I mean some of course and I work for

Dr Christine Payne in the school
of chemistry and biochemistry.

So the topic of my talk is going
to be visualizing chance or

ptosis of low density lipoprotein and
translators so it's just briefly is

the transport of cargo across cells and
many of you probably

already heard of low density lipoprotein
as I would yell or a bad question.

But as an overview.

I'm going to talk briefly about
the significance of translator since

the translight terms of this is very
well known for its blood brain barrier.

The blood brain barrier is very important
because many it's very difficult to

get cargo across the blood brain
barrier and so pharmaceutical companies

are very interested in figuring out how
to translate to us this plays a role in

the blood brain barrier in order to
design better drugs recently transferred

to systems first studied for its role
within a body as mother to baby transport.

The mother delivers in
a body was the fetus

through the process of transfer ptosis
through the cash and test right on track.

Additionally the antibodies
in your adult body

move around your tissues
to translate to assess.

So that's the biological significance and
of chance so to assess the second part

of my talk we will discuss the first
labeling of low density lipoprotein so.

I'm designing a probe that
will be sensitive to a chance.

I took this process and the chance of this
process relies on these sort of physical

techniques of forced or
resonance energy transfer and

freshened quenching Additionally I'm
going to ask say the effectiveness of

these probes using in vitro
degradation assaye and detergent they

stagger nation as well as a magic place
to Gratian so British chance I to

assist chance I toso says the transport of
cargo is an investor call across the cell.

So when you have a blood vessel.

You also have a fairly all sour wall and

that's the real cell wall represents the
main barrier to the rest of your tissues.

So the theory of cells.

Regulate crosses them in order to
distribute the underlying tissue so

this is how your bloodstream and
all the nutrients are distributed

in your whole body so chance I trust
is a very important process and

currently it's not understood
by well that's the corals and

that's of course remembering down
organ animals are able to cross.

And so this is why the study of
translators to extremely important.

So low density lipoprotein it's
often known as bad cholesterol but

in fact in question.

All you absolutely need it for

your cells it maintains
the fluidity in the cell membrane.

And so despite the fact that people
know all the Allahabad question.

It's very highly regulated
the uptake from the blood stream.

So for that reason it's used as an ideal
cargo for the study a chance I to assess.

Cells take up the L.D.L.

particles be a receptor on the cell
membrane surface and they transported

across for delivery to underlying tissues
the medical significance of L.D.L.

is up to the treated to the buildup
of L.D.L. in the blood vessels.

But in fact absolutely
lead out of the L and for

this reason I'm going to use it as
a probe in order to study the chance.

I took this process.

But you should also know that in addition
to the cell take you have L.D.L.

to transport across and

to distribute to underlying tissues cells
also music keep a small amount of L.D.L.

for themselves because they need
the cholesterol within the L.D.L.

particle for their own cell membranes so
part of the L.D.L.

taken up will be transported across by
another part of the aisle certain fraction

or be degraded with and vessels inside the
cell and then it will be distributed for

the cells on personal use and so the key
part in this is that conventionally

people have not been able to distinguish
when I would be Al is being degraded or

when it is being transported across.

And the purpose of my probes is that

they're going to be
sensitive to decoration.

So that I will know specifically
at what time point.

And what place in the cell
they will begin degradation.

And so the way we're going to do
this is with flashlights microscopy

normally what's right.

Microscopy you have an image of a cell and

it's very hard to distinguish what you're
looking at as in this top picture.

It's very difficult to even see
the cellular compartments and

in fact you don't even know exactly what
macromolecules you're looking at but

with fresh microscopy you can tag
a particular macro molecule that

you're interested in and you can watch
exactly where that macromolecule is so

in the case of L.D.L. you could target
with the force and probe and would

be able to see its location in a cell and
we would be better able to resolve

cellular compartments such as the nucleus
or even the edge of the cell membrane.

So.

In our lab we designed the optics for
imaging the dynamic moving things

and misplace it relies on
a microscope that's been

coupled to laser systems and that's
how the flash microscopy works because

inflorescence you have excitation of
the floor for the laser and then you

have an emission of a photon which is what
we're detecting afterwards on our camera.

And so this is Originally it
was a video of trafficking

inside of the cell and
normally the vessels

which would be seen as these tiny
particles are moving microns per second.

And that's a very very fast movement and
for

that reason you have to have
a very efficient protector for

tracking the movement of these particles
but currently with the current

methods of labeling L.D.L. all you're
able to see is the movement of the L.D.L.

you're not able to pinpoint when the
L.D.L. is being degraded inside the cell.

So for this reason my probes are designed
to be sensitive to degradation as I may.

Before.

So the probe specifically
as I mentioned are first

of residence energy transfer based and
fluorescents quenching based.

