WEBVTT

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Welcome Sarah Stanhope for rejoining your
own Biology department.

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Sarah Stanhope is a 2020 Magna *** laude
graduate from the Biology Department at

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Salem State.

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While at Salem State,
she completed a senior honors thesis

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working with Jason Brown,
was awarded a Kathy Murphy Endowed

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Biology Research Scholarship,
and served as the President of the Bio

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Society.

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Sarah is currently APHD candidate in the
Department of Biochemistry at Purdue

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University,
working under the direction of Vicki Week.

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She has presented multiple oral
presentations and posters on her work and

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has published 4 journal articles
including her first authored first first

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authored paper in 2023.

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Sarah has won numerous awards for her
work including an NIHT 32 Drug Discovery

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and Development training grant,
the Bird Stare Award,

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a first place poster presentation award
at Hitchhiker's Guide to the Bio

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Molecular Galaxy in 2023.

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Most excitingly,
Sarah just found out this week that she

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was awarded A prestigious Kurt Stein
NRSAF 31 Individual Predoctoral

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Fellowship.

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We are so happy to welcome Sarah Stone
Stanhope back for her talk entitled

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Prolonged Blue light exposure Alters
Phototransduction Efficiency and one

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Carbon Metabolism Processes in the
Drosophila Eye.

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Welcome, Sarah.

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Thank you so much for that lovely
introduction.

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I'm so excited to be here with you all
today and to share a little bit about my

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work of what I've been doing at Purdue.

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So I apologize about that.

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I'm just going to get the laser pointer
out so you guys can see where I'm

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directing the conversation.

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So today I'm going to be talking to you a
little bit about my thesis reach research

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entitled Prolonged blue light exposure
alters phototransduction efficiency in

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one carbon metabolism processes in
Drosophila eye.

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So just to give you a brief outline today
of what I'm going to be going through is

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I'd like to start talking a little bit
about eye disease and using Drosophila as

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a model Organism in case any of you are
unfamiliar with that.

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I'm then going to touch on some exciting
data that we've recently had come out of

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the lab focusing on oxidation of
phototransduction proteins and machinery.

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And then I'm going to finish off with
sharing a little bit about my thesis

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focus,
which is characterizing an enzyme known

c94435c1-a24b-493e-a262-e02a366509dc-2
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as S adenosyl homocystinase or HCI for
short, as a redox regulated enzyme.

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So to start,
I'd like to introduce my lab and one of

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the major focuses of our research.

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So I'm doing my PhD at Purdue University
in Doctor Vicki Week's lab.

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And one of our focuses is studying aging
and specifically age within the context

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of ocular disease,
primarily retinal degeneration.

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And this is because age is a major risk
factor for lots of types of visual

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impairment.

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So if you look at this graph that I have
displayed on the left along the bottom

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you'll see years and then on the X axis
you'll see percentage of vision loss and

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blindness.

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And what we know as we age is that once
people start to hit around age 60,

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we see a drastic increase in the percent
of vision loss and blindness.

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And as well,
studies done by the National Institute of

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Health have shown that aging associated
ocular disease,

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including age-related macular
degeneration, cataracts,

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diabetic retinopathy and glaucoma are all
predicted to double in occurrence by 2050.

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And now a little bit about Drosophila and
why we use the fruit fly as an aging

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model Organism.

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So one of the biggest advantages of using
Drosophila is that they can age quite

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rapidly as opposed to say,
doing mouse work.

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Whereas if you're studying mice,
aging studies may take up to years.

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While working with Drosophila,
we're comparing this several weeks worth

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of aging.

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So just to Orient you all a little bit
for Drosophila aging,

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we count day 10 as relatively young,
whereas day 30 to 40 are considered to be

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middle-aged and day 50 to 60 are
considered to be late aged Drosophila.

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And some of the things that we study
include visual function.

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And what we know with that is that in
young flies visual function tends to be

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quite good.

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However, as the fly ages,
we see a drastic decline in their visual

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function and that can mean response to
light or the health of their eyes.

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What we also know is that with age we see
increased changes in transcriptional

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changes or transcriptomics and we also
see increased prevalence of retinal

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degeneration.

