Yes every right.

A little bit further down the road over
where the biomedical engineering program.

There was actually yes

yes the nation's work were done.

You know wishing for years microgravity
environment is very very rare.

There are three things

your size laboratory

pictures are major this year.

Thanks so much and
let me thank I B B for inviting me.

I was love this seminar series getting
to hear all of our colleagues speak and

so fully a nice experience for
me to be able to get up here and

talk a little bit about what my lab has
been up to over the last four years.

So the title My talk is designing
cell instructor major cities and

essentially we take bioengineering
approaches to discovery slash I

would say understanding of I'd like to say
that we actually discover new things but

there's very little out there in
the field to be actually discovered we're

really into trying to understand
the phenomenon of the exerciser matrix and

then to deliver deliver either
repair regenerative E.C.M. key.

Use that if we pulled out.

So cell and its extracellular matrix.

Form a very homeostatic system so

in the case of cell E.C.M. cells will
actually transcribe translate to creat and

assemble a federal matrix
comprised predominately of

proteins in pretty Gleick ins with
cell matrix interactions is that

once a cell has created its extra Styler
microenvironment that microenvironment

then exerts specific instructive
power over the cells and

so there's really when we talk about cell
matrix interactions we're really talking

about a battle for control of what
that tissue state is like and

I think in a kooker Mynheer who's
at the Fox Chase Cancer Center

actually she was and
now at the University of Connecticut.

I think demonstrated this extremely
nicely in this study here where

she actually took the form of
an ectopic tumor and subcutaneously.

Allowed it to develop in the mouse.

She then took that tumor and took some
normal tissue of the mouse in an adjacent

area you'll arised those major cities
put them down on a culture dish.

And then asked what what
cell phenotypes do we do

arise when we play the normal
fiber glass on both

a sort of tumor associated matrix
versus a normal matrix and

what she found was that
when you put normal cells

on tumor genic matrix they
actually begin to transform.

So they take on a decimal
plastic nature seen here.

And actually start that
transition towards a cancer cell.

So this is not necessarily new I
think what was really exciting about.

This work was that when she took normal
fiberglass allow them to make an exercise

or matrix and then pulled the tumor
cells out and plated them on a normal

matrix they actually lost their
decimal plastic differentiation.

So they turned more normal.

So we say aha that's it.

All we have to do is fix the Matrix and

we can fix these kinds of path
ologies Unfortunately what

she found was as soon as the cells
could actually turn over the Matrix.

So the cells are continually
both degrading and

reforming the exerciser matrix.

As soon as those tumour cells
were able to reform the matrix.

They went back to their
decimal plastic phenotype So

there is it sets up this paradigm
where the cells are probably

ultimately in control of the situation but
certainly the exercycle

matrix is driving certain cues to
tell the cells what to become.

Another study and this happens to
be her mentor so this is not my

can you model love story but he does have
a lot of good work coming out of his lab

in this case what Ken is looking
at is the role of exercise

the Matrix in particular fiber Nekton
in regulating the branching program.

So I'm in the regenerative medicine field
and one thing we're always trying to

do is figure out how do we get complex
three dimensional tissues to form

to pattern the way we want them to
hear this is the mammary gland.

We actually working along in our lab.

What are the cues that defined
these clefts that are forming

versus these buds that are forming.

And if we can regulate that.

Then perhaps we can realize
the potential regenerative power

of stem cells of the exercise of a matrix.

So unfortunately in the it's
difficult to translate what we see at

the developmental level to the adult and
a main reason for that is that each E.C.M.

maturation in the adult is slightly
different from the embryo.

So you know we might want to
elicit a regenerative and after.

Let's say we cut off a finger.

Well one of the seminal events
when you cut off your finger.

I would suggest that is the formation
of an early provisional

matrix which is comprised predominantly
of the blood clotting protein

fiber in our fibrinogen so
fibrinogen is activated forms of polymer.

In response to bascule or injury and

as a part of that process we cross link in
the exercise or matrix protein that can

you model is interested in fiber
necked and this exercise or matrix

is provisional Matrix actually only exists
for a few days maybe as long as a week.

It's actually rapidly degraded
by cells that are invading that

wound area and they actually then
synthesize what we call a late provisional

matrix which is comprised predominantly
of aligned fiber Nekton fibers

with interspersed
cross-linked college and.

It's this matrix that our lab
is predominately interested in

from a standpoint of cell instruction.

The reason is that a couple of really
interesting findings I would say in

the late ninety's and early two
thousand show that you can't transition

from an early provisional matrix to what
we consider a mature matrix that would be

college and a last and slammin and
without this fiber Nekton matrix.

So if we knock out fiber Nekton in cells
they never form a college a matrix

despite their ability to transcribe
it to translate it into secrete it.

