually very

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similar to the Queen knowns that we saw

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that act as electron shuttles in the

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actual biological system so in the step

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one of this electron transport chain the

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electron is picked up from the

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photocatalytic mineral transferred to

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the poly aromatic hydrocarbon in step

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two the poly aromatic hydrocarbon picks

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up the electron passes it through the

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membrane to an electron mediator in our

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case this is a model compound called

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rhodium by pyridinium which the

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structure is shown over here now this is

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not very pre-buy otic rhodium is not

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very common in the environment but we

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use this because it was it's capable of

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a two electron transfer which is what

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you need for nad to NADH reduction we're

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currently in our lab trying to also work

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now with the iron analog of this which

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will be more probiotics applause about

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and finally in step three of this

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process so when this when this rhodium

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by pyridinium picks up an electron from

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from PAH it's reduced to the plus one

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form from a plus three starting out form

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and this reduced molecule then reduces

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any D the cofactor which is included in

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this vesicle and reduces the nad to NADH

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in the process it itself gets oxidized

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back to the 3 plus form any D is

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prebiotic it has been synthesized by

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Steve Boehner's group in a prebiotic

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chemistry recently last year and the

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structure of nad and NADH again are

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shown over here so this is where the H

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is added to make it an NADH compound so

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we have built this entire system in the

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lab and we followed the reaction whether

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it's actually happening by monitoring

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the production of NADH over time by

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using fluorescence spectroscopy how much

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time do I okay

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so what we're going to do now is going

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to look at a lipid vesicle with various

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different minerals we tested various

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photocatalytic minerals using a serene

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reductant and we are going to look at

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the production of NADH when UV light is

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shown on the system so here are the

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results here is the fluorescence as a as

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a function of at the scanning wavelength

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and you can see that at a certain

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wavelength there is a maximum in the

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peak which tells us that NADH is being

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being produced so after at various time

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points of one hour two hours and three

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hours of irradiation of UV light you can

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see that the peak is increasing that

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means NADH production is increasing in

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our protocell system so we achieve this

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using pyrite and green archite and we

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also use cadmium selenide again this is

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not prebiotic but this is just as a case

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study cadmium selenide or quantum dots

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and they're known to be half auto

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catalytic activity titania and is also

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photo catalytic and produces this NADH

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signal beautifully as well as zinc kite

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we also tried sphalerite and chalcocite

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but they didn't work and it might be

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that these were these were all synthetic

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minerals that we bought so their

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particle size is really small and it's

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in the right

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particle size to be catalytic these ones

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were natural minerals and we had to

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crush them up and we probably didn't get

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down to a you can't crush things down to

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the nanometer size you can only get like

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100 200 micron sized particles which may

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not be in the right size range to be

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photo catalytic so we didn't get the

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reaction with these sulfide minerals now

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so anyway there's a range of different

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minerals which are showing this property

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of NADH production the next thing we

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wanted to test is this is just a bar

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chart that quantifies the amount of NADH

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produced as a function of time and you

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can see that titanium is the most

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efficient and then followed by cadmium

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sulfide zinc oxide cadmium selenide and

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the least efficient actually is pyrite

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but that doesn't matter there was plenty

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of pyrite around on early Earth and by

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three hours

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it's doing a pretty decent job of

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producing the NADH

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now that was using a lipid membrane

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which was based on phospholipids and

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phospholipids have actually been

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synthesized in prebiotic synthesis so

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it's not as prebiotic ly implausible to

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use a phospholipid membrane as some

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might think but just for our

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satisfaction we also tested this with a

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fatty acid membrane so we used oleic

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acid which is a commonly used fatty acid

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and is considered to be more pre-battle

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plausible than a phospholipid and we are

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able to produce NADH in exactly the same

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way using the same system set up with

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serene as the extra vesicular or

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extracellular reductant terminal

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electron donor we also so this compares

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the oleic acid system to the

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phospholipid system that we just looked

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at in the previous slide using serene