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However,
today I'm going to be focusing on a

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secondary model apart from aging.

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And this is because although aging is a
major risk factor,

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it's not the only risk factor for the
development of eye diseases.

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And we have what's called environmental
risk factors.

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These are things outside of your body
that are affecting you and your eye

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health.

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Some of these can include UV radiations
or just natural light exposure, smoking,

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bacteria, pollutants, chemicals,
or even changes in the temperature.

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One thing that environmental risk factors
and aging have in common is the

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accumulation of reactive oxygen species,
abbreviated here as Ros.

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Some of the most common active oxygen
species that you might be familiar with

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could be hydroxyl radicals,
abbreviated as OH,

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or hydrogen peroxide H2O2.

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Now in your cells,
these are naturally present and they're

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oftentimes used as signaling molecules.

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But what can happen is that these can
increase in abundance within your cell

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due to exogenous or endogenous sources.

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Exogenous sources are things outside of
you that are affecting you.

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Light temperature changes is an oxygen
availability,

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whereas endogenous sources are things
that are coming from within your cells.

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This can be like aging,
inflammation or infection.

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But our cells are quite smart and they
developed systems in place that keep

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reactive oxygen species in check or in
balance.

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These are known as antioxidants.

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Antioxidants mainly have two main systems,
either non enzymatic or enzymatic,

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but for the purpose of this talk I would
like to focus on our non enzymatic

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systems,
particularly an antioxidant known as

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glutathione.

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Glutathione is one of the major
antioxidants in your cell and it works to

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combat reactive oxygen species.

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And you can think of antioxidants and
reactive oxygen species as being on a

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scale where in general if you're not in a
stressful condition these are balanced.

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However,
if you're under sources of stress,

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either exogenous or endogenous,
you can get accumulation or buildup of

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reactive oxygen species to the point
where your antioxidants are no longer

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able to balance that.

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And when that happens,
this is known as oxidative stress.

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And oxidative stress can cause damage to
macromolecules.

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Now,
just to focus back on Drosophila and to

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give you an overview of the type of
tissue that we're working with in my lab,

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because we study ocular disease,
we're very focused on stress within the

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Drosophila eye.

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So here on the left,
I'm showing you a picture of a male fruit

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fly with a focus on its eye.

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Now obviously Drosophila in humans have
different types of eyes where insects

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have a compound eye,
and this is what a zoomed in figure of

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the Drosophila eye would look like.

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And as you can see,
these white dots all look like individual

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subunits.

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Each one of these dots is known as an
Amitidium.

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An Amitidium is a single repeat is a
single unit that houses what we call

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Rabdomirs.

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If you're unfamiliar with Rabdomirs,
you can think of them a photoreceptor

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cell for humans.

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And on the outside where you see R1
through R6,

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these are known as the outer Rabd mirrors,
and they're needed for motion vision,

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whereas in the center R7,
this is needed for color vision.

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So each one of these Amitidium have 7
Rabd mirrors, but there's 700,

bdaecb66-5cba-4b57-ac7f-5730f4ef4243-1
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about 750 of these repeating units that
make up the compound eye.

87defb52-3c59-440f-a01e-e4f2f5484fed-0
00:08:34.400 --> 00:08:38.912
And for the first portion of my talk,
I'm going to be introducing some

87defb52-3c59-440f-a01e-e4f2f5484fed-1
00:08:38.912 --> 00:08:43.680
interesting phenomenon that we've seen
within phototransduction machinery.

d1a4e4f7-4d93-44ed-bf18-fe2ec684d1fb-0
00:08:43.920 --> 00:08:47.981
So I'd like to introduce a simplified
version of what the Drosophila

d1a4e4f7-4d93-44ed-bf18-fe2ec684d1fb-1
00:08:47.981 --> 00:08:50.160
phototransduction cascade looks like.

f115dd17-64e3-4a4b-ae5a-fc6b1ae5827a-0
00:08:50.520 --> 00:08:55.978
Now Drosophila phototransduction has been
very well characterized through the years

f115dd17-64e3-4a4b-ae5a-fc6b1ae5827a-1
00:08:55.978 --> 00:09:01.176
because they are they have incredibly
sensitive phototransduction machinery and

f115dd17-64e3-4a4b-ae5a-fc6b1ae5827a-2
00:09:01.176 --> 00:09:04.166
Drosophila can actually sense single
photons,

f115dd17-64e3-4a4b-ae5a-fc6b1ae5827a-3
00:09:04.166 --> 00:09:08.000
which is much faster than our vertebrate
rods in our eyes.