They simply can't assemble it into
a mature form part of that was explained

to due to the fact that fiber Nekton
actually acts as both the template for

immature college and fibers to align and
then fiber necked and

then stabilizes those college in fibers
until they can be cross-linked so

without fiber necked and
we don't develop mature college.

Network for from our perspective.

Fiber Nekton is really attractive
from a cell instructive.

Standpoint due to the the reality
that it's packed with lots

of nice biochemistry.

So this is the molecule it's a dime or
it's a relatively large dimer

it's about four hundred forty kill
adulterants the entire protein.

And what we see on the protein are sites
for Factor thirteen cross-linking sites

this is where it is cross linked into the
provisional matrix that fiber matrix it

binds fiber and it binds heparin it will
also bind college and gelatin So this now

you can begin to see where it's bridging
fiber networks to college and networks.

Additionally regions that we're interested
in are these type three repeats here in

blue and they bind a significant number
of cell surface rest scepter is called

interns so into Greens are the cellular
receptors that bind exerciser matrix and

fiber Nekton is one of the more
one of the molecules in the X.

other matrix that binds the most
diverse number of immigrants.

So if we look at here's a nice little
pictorial representation and we look at

these in a grid binding domain so this is
where the cell instruction comes in this

is measure to assembly cell instruction
and what we see is if we take a slice.

Really the seventh through
the tenth type three repeat.

And this is an inexhaustible list of
the number of cell surface receptors

in a grounds that this molecule
is binding here is listed eight.

But there.

It's been predicted that as many as
twelve different in a grain had a dime or

as may bind fiber Nekton what's
important about this is that

depending on which integrations the cell
interacts with its exercise or matrix.

It will number one elicit very specific
signaling Cascades within the cell.

But number two it actually my.

Modifies the cells response to saw
able factors like growth factors and

cytokines So it's fundamentally
important that not only we

understand which in a grins that the cells
are interacting with for their matrix.

But in the case of trying to engineer
cell instructor major cities this domain

provides a lot of interest for
us to be able to tweak these responses.

So a couple of concepts
I want to get across.

And I'm going to give some props I don't
think is Evans in near here I'm going to

give some props to Evan
here in just a minute.

So a couple of concepts that I need
you guys to be aware of when we

look at the process of fiber Nekton.

Federal Assembly so that is going from
them on America form into a febrile or

matrix it actually requires force.

So we actually bind Here's an integral
it's bound to fiber Nekton.

This elicits particular interest so
you are signaling Cascades.

In general.

Sark in fact activation
which leads to the formation

of an act in stress fibers and
contractility so

the cell begins to form these contacts and
then begins to pull pull on this molecule.

So it pulls on this molecule it
separates the immigrants and

as a result we actually expose buried
cryptic sites within that molecule so

you can think about fiber Nekton is
a wound up rubber band that's all tightly

twisted on itself and literally the cell
is pulling that rubber band out so

that now we expose little domains
that were previously hidden.

Those domains then enable fiber Nekton
to self bind and in this way we pull

assemble pull assemble pull assemble
this is really critical in understanding

two things One is that we can't generate
a fiber neck a matrix without cells.

It's an inherent difficulty in what
we do and we're working on so.

Different methods that I'll
discuss a little bit later

to try to facilitate that process.

But secondly what I want to get across
is that the molecule is highly elastic

the type three repeats that I've
mentioned where immigrants actually bind

are the most flexible and they're held
together completely by hydrogen bonding

and as a consequence when
the cell poles on those domains.

They're actually capable of
undergoing bond slippage.

And then breakage until they
open up if we then let go.

If the cell then lets go of
that fiber nectar molecule.

They then reform in a reversible
manner with very little history so

very little energy loss due
to that refolding event.

So here's my props to Evan.

This is.

Again sort of on the trying to
inspire you guys that that now.

A fiber Nekton rich extracellular matrix
which is this that Evan has stained and

now tracked during embryogenesis
the matrix is not a static entity.

It's actually always moving.

And the cells that are within that matrix.

OK I'm not good at doing that and

the cells that are in that matrix
are pulling and moving and

as a consequence the fiber Nekton
fibers and if you take a look at this.

He draws a grid of squares
are circles here and you can see that

some of the circles are moving apart which
would imply a level of fiber strain and

some are moving together which would
imply compression of those fibers.

So during embryogenesis and during most
repair we're processes in the adult

as cells move in and pull on their
exercise their matrix their physically.

Pulling straining that matrix so
conceptually that's important because.

What we're interested in is
whether those mechanical events.

Are then translated into the biochemistry
of the fiber necked a molecule

unfolding those type three repeats and
resulting in alterations in that

in the immigrant profiles that
are binding binding that molecule.

So we're not quite as fancy
as Evan we don't have any

really cool developmental models just yet
although we're working on them.