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but we also decided to change up the

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extracellular reductant and used three

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different ones we've used serine we've

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used glycine and we've used isopropanol

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and as you can see pretty much any

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simple organic reductant can work in

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this reaction scheme and we get the

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production of nadh ignored these for the

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moment I won't get into the details of

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this if anybody has questions about

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these I can come back to them basically

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these graphs show that the nadh being

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produced is the biologically active

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isomer which is the one six form of NADH

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and not the inactive form which is the

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2/3 version of NADH so we're able to

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generate this protocell with the drive

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the reduction of nad to NADH using a

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photocatalytic mineral with different

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lipid membranes and with different

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reductants so the system is quite robust

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what about the generation of the extras

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the transmembrane pH gradient what we do

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is we enclose this fluorescent molecule

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inside the vesicle and this fluorescent

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molecule is sensitive to changes in pH

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over a period of three hours as we

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irradiate at our system and

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the NAD was being reduced to NADH we saw

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that the the fluorescence intensity

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inside these vesicles was changing and

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by calibrating it to a pH curve we were

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able to determine what was the pH inside

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the vessel we were able to determine

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what was the the pH inside the vesicle

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so at the starting point at time zero

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hours the pH is the same as eight point

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six which is the pH at which the

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vesicles were synthesized so the outside

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and the inside are both at pH eight

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point six when we use the phospholipid

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which is a very imperiled membrane the

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pH doesn't really change much over time

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thank you

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when we use the oleic acid phospholipid

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mixture in a ratio of five is to one the

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pH drops over a period of time and this

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is not surprising because oleic acid

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vesicles fatty acid vesicles are not as

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impermeable so what's happening is that

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the protons are getting carried in from

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the outside where the protons were

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generated by remember that serine when

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it is oxidized it produces the protons

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so those the outside of the vesicle is

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getting acidified and that pH gradient

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is being maintained in the case of the

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popc vesicles but it dissipates over a

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period of three hours because the fatty

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acids shown by these orange molecules

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here do this lipid flip-flop so they're

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initially deprotonated as the exterior

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becomes more and more acidic they pick

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up a proton and they do the lipid

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flip-flop and carry the the the proton

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from the outer leaflet into the inner

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leaflet and release it into the inside

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of the vesicle and so that's why the

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interior starts getting acidifying but

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basically when by the time the evolution

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of phospholipid membranes had come about

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in these protocells

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you would have had a pretty tight

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membrane which was capable of

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maintaining this chemiosmotic potential

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so in conclusion various photocatalytic

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minerals are capable of promoting a

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transmembrane electron transfer chain

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with reduction of NAD+ to NADH and the

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generation of the transmembrane pH

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gradient these reactions utilize an

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extra

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organic compound as the terminal

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electron donor such as serine and

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glycine thus the model represents a

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proto photo heterotrophic metabolism

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because it's utilizing this reduced

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organic compound for the metabolism the

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minerals we are showing actually for the

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you know there there are lots of

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hypotheses that minerals may have played

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the roles of enzymes and proteomic tabal

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ISM but this is one of the first cases

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where there's a demonstration that

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that's actually working and it's

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happening in a photo heterotrophic

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metabolism as membranes evolve towards

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more phospholipid rich compositions this

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new this developed chemiosmotic

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potential would have occurred and as

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evolution proceeded this might have

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eventually been used for the synthesis

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of ATP as the evolution eventually

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produced an ATP synthase so I'd like to

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conclude with thanking my postdoc Poonam

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the ly who did all of this work and was

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assisted with a really amazing graduate

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00:16:44,540 --> 00:16:42,149
student putos Triana and funding from

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the Simons Foundation the National

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Science Foundation grant University of

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Akron start-up funds and very generous

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gift funds from a a person who's just

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interested in origins of life research

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and thank you for your attention

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thank you very much

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[Applause]

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unfortunately we don't have time for

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questions as the session has concluded

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I'd like you to let let's all join