It's always a pleasure.

The only

work

I

am well

OK

So

join

the faculty at a wonderful career so

but also it's all yours.

Thank you Bob.

So to introductory things first of

all about torch tech and
its influence on my career.

Not only did I have a fantastic experience
here is a student that prepared me for

a lot of things in life it's been
a huge influence on me through my

career because of what Bob and
his colleagues have created here at

Georgia Tech has been an incredible
inspiration to all of us in the field.

I came up through the ranks as a junior
faculty member had some hard times and

Bob was always there to talk to fact
we stimulate the field pick you up

when you're on your way and I think all
of you here are very blessed to be here.

It's also been fantastic to see the
incredible growth bioengineering here at

Georgia Tech who was very little research
here I think it was odd at one other

person really had research
labs when I was a student.

Here and it was a chemical engineering and
it was no bioengineering so

you should feel extremely proud
of what you have here now and

also mention I've been very fortunate to
have some Georgia Tech students work in my

lab at MIT so most recently who's here in
the audience material science students but

in are you some work at a fantastic job
less recently but very prominently on

the Platte really came in and shook things
up an arm's length will stem cell laughter

and it's terrific to see him doing so
well here I can finally get it right.

He's doing great.

It's just really great to be back with
the CAN'T of the mind is not on the.

How about that you want to live.

Where.

And the second thing is about the talk
I'm going to get today and the work.

The scope of work that's
going to be presented.

I'm presenting some things I've
never talked about in a seminar and

I in fact got data emailed to me last
night from one of the post docs and

with a lot of goings on this week
the site visit some of you know about for

the S.T.C. I've been spending
a lot of time with Bob in various

locations around the country over
the past week repairing first site

visit I had to channel one of my
colleagues who couldn't be there.

So the talk is not quite as polished
as I normally like to have it but

I really want to talk about this work
because it brings out some issues of

signalling and how we control
receptors and tissue engineering and

I know there are some people in
the audience who are brilliant.

Tristen some of these
may be a lot of facts.

So even though it's not completely
polished I felt it would be really fun

to talk about this.

So please don't hesitate to wave if
there's something that's not really clear.

And so my job.

We had to do a lot of basic by material
science protein engineering as you'll

see today with an eye on some
clinical applications and

three of them are listed here.

Thank you to the folks who came yesterday
to talk about the last topic which

is a very new topic for our lab and the.

Title of my talks a little esoteric and
another way to think about it is far

very much a mass six in the sense that
we want to tame signaling by this

incredibly important family of quote fact
a receptor is the E.G.F. our family and

I'll introduce them to you are going
to get used to one of Motier So

there's clear members of this family will
go through their properties a little later

and for tissue engineering purposes and
little recently for us.

Possibly for cancer therapeutics
we have a tremendous centrists and

controlling spatial localization of
the receptor activation and so all you

have that is the theme throughout the talk
a primary motivation when we started going

on and this is some going to cover some
things that Mani worked on in our lab is

a collaboration with an orthopedic surgeon
George Marshall at the Cleveland Clinic.

This is one of his patients the patient
had a huge tumor taken out and typically

Now there's a replacement process
where you put a cadaver bone in and so

you can see that fixed here graves
over the past approximately

eight to ten years of our cooperation and
then to come up with alternatives to these

kind of cadaver bone class by integrating
both macroscopic scaffold approaches

with microscopic approaches to control
the local microenvironment of the cells so

that you could combine some
source of ostrich an Excel.

With the graft and put it in and
have it heal and the Osteogenesis so.

That most of interest.

If you were just fresh purified Asper it's
going to give a little background on this

just to set the stage of why we
got interested in these problems.

Perceptor dimer sation So one approached.

To healing by one's bone healing wounds
is to take instead you know right

now if any of you has anybody ever had an
autograph of bone like from the hip hip.

So now they typically will take
autographed the bone from the hip which

has it's your harvesting that bone
actually has it's own huge morbidity

issues.

So there's a cost to a way for
now with physicians

who are trying to use marijuana
that can be aspirated interrupted

police instead of actually taking
the bone out they just harvest Mayo and

George has optimized methods to do
this and to operate a pulley so

the gist of it is that when you harvest
marrow about one in thirty thousand

of the nucleated cells have properties
a very only Aust your partners and

maybe true stem cells so there's a mix
of cells in the aspirants that can go

on to form that cartilage and bone and
for today we're interested in this.