What we have done is we've taken
a look at some adult processes

in this case we're looking at.

Here's a normal long
alveolar structure and this

this tissue actually is being repaired and
regenerated every single day of your life.

So every time you breathe in you're
breathing in particulate matter

that particulate matter is
being Indus a toast by cells.

Usually in this region and here.

Those cells are undergoing a pop
ptosis it's causing tissue damage.

They then regenerate.

Every single time you breathe.

So what we want to understand is why in
most human beings we maintain this nice.

Three dimensional architecture of lung and
in some individuals we develop fibrosis.

That being denoted here.

More fundamentally were interested in what
are the E.C.M. cues and in this case.

Biomechanical cues in the exercise or

matrix that are defining this
tissue versus this tissue.

And this work actually harkens all
the way back to my days that you may be

where we actually pulled
out these fiber blasting.

These are hives of highly
proliferated fiber blasts

that are indicative of
pulling our fibrosis.

If we pull fiberglass out of this guy and
we pull fiberglass out of a normal lung.

They display two different phenotypes and
those phenotypes are related to

their expression of a small outer
leaflet glycoprotein called by one.

And we're going to go into a lot of
detail about this but what we found.

Gosh almost ten years ago.

Is that by one becomes
a regulator of row activation.

And row activation leads to sell
contractility focal heat information and

stress fiber so we have these fiber blasts
here that are thought one negative that

lack of this machinery and we have normal
fiber blasts that have this machinery and

what this sets up are two populations of
cells one that's highly contract tile and

pulling on its matrix and the other
that's more homeostatic in nature.

So that sort of sets up our main question
in the lab which is how how might adjacent

and physically different exercise their
major cities determine cell into state

we drew this little simple diagram to try
to help frame what we do in the lab and

fundamentally what we're looking at is.

Here fibrosis associated pulmonary
fiberglass generate fiber neck to major

cities that we know display increased
are experiencing increased strain.

OK so the cells are more contract all
they're pulling on their matrix more and

as a consequence they're going to do two
things one is going to generate a stiff

E.C.M. which has by potential by a
physical effects on the neighboring cells.

So in this case we're looking at
a scenario where the cells stiffness

where the interior of the cell is trying
to match its substrate compliance

with its extracellular environment and
to do that it has to modulator rack and

row signaling the same signaling network
that was altered in our pulmonary

fiberglass So as the pulmonary fiberglass
stiffened their exercise their matrix

these by physical cues are translated
into the neighboring cells

altering their steady
state of contractility and

as a consequence then this may result
in some phenotypic determination.

Consequently if I have a high.

The contract all fiberglass and
it's pulling on its matrix it actually has

the opportunity to unfold those type three
repeats of fiber necked and that we're so

interested in if they unfold those type
three repeats then there's a potential

that the immigrants that are able

to bind that matrix then shift
from one population to another.

So there could be biochemical effects of

this made matrix stiffening here
again stiffness of E.C.M. mediates

the capacity of fiber not going to unfold
in fiber Nekton unfolding controls.

We've been focused on Beta one integrated
binding and Ro rock activation.

OK So this is some really nice work done
by one of my newer grad students vents.

So we're interested in trying to figure
out what's different about this matrix

in a normal long and
this matrix in a fiber optic long.

And unfortunately these images
are not coming out really well but

we can actually stain these tissues for
type one and Type two cells and

we get normal architecture and
what Vince does is actually we instill so

we induce pulmonary fibrosis in one
mouse and inject sailing in the other

after about twenty one days we harvest the
mind and we actually inject soft melting

temperature Agger rose into the lungs to
inflate them but it's then pulls them out.

We embed them in another block of agro.

And then with a vibra time actually make
about one hundred micron thick slices

of living lung tissue so
these cells in this tissue are alive and

they stay alive for
about forty eight hours.

What that allows us to do is then
with the help of Todd Sol check up

in mechanical engineering.

We've gone up and actually done mechanical
measurements at the cellular level

using atomic force microscope and.

So we had a hunch that this
matrix would be stiffer than.

Normal matrix so far broader would
be stiffer than the normal and

what Vince has found is that there's
actually a pretty tight distribution of

when we measure Young's modulus of these
lungs normal lungs being in the realm of

about to kill a Pascal's and
fiber optic lungs ranging from about.

I guess maybe twenty five to
fifteen killer Pascals here but

the box plots sort of show the spread
we do see stiffness values up near

eighty killer Pascals in the lung.

So the big question then is OK so
if we do see

alterations in the physical stiffness of
the exercise or matrix or these tissues.

Does that confer some sort
of altered Sophina time.

So I'm going to back up for
a second if I can.

And the main.