And the difference between these two
processes for the case of force to resume

its energy transfer I'm going to have two
different time molecules and there to be

two different colors Ryle in the flash and
scrunching I'm going to have one color

die but it's going to be at a very high
concentration on my L.D.L. particle and

I'll talk about why these two techniques
make my probe sensitive stagnation.

So for the first one forced a resonance
energy transfer transfer or fret.

You have to have a donor and
except a die molecule and

the reason a carbon donor except
there is in the case of a donor.

We're going to pick a dye that has
absorption seen as the solid lines and

the mission soon as the dash lines in
this region and we selectively die.

Exactly so that the mission of the donor

happens to of Iraq with the absorption
of the except a molecule.

And so

if I draw this out when I excite the donor
with a certain wavelength of light.

There's going to be a specific
kind of energy transfer and

it's known as Forster
resonance energy transfer and

it's going to transfer excited
energy from the donor to accept or.

And this results in a mission of
the accept or die molecule and

this is extremely important
because you're having a very large

shift in the detected by blanks.

But it should also be noted that this
energy transfer process is highly distance

dependent and it can only undergo energy
transfer if it's less than ten nanometers.

So what happens when you have a larger
distance between the two die molecules is

when you shine the excitation right.

The distance is too large for
energy transfer to occur.

So rather than transfer the energy
to accept or the donor only has

the option of emitting a photon in
its own characteristic wavelength.

And so what this translates as if you are.

Reading the fluorescence in this case
when the two particles are very close

together you'll see a very high intensity
in the except or wavelength region.

When the two dives are far apart.

You'll see a higher intensity
in the donor region.

And this is extremely important because
it seems this is a large shift in

the wavelength and so
I can easily detect in this case red light

is being detected and in this case
green light is being attacked and and

so you get this distance dependence and
it's very very high resolution.

So how does this relate
back to the all particle.

So reality of particle is a sphere around
twenty nanometers in diameter and it has

a protein that her together and B L D L
itself is primarily made up of question.

So what I've done is saturated the
particle with don't accept your dyes and

these are going to reside on
the surface of the L.D.L.

particle in this case I'm going to have
a pretty low surface area to volume ratio.

So in this case the guy in the except or

molecules are going to have a high
probability of encountering each other and

the higher the probability
of incoming chair there.

The higher the probability for
undergoing threat and so

what I'll see is a higher
emission in in the red region.

Whereas when I do grade this I will
be all particle my fears are going to

get smaller and my surface area
to volume ratio will increase.

So in fact there's going to be
a fewer number of DI particles on

the surface of these smaller particles and
I'm going.

There's going to be a lower probability
of them encountering each other and

due to that lower probability.

I'm going to see greater emission
in the green region and so for

this reason when my L.D.L.
particles be integrated.

I'll be able to detect a shift
in the emission wavelength and

that's how I can know when
my particles being degraded.

So.

When I tried this on an innovator I say
there are many ways to degrade these

particles and one of them is via the
Trojan you know you use detergent on your

hands to break up the dirt particles and
it will also break up L.D.L. particles.

So when I incubate with detergent and
I check for a change in the ratio.

In this case.

When the particles intact.

I have the red and

I have a lower ratio a small value
here and in this case I'm going to

have a high ratio because I have greater
emission in the green wavelength region.

So what you consider is where is one
hundred percent was the starting fret

ratio.

I have an increase in the threat
ratio which is what I would expect.

And so I can I can detect this change in
the fret ratio in this case around one

hundred fifty and around two
hundred twenty five percent so that

that is excuse if I can change and that
is easily detected via our camera system.

So the detergent I say worked very well
for prob number one the fret probe.

Rod Number two the question based
probe relies on the fact that there

are many many die particles on the surface
of the L.D.L. particle when there are so

many die particles they are actually
not able to emit photons as they would

normally when they're being excited
Instead the energy is dissipated because

there are too many die particles and when
the energy is dissipated like that you

just have little fluorescence and you
see a very small peak in the red region.

When you degrade and you have the higher
surface area to volume ratio and

so for that fact there are fewer
die molecules on the surface and

you will see that high fluorescents were
stored in you you can measure that as

a huge increase in the flush and
sometimes City after degradation.

And so when I did my controlled by
degrading the particle it detergent.

You can see was one hundred percent
would have been the base of value.

I had a thousand percent
magnitude increase and freshness.

Which is a very significant increase and
this show.

Is that it's very very sensitive to
generation which is exactly what we would

hope for and a probe that would be able to
distinguish between chance I took this and

declaration and so.

The reason we have ph of seven mph of five

is because the blood in your
body is not a PH of seven.