OK And I'm being very careful to
call these projects under cells and

not strictly stem cells but you always get
a mix of the assets we do do not guarantee

stem this despite the popular use in the
literature and Conall stem cell as a term.

It's been shown that if you take
that marrow and pass it over a graft

bone etc shown very quickly here there's
actually machine now that does this.

So you pass it over the crap
the stem cells can be selected and

this can be enhanced by Moon healing and
so

has been big interested in this idea that
passing the scaffolding a couple of them.

He's use are shown here by mechanisms
not well understood you can enhance

the pretender cell concentration and
prevalence in these graphs and

this is work that's been published.

So we've been interested in
figuring out how to do this and

how to actually use it in the clinic.

You don't always have to understand
everything before it's useful in

the clinic right.

And it's OK and so together we've worked
on building scaffolds to can entrap these

cells working together with their company
that has commercialize this three D.

printing process to make scaffold.

So we designed a scaffold set
would we hope trap cells and

in fact showed that the scaffold that we
designed will track trap these connective

tissue pretenders as well as demon
analyzed bone matrix and as we think about

translating these things into a clinic so
there's an issue of this microenvironment

if we're going into a really large defect
where there aren't that many stem cells.

We may need every stem
cell to do a huge job.

So the problem we face is that different
patients have different problems to

stem cells so these are the same exact
number of nucleated cells from five

different patients that are plated
in what's called a colony Asser So

you plate the marrow half a million
nucleated cells the cells that attach and

form colonies are the ones that
are believed to be the projector so

you can see the incredible
diversity in problem and so

this this patient has very few and
this patient has a conciliate of them.

OK And again you can see they had
originated in colonies another area of

interest in the lab not want to talk about
today is building a better colony assayers

OK So the challenge is now these cells
are going to go into this really hostile

microenvironment right there's all kind of
pock circuits filled with cytokines and

so my slot is cut off but
with that it's filled with cytokines

if you have access to stem cells like
if you're doing a transplant in a mass.

You might put in one hundred
times more cells than you need.

So you see up down rows.

OK even though maybe a lot of the cells
are killed but in a human defect that's

why you may only have close to
the number you actually minimally need.

So we have to worry about whether some
of these cells are going to be killed.

So we started thinking about ways that we
could influence the different steps in

this process and in particular if we look
at the stem cell opportunity are going

to come from Excel and not known
about different factors that act.

Different stages of development.

OK And in particular the morphogenetic
proteins have a widely used now in

the clinic to facilitate bone healing and
spinal fusion in factual repair and

a few other applications tired
hormones used also to stimulate.

Process.

But these really drive it to
point a committee progenitors and

they don't necessarily cause
political ation of those pretenders.

And so we draw up a hypothesis is that
maybe approaches in these large defects

have not been successful because we drive
premature differentiation What if we

go further back and try to drive political
ation of this compartment and yet

also influence downstream processes.

So there's a number of
factors known to act here.

One of them.

Interestingly it's happened on grass
factor it's signalling by E.T.F. receptor

is involved in many stages of bone
development and homeostasis in healing.

So as boring as many people
think you have perceptive is

first growth factor discovered there we
have it as an interesting molecule So

our question is can we
stimulate the survival.

And growth of.

These cells that we transplant using each.

It's attractive.

It's been around a long time and
we know a lot about it but

it's never really been developed into
a successful clinical product and

part of that you know is we started
thinking about this many years ago so

I think you know rather didn't Johnson and
Johnson or

anybody actually ever have a clinical
product and come to that and then it.

It turns out there is actually
a commercial product.

If you inject soluble E.T.F.
into sheep it circulates with a half life

of a couple of days and
the sheep when the hymn grows out.

It's very thin and you can actually pull
the wool off the sheep as you see here and

in Australia kids on the farm kids want
to stay on the sheep shearing is very

skilled So

having this is actually a now commercially
viable operation in Australia.

There's also if you go on e-bay.

Google E.T.F. you can find these
facial anti-aging products they

cost like two hundred dollars for a little
while you can barely see and says F.D.A.

doesn't prove that these right but
I'm eighty five and I use these products.

So you can decide for yourself.

OK So hypothesis so
we went back and we looked at so

let's look at physiology
of each after supper.

There was a law against it for
this particular super scepter it's for

those of you not familiar with it.

