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hello everyone i'm paula and i'm a

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second year phd student at the earth

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life science institute at tokyo tech i

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work in the group of sean mcglenn at lc

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and my research interests are in the

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fields of extremified microbiology and

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computational biology

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the title of my talk today is single

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cell and genomically resolved analytical

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techniques elucidate metabolic

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adaptations of methanogenic ikea to

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varying temperatures i will walk you

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through this step by step

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looking at my title the first thing you

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might be wondering is why should we even

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be interested in methanogens well

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methanogens may have been some of the

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earliest life forms on earth

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looking at some of the major events that

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led to life as we know it today starting

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with the moon-forming impact 4.5 billion

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years ago to ocean formation and the

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first isotopic signatures of life 4.1

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billion years ago methanogenesis and

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sulphate reduction are the earliest

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metabolism for which we have direct

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isotopic evidence at 3.5 billion years

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ago before we can find signs of

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photosynthesis and later on the great

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oxidation event

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looking at the evolution of biological

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processes and atmospheric oxygen and

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methane on an early earth without oxygen

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and an environment that is hostile to

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most modern life forms methanogenesis

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enabled other non-photosynthetic

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prokaryotic metabolisms such as

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methanotrophy to thrive which then in

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turn pave the way for photosynthetic

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primary producers and later on

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but what exactly is methanogenesis in

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simple terms it is the biological

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production of methane through one of

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three major pathways

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first we have hydrogenotropic

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methanogenesis which uses carbon dioxide

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as substrate and hydrogenous electron

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donor resulting in methane and water

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formation

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second acetyl-clastic methanogenesis

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where acetate is split by oxidizing the

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carboxyl group to carbon dioxide and

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reducing the methyl group to methane and

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third methylotrophic methanogenesis with

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methanol regular means as substrates

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where electrons are gained through the

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oxidation of an additional methyl group

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to carbon dioxide so with four methyl

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groups as input you will end up with

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three molecules of methane

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today methanogens are found in various

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environments such as animal rumens

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c-flow sediments subglacial lakes or

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industrial plants and in recent years in

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addition to the substrates mentioned

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here more and more additional substrates

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such as items have been identified for

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use by methanogens but that is a

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different story

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in addition to the white substrate

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utilization shown in part on the

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previous slide methanogens also have

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wide growth temperature ranges

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for my project i have classified them

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into three temperature groups

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we have psycho-tolerant organisms with a

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minimum growth temperature of 15 degrees

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celsius or less which are shown to the

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left of or touching the blue line in

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this plot thermo-tolerant organisms with

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a maximum growth temperature equal to

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above 45 degrees celsius which are

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either to the right of the red line or

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touching the red line and then finally

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mesospheric organisms which are all

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organisms in between the two lines and

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everything touching both lines

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the total temperature range is from

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minus 2.5 degrees celsius observed in an

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antarctic lake organism to 120 degrees

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celsius observed in an

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organism isolated from a black smoker

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wall

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it is worth mentioning here that there

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are no universal definitions for

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like tolerant thermotolerant or

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thermophilic temperature cutoffs and the

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thresholds here were set based on the

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overall distributions within this group

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of organisms

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now let's have a look at the phylogeny

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of the organisms i define methanogens as

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every archaem containing methyl coenzyme

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and reductase short mcr alpha beta and

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gamma subunits which are a total of 295

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organisms this here is a subset of those

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that have temperature data available

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which is a set i'm showing in the

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following analyses as well

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there are a total of 86 organisms the

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shapes in the tree indicate the

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different substrates where the collard

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boxes represent the temperature classes

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we can see that temperatures and

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substrates are distributed throughout

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the phylogenetic tree and that the only

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real trend that we can observe is that

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there are no acidic clastic

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psycho-tolerant organisms

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when seeing these wide temperature

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ranges you might be wondering what

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mechanisms allow methanogens to inhabit

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such a wide range of environments and

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how can we find those mechanisms in my

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thesis work i'm looking at methanogens

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genomes with respect to their

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composition function and structure

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this is what i'm going to talk about for

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the rest of this presentation

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i'm also interested in the physiological

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adaptations and i'm doing single cell

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analysis of organisms grown at different

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temperatures to investigate to what

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extent individual cells in the

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population differ from one another

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so let's have a look at the genome data

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and if you're interested in the

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physiological physiology part please