But degradation of the L.D.L.
particles actually occurs an apartment

in a compartment inside the cell
that is around a piece of five.

So we just want to see how ph affected
the degradation of the smaller kewl.

And as you can see that your nation
is enhanced with the regular Ph.

So the next step was to perform
degradation with isolated enzymes and

in the case of that base probe.

I degraded with immense I'm quite trips
and an enzyme called CAPS in beauty and

chips and just a normal control enzyme
that's commonly used in the occupied by

a logical steps and B.
is the main Proteus of L.D.L.

And so this was normally what L.D.L.
is degraded by and

when I include by my L.D.L. particles
which are something that somebody

before I was expecting an increase
in the flat ratio and so

as you can see there's a decrease
in the case of troops and.

And only a slight increase in
the case except that some being.

For quenching.

Previously we saw a thousand percent
increase in the freshman's activity and

this is barely significant Richardson and.

Definitely not as significant as
the two thousand percent increase.

We saw before with the Turgeon.

And a Ph of five so for

this reason there was this question
about activity of the enzymes and

whether they're able to officially
break down the all the all particle so

to checklists you can run in the current
of my gel and a kernel Nigella's

what they do is they separate molecules
according to their molecular weight.

So the larger the size the molecule
the higher in this gel they are and

the smaller the size.

As measured in code owns the realm
where they are in the middle and

so what you can see here is
when I just run the L.D.L.

on the gel with no enzyme I have
a very large band at the beginning.

When I add enzymes the L.D.L.
particle you see the formation of

smaller bands which indicates
the enzymes are degrading the L.D.L..

When I have the labeled L.D.L..

You see the same degradation products
because there is a concern that the flesh

and dyes may be inhibiting their Gratian
the particle that we check for this.

So these isolated enzymes.

Are able to cleave reality L. particle.

But now there's this question about why

why the lower efficiency eventually
put a target there going to action.

So I had thought the size that perhaps of
the enzymes only a fraction of the L.D.L.

is being degraded there was still a large
portion of the L.D.L. that was remaining

at the top of the gel indicating that
not only are they always degraded.

Additionally there may only be a small
change in the size of the L.D.L.

particle after declaration and

this would also give me smaller values
of what I expect with the enzymes and.

In conclusion through the two probes they
were successful they were very successful.

Based on a target integration assaying and

you can see that they
are sensitive to being broken up.

Both with the front probe and
the quenching probe.

So then it becomes in
this case the next step

is to test the fluorescence
after degradation and cells and

this is because our in Beecher enzyme acid
is there using isolated and zines and

it's not representative of what's actually
happening inside of a cell and so

the next step is to incubate our L.D.L.
particle with the real cell extract and

then we can actually see the change
inflections in a more real manner and so

the next step would be to use these probes
to actually track translators in this and

discern degradation which is the chance.

I took this process.

So I'd like to acknowledge that.

Undergraduate research office and
pharmaceuticals.

And also part of this research was done in
part of the bio chemistry lab course with

Dr Mae peek and their Buchan's and
the rabbi work and

Dr Christine pain and
I hope you supplied many of the images and

videos and these are the other guy just
it's my lab who have helped me a lot.

And questions.

This

chance I was it's important to all
multicellular organisms because

it's relevant to
the distribution of nutrients.

So you know like you always have blood
vessels within a few millimeters

of any cell and so for that reason
nutrients are always going to have to

pass through a cell layer and the raid
that they pass through the cell layers

through translate ptosis So that's
that's the fundamental biology issue but

then you have pharmaceutical companies are
interested in drug design everyone wants

to get through the blood brain barrier
to treat you know no logical disorders.

Potentially you could have you
know drugs being crossed or

Gene the liberty of actors in
the case of genetic engineering.

So there's a lot of potential for
riots and point to study translators this.

This when you're going.

So much.

And we're only in this

case we actually have isolated I'll be so
it's there should be nothing else and

if we re clean it up the size
exclusion kind of target the column.

So the L.D.L. has been separated from
any kind of cellular components and

it's just literally putting
out of the on a test tube and

a little bit of detergent and a little
bit of buffer just to keep everything

constant and
then I track the questions about turning.

Workers.

Yeah there would be no way to
treat cells with detergent without

just dissolving their membrane.

This.

Well in this case the cells beneath
role take it up themselves.

It's a chance.

I just this is most important and

that they will sell later because that's
had an insurance or to distribute it in

for the most part tissues underneath
don't really undergo translate this and

they take it up and all they do is degrade
the L.D.L. particle and since the L.D.L.

is composed of both questions and protein
the person is degraded and the question is

actually recycled back to the membranes
of the cell in a different cell right.

But our cell culture is only
on a single model layer.

So there should not be any cell
in Smith thank you thank you.