It's got an extra cellular domain
the pines one of several a trans membrane

domain and then enter Sigler domain the
transmit signals will come into this part.

More later.

And so it activates it's a receptor
Tyrus in kinase that initiates several

cascades of signaling through
its Tyrus in kind a stem main So

we asked the question you
know most of the audience

actually are somehow restrained
in the extracellular matrix and

may actually signal in a more tethered
fashion than in a cycle fashion so

we started thinking about this concept
of delivering from the Matrix and

this brought in some to design issues
because the E.G.F. Perceptor have to

die more eyes in order to signal and there
is some controversy still remaining but

the evidence is overwhelmingly in
favor of the receptor kind of system

Mainz needing to come together to foster
to initiate hospitalization Cascades.

So there's been crystal structures
published of the extracellular domain in

complex with the like and this happens
to be teaching at Alpha and so one

could imagine tethering this molecule via
its terminus there's only one primary me.

To a substrate and so we initially
decided to do this using polyethylene

oxide very simple approach and
space the so that we could get

clustering dimer sation of the receptor so
and work that actually did over.

Contributed to during his stay since we
designed a polymer system that would let

us present little costers E.G.F. receptor
on the surface or other like hands

shown schematically here hydrophobic
backbone polyethylene oxide side chains.

When you cast these on a surface they
make the same my interpenetrating discs.

So you can make little clusters of Y.

Ganz by mixing modified polymer with
an unmodified polymer and couple in here.

So you can get a structure like that.

And so then if we want to examine
how this affects M.S. C.S.

We could speculate that it will initiate
signaling cascades because he finds

that the surface dimer station occurs and
then typically the receptor Mike and

I are in the side toast and traffic
through intracellular compartments.

But signalling is initiated at a service
of the cell so you have E.T.F.

Perceptor Don lies here and

there she ating several different
signalling Cascades P I three kinase.

And we'll see gamma which
involved a motel ot So

one question we speculate is maybe
tethered yet would actually act better

than soluble in protecting
cells against proud signals so

tethered E.T.F. would stimulate the
receptor protect against protest signals

because the signals tend to be
preferentially on the membrane.

And so it worked.

That didn't fan did in her thesis.

We looked at whether to tell
that he had had any effect on

protecting cells in the kind of
environment they would experience.

So these are primarily human mesenchymal
stem cells we get ourselves from

facility he's now in Texas.

These are very low passage passage
through your passage for cells but

there was a stem cell maelstrom
of cells will call them.

So no treatment if we add fast like
you have we kill the cells but

if the cells are cultured in the present.

That E.G.F.
in fact they look protected and

quantifying this if we look at
the percent of cells that are dead.

This is the control so
very little cell death.

OK add this Gand pro-inflammatory
a protest cytokine and

you see a lot of cells die.

Sorry but you have actually potentiates
this and this we think may be due to

interruption of adequate bloops we're
investigating that now but tethered.

Protects against that cell death so that
it's interesting there's an important way

we might influence the survival of
these cells and so we went on and

asked well doesn't enhance Komi
formation and here this is

again from giving us work to showing
plaiting cells early on soluble E.T.F.

compared to tether tethered
actually enhances adhesion

probably through a localized actin
Plimer station going through and

last coherent pathway for those of
you who read the signalling stuff but

you can see dramatically in advance of
this and it's actually dependent on

activation of the erk signaling cascade
we showed so the knickknack can Tonio

in his thesis work investigated whether
primary cells colony formation for

Melo plated would be enhanced by
the smaller kill and in fact what we found

looking at different substrates an
environment where there is only absorbed.

Serum proteins one where
we use a synthetic R G D

that has the synergy site from five or
not.

Ten were fiber nets in itself.

So those were three it he said backgrounds
and now we add tethered E.G.F. and

in each case we see color information is
enhanced so with out with have an E.T.F..

With with tethered or
fiber Nekton interestingly

soluble E.T.F.
inhibited compared to the control room or

for fiber necked and so
there are all effects on these cells and

another suit in the lab rather well as he
just a friend of his thesis went on to say

let's see if we can develop ways to put
these on scaffolds that are clinically.

Used.