Phenotype that we're interested
in studying here then

becomes this regenerative event
whereby when the lung is injured so

that space is injured
the alveolar precursor cell

known as the eighty two cell
has to differentiate into this.

Gas permeable cell here eighty one.

So what we're concerned about and

what there is some growing evidence is
that in fiber optic lungs these eighty two

cells instead of differentiating down the
normal pathway terminally differentiating

into epithelial cells they actually
undergo a trance differentiation to

a phenotype invade the matrix and
then synthesize more extracellular matrix.

Resulting in sort of this
positive feedback loop whereby

you generate these fiberglass to focus on.

So this event of normal differentiation
versus trance differentiation what we

call epithelium the sink will transition
is a really critical cell behavior.

Certainly for fibrosis.

But certainly for the formation
of three dimensional tissues for

cancer metastasis etc etc So
we wanted to ask.

And there are these different environments
are listing different responses

we model that with a really simple system.

We form public role in my gels very
thin public gels surface immobilize

extracellular matrix proteins seed our
cells on top of those major cities in asks

What are the phenotypic changes that
occur here what we're looking at is

simply proliferation and what you can see
this is some work by actually Carson.

Scuse me actually Brown that shows
that in this case type two or

epithelial cells undergo
a stiff stiffness dependent.

Proliferative response up to about well
what do you know twenty kill a passable

right around the range that we see in
the normal in the fiber optic along

this response then actually
tails off on stiffer materials.

Here thirty six killer Pascoe's and

comes actually significantly
down on tissue culture plastic.

What she'd done then was obviously we're
interested in this dynamic whereby

the stiff extracellular matrix modifies
the interior stress of the cell and

I had mentioned that that's mediated
primarily through activation of RO

signaling and so contractility So
what actually did here.

I'm sorry for the missing the labels.

She actually added a row inhibitor
actually technically a rock inhibitor

that inhibits ro signaling and

she says that you can abrogate those
stiffness dependent responses.

Again I'm sorry for the.

Projection of the images what we've
been wanted to do is ask whether or

not normal epithelium cells these
eighty two cells actually take on

an altered morphology so normally
you'd like to see eighty two cells

in a nice cuboid cuboid will form
lots of cells so contacts and

not in a highly spread state again
what Ashley shown here with.

Acton staining and

Alpha smooth muscle actin is that
you have to trust me on this.

Here's the numbers if you if you don't.

Is that increasingly stiff.

Substrates the cells actually go from
a more epithelial phenotypes and

more cuboid will into a much more spread
fiber Blastoderm a zinc a mole type

of phenotype we can maintain even on
this is on both fiber Nekton lamb and

controls are both on glass or plastic.

You can actually maintain Epatha a little
phenotypes with lemon and so that's our

control here and what she showed
here was in the first panel are just

the cells in the second panel is the row
inhibitor or row signalling inhibitor.

And the same paradigm here in
Alpha smooth muscle actin and

what you see is again with increasing
stiffness we see a loss of cell

circularity so the cells are going for
a round to spread.

Here's the lemon in control and
if we add the row inhibitor.

And then we aggregate
some of these responses.

OK So cells are proliferating more

on stiffer materials as we go from
normal to more fiber optic physicalities

the big question then becomes And
and they're spreading more but

this doesn't necessarily
indicate that they're becoming.

So what we want to do then is actually
look at specifically at epithelium

the expression of epithelium markers and

the expression of material
markers here we've done P.C.R..

On both the cat here and which is a cell
so junction protein so facon protein C.

is actually a protein that specific for
alveolar type two cells and

what we see is that on increasingly
rigid substrates we actually begin to

lose the expression of our epithelium
marker we lose significantly and

so you'll have to look at
the breaks in these graphs.

This is one hundred fold decrease from.

Eight point five kilohertz goes
to thirteen killer past goals.

Loss of expression of so fact and
pro can see here we're actually gaining

the expression of musical
markers in cat here and and

by Menton and
again in the red what you can see

is that when we add that rock inhibitor
we aggregate some of these responses.

So what it's looking like it's that.

As the as potentially as fiberglass
begins to modify this interstitial space.

Making it more stiff by condensing
the matter in that area

we get a stiffer environment and
that's actually affecting

the peripheral Epatha illegal cells that
are lining those fiberglass to high.

So going to switch gears and

now talk a little bit about our work
on then if cells or if I blast or.

Contracting their exercise or
their matrix there's the potential for

biochemical alterations in the X. or so
the Matrix and in particular our focus is

again on this unfolding of fiber
next in type three repeats and

the loss of certain inner grins
capacity to bind that matrix.

So so first we have to ask the question.

OK well if a cell is altered in its
row activation do we actually see

that we get differences in fiber Nekton
unfolding and so what we've done is.

This is actually work done with assistance
of Vogel who's now at the inn in Zurich.