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contact me later

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i started by splitting the genome of

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each of the 86 species into their core

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and pan genomes

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the core is shared by all organisms in

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this analysis and appears in blue in the

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third while the pen genome consists of

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genes that are not present in every

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single organism but in some of them

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the pendulum can further be divided into

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unique genes that only appear in exactly

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one species and are in green here in

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this plot and shared genes which appear

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in at least two but not all of the

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species and which are the orange

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fraction

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in this plot each bar represents one

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organism and the core of all of the

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organisms consists of 225 genes

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which in size of the whole genome is

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between 5

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and 15

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that is actually relatively small

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so where are the adaptive mechanisms

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that we are looking for are they in the

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core and the pen genome

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we next have a look at the composition

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and structure of proteins of each core

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shared and unique gene groups to find

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out more since we cannot see a clear

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trend in the phylogeny from the previous

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slide or the genome composition on this

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slide

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i will first show you amino acid

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compositions of each of the fractions we

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are looking at amino acid substitutions

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because they cause structural changes

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the proteins that can cause an increase

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of decrease in flexibility and stability

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of the protein which is important in

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different temperature environments

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so for example cyclophilic proteins have

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a higher flexibility and lower thermal

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stability compared to their mesophilic

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counterparts

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we found that the observed substitutions

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follow the expectations that we get from

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the literature with the conserved core

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and variable unique proteins

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so let me walk you through each of the

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factions one by one starting with the

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overall genome composition

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this plot shows the differences in

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thermo-tolerant and psycho-tolerant

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species with respect to individual amino

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acids calculated from the overall mean

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appearance of an amino acid through all

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thermo-tolerant cyclotron species

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respectively

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a positive value means that the amino

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acid is more than cycle tolerant forms

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while a negative value means that the

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amino acid is more abundant in

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thermo-tolerant organisms

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looking at all genes together the core

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plus pen genomes so this is the black

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bar here

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we see some differences which are not

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too pronounced the main differences are

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higher glutamic acid and lysine and

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thermals and higher serine and uranium

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and psychotron species

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but those differences are mostly within

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the one percent

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range next let's look at the individual

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genome components starting with the core

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the differences are similar to that of

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the overall

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genome even a bit less pronounced than

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in the whole genome

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the trend for the shared fraction of the

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genome is very similar as well

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so finally let's look at the uniques and

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here we can see that the differences are

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more pronounced than the other fractions

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that we looked at before

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when we compare all of this data side by

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side we can see that indeed the unique

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fraction has the most pronounced

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differences in amino acid composition

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sometimes even a completely opposite

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trend as the other genome fractions

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let's see if we can observe a similar

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trend in protein domain architectures

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the architectures will tell us about the

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orientation of secondary structures of

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protein faults the core here is very

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very conserved which you can see in the

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absence of the blue bars

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there are some differences in the shared

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fraction but the most pronounced

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differences again and the unique

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fraction

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and in addition to this plot this is not

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displayed here but

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one important thing to note is that when

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it comes to the mapping coverage of the

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genes

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mapped only a small fraction of them had

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an architecture mapping onto them which

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means that they need more attention if

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we want to find out more about their

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structure and function

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so finally what does this data tell us

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basically it tells us that the core is

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very conserved when it comes to amino

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acid composition meaning flexibility and

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rigidity and protein domain

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architectures meaning secondary

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structures

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we know very little about the unique

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genes even though some of them might

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hold keys to the thermal adaptation

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mechanisms of methanogens

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so if you want to find out more about

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those we will need molecular biology

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studies

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and then finally what is next for me

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i have a bit more data to go through in

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this project such as gene phylogenies

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for the core genes from which we can

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relate the connection between

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temperatures and evolutionary rates and

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i'm also interested in the hypothesis of

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thermal reduction which was first

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introduced by forte more than 20 years

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ago

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as i mentioned in the beginning i'm also

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looking at the physiology of individual

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cells through laboratory experiments and

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ultimately my plan is to link the genome

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potential with the observed phenotypic

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variations

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if you're interested in any of this i'd

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be happy to meet up virtually during or

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after the conference as well

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on this slide i've just listed some of

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the references i used for my figures if

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you're interested you can read further

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here

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thank you for watching my presentation

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until the end you can find me during the

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conference either on gather town during

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the japanese afternoon session or

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message me through discord at any time