So the company that's commercial is three
dimensional printing three the other

has a line of products based on Tri
calcium phosphate and this is just showing

pictures of it so they do really well in
the clinic and this is one substrate we

have vision using either alone or
in combination with a polymer so little

working with some undergraduates in the
lab developed an approach to tether each

to the substrate will talk more about the
protein engineering later in the talk but

the con conceptual part here we
use Project display to identify

a peptide sequence that binds with very
high finity to try calcium phosphate and

this is just some data showing that
there's a specific binding and then E.T.F.

is here and you can just view this for now
as a spacer OK So he's got this he puts

the scaffold in a solution of this
molecule it absorbs on the surface and

now we've got tethered OK so if there's no

binding the main you don't get binding so
here there are specific binding here.

There's none so it's pretty tightly
immobilized on the surface and

this is just showing the construct
there's you do Fosse display.

We've got a consensus sequence
shown here using several different

kinds of substrates for screening so this
is a manuscript that's in preparation from

Lou Alvarez's they says OK so now we
take scaffolds treated with tethered.

And look at the effects of
this on primary stem cell.

Plus relation and so what we see is that
in this just looking at a number of cells.

What we see is by day seven.

This isn't serum containing media so

you've got all kind of growth factors
there and even in serum containing medium.

You can see there's a tremendous increase
in cell number at this early time

point and even at fourteen days they
are statistically significant increase and

also in serum for medium we can see that
these enhanced survival of the cells and

Manu has a couple of papers that came
out that also investigate further.

Signalling properties of tethered E.G.F.
in our original format.

And I'm sure he's talked
about those here but

the basic question comes up putting up
on the surface in this way we wouldn't

expect it necessarily to be close enough
to time arrives very efficiently.

So there's a stochastic presentation
on this or near stochastic so

we can ask the question is this really
acting through an E.T.F. receptor we can

inhibit the Perceptor combination in
aggregate effects I just showed you.

So it will convince just acting through
E.T.F. receptor but is it acting through

homo dimer sation is really the question
because we would predict that you know

that wouldn't really be that much
completely correctly positioned to hold

on to my so now we're getting in to this
question why would we get the signal so

let's go back and look more carefully at
the E.G.F. receptor family I have a couple

of slides in sequence to take you through
because it's a little bit complex and

I took a picture out of the literature and
let me take you through it.

So there are four family members
the studies showing raggy ends for

the family members.

It's showing the receptors and the
billions kinds of dimer sation they can

ensue downstream signaling pathways and
transcription control and influence

these things so it's a comprehensive
one thing it's a comprehensive picture.

OK so what you see here if
we plow it up is that E.G.F.

Perceptor can hetero timelines it
can have a time arise shown here but

it can also had a hard time
arising notably put it right.

OK.

Are they actually.

Famous.

That you talked to was going on and

for so if anybody has heard
of Herceptin the entire

breast cancer drug it targets her to go to
is overexpressed a memory tumor cells and

so it's a very potent signaling and it and

her two does not have a lot of cancer
her two has to partner with someone or

two has to join up with each
Yeah Perceptor one of the other ones and

it maps to join up with
the Perceptor can also join the sky.

So it has to partner someone to signal
an E.T.F. receptor can heterodyne arise.

So miserable stem cells have zero two and

they have her three
because they don't have.

There's another guy or
they don't have her for her.

So they have all of the siblings or

potential partners that you could
imagine are present in a messy.

So it's entirely conceivable
that R M S C S When

they bind one of America's E.T.F. groups.

Ron Americans Yep they're making
these kind of header timers and

these are very potent signalling for
survival.

So that could be what's happening on
the scaffold so I just showed you.

OK so we could then say that more
American each office acting through these

heterodyne hours and it raises a question
what if we don't want those head or

dollars to form actually what if we would
imagine in cancer cells keeping her or

two from joining up with her
one if we could put her two out

of the party by forcing these
two receptors to homo Dom arise.

OK So that's one interesting thing so

no other words here again just
showing a little cleaner.

We've got.

E.G.F. receptor her to head a time or
each after sector home.

Three had a timer and
showing her three so this is in a C.S.

and then they cross talk with immigrants
we bind E.G.F. each He has very

high affinity for each you have
Perceptor not so much for her three.

And if we want to completely
manipulate this entire family

we can look at my dance for her of three.

OK Her three can also
heterodyne more eyes with

her to go to doesn't have a walk
in that supposed to be a blind.

So the regular end is
a molecule that binds here.

OK so if we really think about
manipulating this system we might also

be able to keep her own three hundred two
head of a dime or some form with basically

agree with the scheme is exclude her to
keep her two from coming to the party.

So this is schematic.