We actually go through here schematic
of fiber Nekton we actually

then label the fiber Nekton in very
specific spots both within except or

in a donor floor four and
then as the cells begin to pull and

unfold the molecular structure of
fiber Nekton we get the donor and.

Scepter floor four is moving apart and

we actually observe a decrease in
our resonance energy transfer.

So we get a loss of fret signal as this
molecule goes from a compact conformation

into a partially unfolded confirmation.

So we generate heat maps like this.

This is just a standard fiberglass
culture and here what you can see is the.

The quantity of our loss of
fret with actual unfolding or

let's say strain of fiber Nekton fibers.

So what we've done here is this
is some work that I've done.

Actually my postdoc that set up some
of the some of things we're doing

in the lab where you have a normal
fiberglass this is a pulmonary fiberglass

that's wild type and

in this case we've knocked out a protein
that's essential for its ability to.

To contract its matrix.

So it actually interrupts that
cell site a skeleton machinery and

what we see is we get gross differences in
fiber Nekton assembly but perhaps what's

more important if you look at the blue
versus the red line is that normal fiber

blasts unfold fiber Nekton observed as
a loss of fresh signal to a significantly

greater extent than cells where we have
interrupted their contract all signaling.

OK so that's a that's a transgenic
where we've knock something out.

It's hardly physiologically relevant in
that sense so what about this scenario

if we go back to this idea that
normal fiber Blaster expressing

thought I won abnormal fiberglass
are have lost the expression of Taiwan.

If we look at them.

Pictorially Here's some images
from Rick Phipps lab who

actually initially described
these fiberglass up populations.

And what we see is that
the profile brought excels by one

negative cells actually
strain fiber nectar.

Fibers to a greater extent
than one positive self so

that's normal normal fiberglass here and
five brought it.

Fiberglass here.

So we're still following that work up and
Vince is following up on that trail.

Looking at the dynamics
between fi one expression and

surface substrate rigidity
on fiber Nekton unfolding.

What actually had tried to do this was in
our very first years she's probably having

bad flashbacks right now of this work but
so

we actually asked the question well if we
pull fiberglass out of these mines and

we try to physically manipulate
the matrix without the cells right.

So I mentioned an important
feature is that unfortunately

we can't generate a fiber
neck to matrix without cells.

So we have to pull cells out
of the lung we put them here.

This is played on P.M.S. sheets.

So that we can actually
strain that system.

We then go through
the settlers ation process and

a characterization process and here you
see those major cities in this last panel

are almost exclusively fiber necked and
we have a very little bit of lemon and

no college in one or
three in the college and four.

We can actually then label that with
beads to try to map the strain elements.

As we begin to apply
forces to that matrix.

So if we do that we have this nice
little strain device that allows us to

Rich on that fiber neck a matrix.

That's been assembled on this
little thin P.M.S. sheet.

We can actually get these rearrangements
and fiber neck to Matrix and

when we see the other type two
epithelium cells on these we see

when we strain that matrix fifty
seventy five or one hundred percent.

We get began to see the expression and
this is at the protein level of musical

markers like environmental OK we actually
also see very similar to what we'd see.

Before sort of a by Facebook
response of proliferation.

To strain in the Matrix.

Now the unfortunate part of this
is that you can imagine if you

have a matrix that looks very
heterogeneous like this you have fibers in

many different orientations
the fibers are different thicknesses.

And probably molecularly they're
packed slightly differently.

You can imagine I apply a strain and

I cannot determine the difference in my
cellular responses due to fiber alignment.

So if I pull on the fibers I'll
go from a random orientation to

an aligned orientation
versus fiber unfolding.

So that's domain unfolding.

So unfortunately we can't really make
definitive claims regarding mechanism

with this data.

So we actually decided to and so
I guess I should point this out.

We asked ourselves a question are observed
to be changes due to the exposure of

cryptic sites or the disruption
of immigrant binding motifs and

we simply lack the necessary
Ria agents to figure that out.

So we actually switch gears and went
more biotech on this particular project.

Again looking at fiber Nekton here and
zoning zooming in on the cell binding

domain which is comprised of these type
three repeats what we decided to do was

instead if we want to test the hypothesis
that fiber Nekton domain unfolding.

Results in an integrated switching and

alterations in cell phenotype
let's just make this work.

Ominously.

So that's what Ashley did.

This is based on some fundamental work
again by Viola Vogel some great work by

Chengdu here at Georgia Tech and
Klaus Shelton up in Illinois.

What we do know about these two domains
This is the binding domain here is that if

we apply a force on this type ten repeat
this ten type to repeat we actually.

Elicit the sort of unraveling
events we can actually then

began to modify this by
regulating the amino acid

structures in these two domains
to perturb physically perturbed

those hydrogen bondings events that
are holding these two domains together.