So if we come back.

Now we can say we want to control this
time or station and how could we do that.

So the idea is could we take a cell
that expresses multiple receptors.

If we could it not America we have no
control over the diners nation partners

but now let's make a bad we don't like and
let's make a tether the two together and

now can we buy us time or station so that
we only get a particular receptor pair.

OK now you can imagine this may not work
because you may get these to act just like

a lot of a lot like than one might
find in the other thing is free.

There's a lot of things that can happen so
we have to test it.

So we've made a family of these timers and
I'll show you how to moment and

show you some data.

So we you know we're so
perceptive we have E.T.F.

know Raglan our main two players and
then we can make various combinations

like in timers if you have a regular
right on a regular line and

theoretically at least drive
different kinds of time or station.

Hopefully to the exclusion of
the header timers we don't care reform.

OK in order to design these there was
a fair amount of protein engineering that

new album had stayed in this was in
collaboration with Richard Lee who said

Brigham and Women's and
weekend starting with the.

Crystal structure thinking about these
many design issues going in and looking

at the structure of the molecules so
this is each yap showing the three bonds.

There's a lot of information in
the literature about each act Perceptor

interacting with its live hands because
it is such an important system and so

we know what will likely happen if we
manipulate the various ins they're

not really part involve much binding and
we also know a lot about the affinities so

we could go in and say it's probably
OK to manipulate either the C.

or in Terminus and in fact we went in and
showed it doesn't really matter.

You can manipulate these so
we designed a fast cycle so

since we had a smirk at combinations this
wouldn't necessarily be what we do use in

the clinic but
it made it very easy to do an experiment.

There's a so this is the essential design
shown here a quote unquote main is used

to link two different dance together
the square where the main was published.

It's got a very low pick a mole or
binding affinity and

we believe that there is a very high
affinity and we make these like and so

now we have the square clear of the main
and here we can actually link something to

it that would let us stick this on
the surface and actually we could do that.

We could do a biotin we could do that
the binding protein etc So this is a place

that we can stick it to the surface or we
can use it and so I will form so this is

simply the design of the constructs
of the quilt call are held together.

This is a spacer region designed
by Bob Sauer slab that is

persistent to produce a segregationist so
that we can keep that intact.

OK So we go into we characterized as a
person we do we make the slide Hansen ask

are the right hands in there want to
be a long form similar to those that

are the native you know that the thing
that you would buy from protect you know

a collaborative research this is E.T.F.
that you buy this is are like

can we do the same thing with regular And
and when and in every case.

We can activate these are he were cells
which have approximately the same number

of each epicenter and
her to about fifty thousand and

seven seventh's have her three in her
poor predominantly with some Her two.

So we can activate with all of
the individual components and

inhibit this with an inhibitor of
the E.T.F. or separate or specific.

OK So these are just prove to us that
these are active then we can go a way to

characterize this finding we did a whole
lot of different kinds of assayers so

simply showing one which is
a binding two to a surface and

we get about an eight peak or more
affinity isothermal titration calorimetry

experiments verify this sort of
number of different be a core.

Measurement very high affinity finding So
at the concentrations we would use

them they were sent to all time highs
which very very little pre like and.

We have what I want to finish up to talk
with these two things some phenotypic data

that has gotten so excited about this
as well as some signaling data that

are starting to explain the phenotypic
data and supporting our idea that we

are saying time or station of these
receptors with these like hands.

OK And we predominately have used actually
three cell types once not shown here

these stem cells.

What's notable about
them they have very few.

Number of receptors so
about ten thousand E.T.F.

receptors a few her to this is work.

Manu characterized during
his see his posts.

They do have her three but know her for
M C F seven cells are a very

widely used breast cancer cell line and
they express a lot of her three and

her four and very little Perceptor or
her two and her three and hope for

harder to characterize American
just because religions but

this is what they do we also not shown had
as you saw a moment ago you see no cells.

He will.
Cells are and also primary cells so

he will cells for cervical there were
actually very close widely use outline and

they have each year for
such her and her to place so

let's just look at some
phenotypic data and.

So searing pretty culture
a Muslim commits stem cell line.

It's more to life with a church human race
and culture over twenty three days and

so these are it's true that more lies
a church helps is pro-survival and

inhibits some of the atomic pathways.

So even when you don't add
these are plated in serum

allowed to establish from one day in
serum that we take the same amount.

So there are cells that survive even
know their played a high density they'll

survive these are really
pretty tough cells.

OK if we add E.G.F. there's actually
the quantitative data show that there's

not quite as good survival of these cells
as Count little bit hard to see here but

there's a little bit worse actually than
here and that could be because E.G.F.

may be inhibiting adequate loops that are
pro-survival we have special talked about

this with Manu interesting a lot of
the E.G.F. Perceptor hundred time or.

You see really terrible So
in fact very few cells survive so

there's some huge difference
between these guys.

OK the regular one is you
know manna from heaven for

these guys you could see their credibly
able to survive in the presence in

Iraq in one and both really interesting
only her three doesn't have a kind.

So it can't act by itself but
when it had time arises with her too

it sends a very very strong signal
through the whole K.T. pathway.

So this is consistent with what we would
expect from a pro-survival signaling.

This homo timer.

Ahead ahead of a time or an E.T.F.
and Raglan also pro-survival But

interestingly So
this is native New regulated.

This is the time or very poor survival.

OK These results are actually what
we would predict based on if we

first thing E.G.F. homo diverse we
get more survival because we've taken

her two out of the party and her two was a
more potent pro-survival signalling here.

If we've taken her three
out of the party her three

shutting it off again
we get poor survival.

So it actually is consistent
with what we expect.

It another very important
phenotype both for

tissue regeneration of cancer is migration
and we follow this a number of ways.

One is just simpler to do my creation
experiments random some motel O.T.

which you can follow with time
lapse video of my cross Skippy.

This is an image that shows taken by Kevin
Coren as some are YOU student from U.T.

Austin in collaboration with Edgar Sanchez
who's currently writing his thesis

proposal in my group and so what we have
here is we have you can see these cells so

this is a single field and this shows
the trace of where the cell wanders

during the twelve hours we have serve it
so the cell starts here of Wanderers here

the cell starts here wanders over here and
there's then there's various things that

can happen the cell can divide it could
die it can wander off the screen a few

percent of cells have these kind of weird
events and we take them out they don't.

Statistically change the migration
analysis that we did.

Because so we can do these kind of
experiments on a lot of cells and

this is a little bit faint but.

The tracks didn't show up.

So while E.T.F. has pretty much
the behavior that was shown here

had the tracks are not showing up so well
so you can see these guys are very very

active but when we have the regular to
regular in the cells are just spinning

spinning around in their little things and
I have for some reason on the screen.

We're not really seeing it.

If we summarize their different migration
behaviors and do spike a lot of hands.

So essentially some cells probably
go anywhere they spend in circles or

they just are not mo tile and this is
observed particularly with the sly games

here then phenotypes or
the cells move really fast and

really persistent or
observed in other conditions.

OK now I'm going to try to yes show

you some data not not a very
pretty sad but one way.

There's a lot of different ways
to interpret migration data and

we are sorting through how to
compare different modes but

one way is to say what fraction of
your initial population moved total.

More than fifty microns from where it
started and so if we look there and

this is the concentration
of the various live dance.

If there is nothing there just by
happenstance about half the cells

move fifty microns that's just by
happenstance I have to be fifty percent.

If we have E.G. half.

There's an interesting kind of slightly by
face of behavior it's similar to not but

then we start to inhibit migration here.

The head of time or
I mean sorry the homo time are tremendous.

At first it inhibits at low concentrations
there's a tremendous by phasing effect

where it's very stimulatory here and
then comes back down the regular and.

Is inhibitory at low concentration
starts to stimulate as we go higher and

higher and the whole no diamond though
completely opposite behavior so

this is this is an irregular and
this is the homo

dimer it's essentially shutting we
think shutting off signalling and so.

The point of the side is to
show you that quantitative

effects are very different when we have a
monologue in versus a time or like and so

we can monitor here dimer here really
opposite effect of what might be going on.

OK And then another kind of asked say is
Kim attacks a Cinna Transmetal assaye So

this was not me some stem cells.

This is a that has traditionally been
known as suppressed cancer when it's

actually a melanoma line and

this was from Jepson got that single slab
he has supposed to express themselves so

that her two hundred three had or
timers are very potent for immigration so

they're doing that a static cancer cells
and if they've got models and now if

we take these cells and set them up in
a chemo tactics experiment where they're

undergoing chemo taxes to regen which is
something they normally Rob to do they

live to migrate to the regular end and we
add a constant concentration about time or

to post sides of the the assaye we can
see that regular in the regular one and.