So basically what we can do is we can
make the domain stiffer so that it's

it's harder for cells to actually unfold
those domains or we can make them floppy.

So then they begin to wobble around.

OK So this is the hypothesis that if we
have wildly floppy fiber Nekton domains.

Then we may get an inner grin that
would normally bind now in an off state

because it simply cannot
recognize that motif.

Whereas when it's in
its normal conformation

in this type confirmation
that immigrant combined.

So again actually looked at we stabilize
the ninth and tenth type three repeats and

we show that it significantly
induces influences so morphology so

this is pretty pretty striking we're
actually able to keep the empathy a little

phenotype on fiber neck and
this was for forty eight hours but

we've shown actually in this case she
went all the way out to two twelve days

around five days we can keep that
epithet a little morphology and

I think we're keeping some of
it as far out as ten days.

Give me twelve days

the remarkable thing about this is
that every piece of literature.

So far states that if you place an epithet
a little cell on fiber neck and

it will undergo E.M.T.
within three days and

so what we're seeing here is that
perhaps that's not the full story.

Perhaps there's an issue where in
experiments people are plating fiber

necked and surface coating fiber nectar
and and they're actually getting molecular

unfolding of those domains which is
the listening these types of responses.

What we did find was that if.

We lopped off the ninth type to repeat so
we have one domain that stabilized.

We have one where we just simply
cut off the nine Type three repeat

the importance about that is that
those two domains actually work in

synergy together to bind
a specific subset of anagrams.

Whereas only the tenth type three peat
binds a different subset of anagrams.

So if we actually now push

the cells to interact with one
integrated set versus another.

Then what we can do is we can maintain
these epithelium phenotypes or

we can actually drive these
are predominately miserable cells at this

stage now it should be noted
that we can we can overcome

any of these major effect by adding
some master regulator like T.G.F.

beta T.G.F. beta for the fibrosis
world is the bane of our existence.

It will turn any empathy a little cell
into a minimal cell in here we're showing

that we're not we're not seeing anything
out of this world T.G.F. beta drives

all of these cells to a missing
phenotype regardless of their substrate.

So you know I'm doing on time.

OK so

what we wanted to understand was a little
bit about the mechanism so OK so what.

So we modify these fiber Nekton domains
and we get one cell phenotype over

the other but the big question is
why can we really explain this.

So what we wanted to do was look
at what the immigrant profile is

that that cells are engaging these
fiber Nekton fragments with and

what we found was when we do an attachment
assaye here to our stabilised mutant

we see that it's predominantly
a result of Alpha three.

Immigrant binding alpha
beta six they do one and

Alpha five Beta three and Alpha V.

which were predicted did not list
that a huge binding response

to this fragment the converse
is when we lock off.

The synergy domain.

This one type three repeat Alpha three
completely lacks any functionality

with cells mining to that domain.

We still see out of the beta
six a little bit of Alpha.

Scuse me a Beta one but none significantly
and the mouse is sitting right in the way

here a little bit more beta three but
now out of the begins to dominate so

you can see now the shift from
interaction with out for three and

Alpha five anagrams to interaction
with out of the inner grins and so

we're beginning to think that that's one
of the regulators of what's going on here.

What this is simply showing is that
the responses are predictably.

Sensitive to the addition of saw uble.

In a grand ligand.

This in the form of our G.D.P..

This is our synergy domain so this is the
domain on the ninth type three repeat that

synergize is R G D binding and then
together we completely knock out any spell

binding to those fragments as we
predicted on the case of five and

I can type the ten type three or
P It's predominantly mediated by R G D.

And almost non by by synergy.

So we were moving more
quantitative with this work.

Now this is still work in progress.

With actually.

And what we've begun to do is actually
measure quantitatively integrated

binding to our fiber neck and fragments
using surface plasma resonance and so

this is a methodology whereby this
whole process is to immobilize

we mobilize in a grin
on a gold substrate and

through light diffraction we can actually
measure mass additions to the surface

due to this plasma resonance effect.

And so what happens is then we
immobilize are immigrants and we flow.

Are fiber neck and
fragment across these surfaces and we X.

Do we see binding events or not and what
is the magnitude of those mining events.

So if we do our homework and we do our
nice little dose response curve here.

And we do it under the proper
conditions low flow it cetera.

Then we can actually model this data and
back out binding kinetics

this here is actually
the dissociation constant for

our different fiber neck in fragments and
this case two Alpha three beta one.

And so what we're showing is that.

The binding to our stabilised mutant is
about twenty three nanometers animals and

we're approaching that of the traditional
Alpha three Beta one in a grand

binding domain which is laminate So
that still work in progress.

If we now take those
alveolar type two cells and

then plate them on these different
fragments we see very similar things to

what we've seen before we actually
when we lose energy we lose

when we lose the center just a binding
between the ninth and tenth three peat we

actually begin to lose expression of our
epithelium markers and we begin to gain

expression of a marker so the unfolding
events at least at this point time.

What we can say is that the presence or
absence of synergy.

Is driving or
maint I should say maintaining

an epithelium phenotype we're not yet
to the point where we can say molecular

unfolding is resulting in E.M.T.
or working on that.

OK So

this is just trying to figure out what
what maybe some of the mechanism involved.

So we know which in a grins or
binding these fragments.

But it doesn't really it still doesn't
tell us how these things are becoming

thinkable cells.

This is some we're looking at
PI one expression Pi one is a.

Immediate downstream.

Target of the T.G.F.
beta signalling pathway.

So an activation or

an expression enhanced expression of
Pi one is indicative of enhanced T.G.F.

beta activation and signaling So
what we see is in this case.

Here's the normal response and
this is pretty normal on fiber Nekton

in the presence of fiber Nekton and
T.G.F. beta.

We obviously see a nice whopping
expression on lemon and T.G.F.

beta does not elicit a pile one response.

So those are negative control and
over here what you see is that on our

stabilized mutant we actually if we
look at fold expression with respect

to that we see that we get a doubling
of the expression of pile one when we

remove that synergy domain so
this is still far from conclusive but

what we're thinking is happening is that
as the immigrant shift from Alpha three

Beta one to Alpha Vienna gran's they're
actually taking on a more contract

all phenotype which is enabling enabling
their activation of T.G.F. beta.

Which is an inherently
mechanical mechanical then.

OK so where we're really trying
to go with this work and

I'm not going to talk about this except
to kind of give you some teasers

hopefully maybe a year from now we'll
be able to give a talk on this.

So one of the things we're really trying
to do is figure out how can we generate

fiber Nekton fiber so that we can do these
studies on strange fibers without cells.

So a nice nice work by spots and
been a Geiger from Germany

showed that you can actually
generate a cellular fiber and

fires by applying force to fiber necked
in sheets and K. so what they've done

is they formed a fiber Nekton film
across micro pillars they wetted

the pillars which applied a force to the
tips of the pillars and that application.

A force then formed of fiber.

So we actually do.

Fundamentally the same thing and we do it
at the air liquid interface of a really

small droplet So we actually can form
a really small droplet on a hydrophone

surface and
with a pipette tip then dip it in.

And literally pull out a fiber neck and
fiber similar to how you might do

like nylon and so
in that way we can actually now begin to

pattern fibers fiber Nekton fibers in
this case they were doubly labeled for

fret analysis and
we see that here is the direction of

the substrate being stretched we lose our
friends signal and perpendicular because

we get a compression we actually enhanced
the for a signal and here are just

some pictures showing that we think will
be able to seed cells on these fibers.

OK so I have a few more minutes and

I'm going to whiz through the translation
of some of this technology but

the conclusions with our work on fiber
Nekton is that we real really still don't

fully understand the effect of force
on fires and how that's regulating so

phenotype we still are trying to
figure out whether forces expose or

exposing new cryptic sites for
new receptors on the cell surface or

whether these forces are simply disrupting
or decoupling synergistic binding events.

You know our evidence is beginning to lean
this way but we can't rule out rule out

the top either what we do know is that
if we mimic different structural States

can type three repeats we do seem to
be able to gain some control over

which in a green cells interact with
their exercising their matrix and so

in this case when we stabilize
the central cell binding domain.

Specifically the ninth and tenth type
three repeats we can actually enhance

the apparent binding affinity and I didn't
talk a lot about it but of Alpha Phi Beta

one and likely Alpha three Beta one but
this is somewhat still controversial.

What we do think is that we can direct by
directing immigrant specific engagement.

We can begin to allow
some relative control and

I'm very cautious about this term.

I'd love to say I could tell a to
become B. and I'm not entirely sure

that the E.C.M. can do that in
general what we see is that the E.C.M.

is really good at hitting the brake or
hitting the gas and so

that's probably where we are really
the paradigm that we're working with so

the big question then becomes in
the last say five minutes or so.

Is how do we then translate
this to the next level again.

How is going to how is one going to
develop exerciser matrices that instruct

sell into state again
operating in this paradigm.

And what I'm going to do is go back
actually to the early provisional matrix

and say that this is a nice opportunity
to begin to engineer some of

the features we see here at
this stage the reason is that.

No matter we're all engineers were and

most of us are trying to develop
technologies that are probably going to

adapt with the body or
be implanted in the body or

have to integrate with the body and
in any point in time where you puncture.

A layer of skin including
a purposeful incision and

placement of an implant you cannot
avoid the formation of fiber and so for

us it's a nice avenue to say well you're
going to get it anyway let's reinjure near

that so that it looks more regenerative as
opposed to more scar and wound healing.