So there should be at the time of
prostration when there's nothing.

You get.

These are actually dime
a concentration here in this city.

So when there's nothing you
get really great migration but

the regular regular and hard piece
are with a tactic agent added So

these were arts remark on trails he got
cut off your sister to rag on got cut off.

So this was with No no.

Right on presence so this is our control.

There's no queue Metaxas essentially
if we add now than the regular And

we get chemo taxes that
we talked about why can't

the regular rag on how much time or
completely shuts it off.

So it's consistent with our hypothesis.

Now that might that be happening
in fact we are now going

to start looking at signalling for
example activation as measured

by Fosco arc we can see
a regular on activates arc.

OK And it does.

Dependent manner by Bill in America when.

Very modest activation and if you go to
higher concentrations it shuts it off.

So this is completely consistent
with the view that with so

I mean these homo timers of one three
three doesn't have a kind of a so

the how much time was just sit there they
can't do anything and so nothing happens.

OK that actually could be very good for
cancer cells.

OK So let's think about an experiment
in these M.C.F. seven cells which

actually have all of the family members
notably they have her three and

her force we're going to have to do
a kind of tricky experiment with this.

To observe it.

Again on her three hundred
on her shouldn't signal but

the regular and
binds to both her three and her four.

So in this experiment we're going
to do some controls where we

block the whole floor access by using
just an antibody that binds to her for

OK so we're going to if we add.

These don't signal this one signal.

OK.

This would signal but
this wouldn't signal.

Now what for our farmers can.

So we can block that if we add anti
her forces we have anti before

we knock this out and this is silent.

Anyway so we should be able to block.

OK So we do the experiment.

Here there's nothing added here we add
an anti to rag on anybody to block her for

we put in our law again.

OK So then we put in our by mail and

right in line and
when we stimulate with no regular OK so

if everything is hunky dory and
regular because the cells to signal right.

OK but if the Babylon is inhibiting we'll
see a DOS dependent inhibition OK So

in fact again this is just showing
a native irregular and you stimulate.

It's highly stimulated
again they have both for

three or four so
this is completely expected.

OK now if we do this experiment.

OK we we add we stimulate.

You know we add either plus or
minus the any body.

We then put out by Vaillant like India and
then we stimulate with a regular one and

this is a dose response would
be a lot of doses no regular and

is able to stimulate these cells case and
rego is doing great group great.

And now as we add continue
to add this higher and

higher concentrations of by
the law again we shut off again.

And so this is if we inhibit her through
our if we don't inhibit her for more.

And this is kind of expected you would
rather just looks like in these cells.

We are in fact shutting off this axis
of signaling and in fact it causes

the cells to undergo a photo system we do
in a photo SAS a percent just dead cells.

When we go in and
again look at a phenotype So

this is actually pretty exciting because
it offers an opportunity different way and

who would think to shut off the signaling
by this terrible amount of cancer.

If you've got the supposed three hundred
two hundred timer solution that's causing

a lot of problems.

Anybody therapies are very difficult
because any problems are big and

they actually have a hard time getting
into Murs But we're finding with this.

These are very small.

Molecules So there's potentially a lot
of therapeutic potential you know who

would think you have a wide and from what
you're trying to shut off to shut it off.

So my lab is in a cancer lab we're working
with some other labs now to pursue this.

OK just to finish up real quick
on the M.S. see the signaling

is sort of the same story I focus there
on her three hundred one hundred two

timers again we can control the signaling
or drive the you know the signaling data.

In terms of.

If we have by they want E.T.F.

we can get those responses are relative
increase relative to one of pale an E.T.F.

receptor phosphorylation but
we see a decrease in her too so

the signaling data shows a shift into
each after Sept her that we'd expect.

OK so what I tried to do today
is present you a motivation for

why we go down this path of this protein
engineering looking at receptor dynamics.

So initial data that this could be
very interesting way manipulate

this receptor family and I'll close with
when I go see garden it's really nice

photos of the complexity of
this pathway all the way from.

Production of the live hands
by shedding pretty little

shedding from the cell surface
finding timers ation and

effectors can even be clearly
influencing all these pap place.

What I hope we've done is not only about
things that may be useful in the clinic

translation therapeutics but
also as tools to help us sort out

what some of the signalling pathways
are in that you have Perceptor system.