So we actually use some again
I'll steal some terminology

from Jeff Hubbell we're going to
hijack some of the native biology

of fiber employers ation
specifically these two sets of.

Fiber knobs which have inherent
binding affinity to fiber and

pockets are now we're
calling them fiber and holes.

So the field has now educated me on and
so what we're going to do is

exploit these this inherent fiber in
assembly specifically fiber not peptides.

So these little knobs that are exposed
when Brahman activates the protein to then

begin self-assembly we know that we
can synthesize these synthetic knobs.

Here's one G P R V.

Which is the human a knob.

Versus this one which is
a synthetic analog of that and

we know they bind with relatively high
affinity to these fibrinogen holes.

What we wanted to ask was this has
been out in the literature for

about thirty years.

What we wanted to ask was can we.

And can we understand what the design
principles are for building better knobs.

And.

And can we do that.

And so what we did was we actually
designed a number of different sort of not

variants this one being completely unique
to the literature and what we found

was that the binding affinity is dependent
on the peptide residue properties

specifically from some molecular dynamics
work that Sarah Statman felted done.

We found that it's actually
the binding dynamics

actually depend on the stability
of this backbone chain and

actually this position of the side chain
of the third Argentine So depending

on whether it takes this takes the trans
or trans or gauche plus confirmation.

We get different binding affinities and
if we build in salt bridges and

between side change we can actually
begin to modify that affinity someone.

OK So the big clue for us was OK Now
how do we begin to engineer in

some of those bio physical properties that
we want when to modify the bio physics.

And the physical properties
of the matrix and

the bio chemical properties
of the matrix and

I'm going to touch on two different
two different methods to do each one.

So the first was if we express.

Or I should say present a fiber not
domain on a synthetic molecule.

Synthetic polymer like polyethylene glycol
can we occupy those fibrin holes and

stare Clee hinder fiber and
fiber in self-assembly and

can we regulate the dynamics of assembly
and as a consequence of regulating

the dynamics of assembly regulate the
polymer structure and material properties.

So this is just some homework on
looking at different peg sizes and

different peptide motifs and their
effects on both percent Claud ability.

That's our measure of activity of fibrin
and what we find is that as we expected.

We get somewhat of a distribution.

Now if we add those some

of the better products at really
really low concentrations.

So we're adding them at low
enough concentration so

that we're not inhibiting fibrin
Polemarchus ation as you can see here.

So this is the activity of
the native protein which sits

around ninety five percent and
we're modifying the polymer.

In such a way that we always achieve
at least ninety percent activity.

Somewhere between nine hundred
ninety five percent activity.

The blue is our control the red is
our our unique peptide sequence that

we discovered and then the orange
winds up in this case being

a synthetic analog of the bee
knob instead of a knob and

there's some evidence that that
elicits very different responses.

So what we see is that we can
maintain its activity but

we significantly alter the effect
of diffusion coefficient so we can

actually enhanced diffusion of molecules
through the polymer while still maint.

Painting the same mass of protein in
that polymer which is pretty amazing.

So you're thinking OK we've
modified pour structure in and

that's really what we've done.

Here's the control this is what
native fiber intends to look like if

we add a knob memetic which is
something that's probably not

overtly obvious as we get a lot of
either fiber breaking or fiber you

know sort of blunt in fibers so fibers are
growing and then they are trying Kate off.

What's interesting about the bee
knob is you actually get

what to me look more like a tape like
fibers that there are a lot thinner and

a lot finer and so
then the question is do we alter

mechanical properties and we saw some
really interesting things which is that.

Here's a normal polymer here when we when
we have this scenario as you might expect.

We actually decrease the complex modulus
But what was shocking to us is that if

you look at these two here I'd say
the complex modulus is going to be less

cure as well but in fact we can actually
push that in the other direction.

OK so the last little bit of this is
we've now expressed these fiber knobs on

recommit proteins and in this case shown
unfortunately again it's not projecting

well that we can actually target these
products these were common protein

products in a fiber in major cities and
their retained in fiber major cities.

Even during a profusion event.

OK And so we're going with this is where
actually beginning to try to quantify

polymer structure and predict cellular
responses based on the architecture of

the polymer through
computational models and

I will I will in there definitely
thinking my lab this is a lot of

work that we've done and I've got a lot of
bright kids in the excuse me young adult.

In the.

In the lab my postdocs Dr Sarah stem and

fell Dr Kelly Claus and
Dr Rodney Everett are senior scientist.

Sarah Jordan.

And our current graduate
students Ashley Carson.

Allison soon.

Vince Fiorina Lee J. K. L.
and our collaborators in funding and

thank you very much would you.