I had tried to mention everyone involved
in the work which really was lose

bias or it was mainly the mark of him.

Seymour who was from E.P. F.L.G.

problem only swore it sent him over to
do his masters and Linda Stockdale's her

technician who makes a lot of the Palmers
Edgar Sanchez did the video tracking

and eggers on there twice I
guess you do a lot of work but

yeah he did do a lot of
work actually actual.

Undergraduates.

You know I want to hear.

We have two tracks yeah maybe we
can give him one of both tracks.

OK so one to share with you.

Just some facts about this family and
we have the answering questions.

Thank you.

OK so

what I'm trying.

We try

to see what

we're doing not right now part of
it is you know different research

projects take on a life of their own
the molecular stem cells have so

few that trying to do the initial attempts
to do that was not very successful.

So it met bringing up some of these
cancer cell lines where there

is a higher receptor expression level so
we're in

the process of doing that in particular
because they have a biotin linkage site so

you can put that on so Robin prince
who unfortunately her name's not here.

She did the same attacks experiment
she's just doing a short term post-op

to sort out those issues.

So we're in the process of doing
that those experiments can see where

they can have a lot of artifacts so
it's tricky to get them from work that

we should be a particular cause
we had that robins in and

that's actually one of her projects right
now is to actually get that another thing

we're doing in this part of the S.T.C.
is we're labeling both of them with.

Floors that are non fluorescent and
then become for

us when they bind in this is probably in
per jali so that we can actually measure

the binding by increasing porosity upon
binding of the likeness of we just got

a post-op to start on that these methods
to prove the physical sociate are really.

The key to get to work as you.

The really tricky to get more.

OK.

And I'm happy to say I'm
talking firmly to anybody.

Afterward.

Yeah.

That we haven't we haven't
done that we really

based on our space are like off from work
that we had done with the system and

what we thought was effective
the facts on the docket to

be went back to the Post stock
that we had with Barbara's lab.

She's got a method that we really want to
do is NOT have them joined by the quote

but actually have them as one protein
if you express just one protein

no you get a giant mess because the three
bites you know each has three dots

you get a lot of blood.

So you can do those you can
make each one separately and

Reigate them and this is just not stuff I
do in my lab but probably Imperioli slab.

So she's going to rub neither of us
are going to do any of this Andrew

is going to do it all and he was going to
do exactly that make a series something

quite systematic and during spacing
them so we have a single molecule.

Probably a little clearly
resistant space or

that we could do exactly what you said
yes and those people are interesting.

I tend to be you know there's a fine line
between doing a lot of bio physics and

focusing on the phenotype so that this is
by physics it's not that hard to do and

that we should do so
well the great question.

Of course.

We haven't we haven't
the experience like that.

I think partly I tend not to think
about activate you say have to

be an active bodies.

We used to have two to
five very frequently to

inhibit body why can't you have
Perceptor effect have some

data showing that we can have the effect
we see with diamond use of five.

You mean like I guess we could you know
you could do the same thing with a herd

herd but we have we haven't done that.

I'm not sure what it was
what it would say exactly.

But yeah yeah.

That was a solution.

And I wasn't none of the final stuff
none of the time a work was tethered and

I'm six one five.

OK So
there's a lot of play after the first

of all in solution we get so solution.

Things can be internalized what I what
we haven't done yet or done a very good

job of with there that I would show
here is look at receptor traffic.

So if you give us all you pull
them regulate the receptor

because it's internalized and degraded.

If you get tethered that it turns
out you don't completely shut it off

because this takes place
against a background like so

the front now has to understand how and
why.

And this is about as
a little terrified all

of the cells are definitely yeah right

like it's now some of the upper life
is a signal from the surface primarily

expression that when someone gets so
nervous.

My belief the hypothesis
is they're doing some help.

Now can't lie.

So here goes.

Why was this.

When was that really the efforts of
her family this year not as a problem

but the big picture is it probably changes
the dynamics of availability or separate

the surface and there allow signalling so
that your kids get a service.

You're also heard to her one head or
a time or trafficked way more slowly

than her once so you're keeping it
at the surface is one explanation.

You keep Perceptor surface and you have so
it's all your it's hard to have lost it.

You know there's a lot of ways ties it and
we're you know we have

a point I want to make but
there's a lot less of me in the system.

We're building tools that will let
us dissect out some of these facts.

OK And the other question I

think we should give Linda
another hand print it.