593ed4c4-b965-4bf2-8997-45ff0cd618f4-0
00:09:08.600 --> 00:09:13.922
But how phototransduction works within
Drosophila is that it is initiated by

593ed4c4-b965-4bf2-8997-45ff0cd618f4-1
00:09:13.922 --> 00:09:17.240
light and specifically a blue light
wavelength.

f26b6649-fd5b-4dc0-9642-e5c40db17f28-0
00:09:17.520 --> 00:09:22.960
So the blue light wavelength will
activate a light sensing protein,

f26b6649-fd5b-4dc0-9642-e5c40db17f28-1
00:09:22.960 --> 00:09:25.120
abbreviated here as RH one.

9dc8b8d8-6edd-4db2-9f89-ae17599fdf57-0
00:09:25.360 --> 00:09:29.120
If any of you have taken upper level
biology classes,

9dc8b8d8-6edd-4db2-9f89-ae17599fdf57-1
00:09:29.120 --> 00:09:34.691
you may have heard of a type of protein
called AG Protein Coupled Receptor or a

9dc8b8d8-6edd-4db2-9f89-ae17599fdf57-2
00:09:34.691 --> 00:09:35.039
GPCR.

3ce1b9dd-e649-4b62-936f-257f0cf958ce-0
00:09:35.400 --> 00:09:39.349
This is characterized by having a
transmembrane as well as the three

3ce1b9dd-e649-4b62-936f-257f0cf958ce-1
00:09:39.349 --> 00:09:42.040
subunits on the bottom, alpha,
beta and gamma.

0536e230-dbf3-4a8d-a2ee-ba5047e0af21-0
00:09:42.600 --> 00:09:46.320
So what happens when light activates RH
One,

0536e230-dbf3-4a8d-a2ee-ba5047e0af21-1
00:09:46.320 --> 00:09:51.280
its alpha subunit will disassociate and
bind to a PLC beta.

1c87d10c-1f27-4e56-892c-ba5565991c8d-0
00:09:51.480 --> 00:09:56.680
This stands for phospholipase C beta,
and in Drosophila we call it Norp A.

538def23-43b4-4cb1-89d4-12e7415468f9-0
00:09:57.040 --> 00:10:03.200
Now Norp A breaks down lipid molecules
like PIP 2 into a secondary messenger

538def23-43b4-4cb1-89d4-12e7415468f9-1
00:10:03.200 --> 00:10:04.240
known as DAG.

5b9aa1cd-5996-47f5-893c-1f20637f59dc-0
00:10:05.000 --> 00:10:10.191
Another important protein within the
phototransduction cascade is known as Ena

5b9aa1cd-5996-47f5-893c-1f20637f59dc-1
00:10:10.191 --> 00:10:12.360
D Ena D is a scaffolding protein.

e641d98e-6221-4630-ba5f-17d1030060c3-0
00:10:12.600 --> 00:10:16.837
Scaffolding proteins are important
because they hold a lot of proteins in

e641d98e-6221-4630-ba5f-17d1030060c3-1
00:10:16.837 --> 00:10:20.560
close proximity to each other,
so they're able to work together.

ad9c136f-b8b5-4794-b0d9-eb1a3d8772ec-0
00:10:20.840 --> 00:10:27.320
So Ena D can bind to NORP A as well as
this channel right here known as TRIP.

b22c2f47-bee1-4226-a901-ffe32919e887-0
00:10:27.560 --> 00:10:33.912
The trip channel is a calcium ion channel,
so when open it allows for calcium to

b22c2f47-bee1-4226-a901-ffe32919e887-1
00:10:33.912 --> 00:10:35.560
influx into the cell.

b0deb124-7761-4610-a8ba-ac36c8a2af59-0
00:10:36.240 --> 00:10:40.234
So this would be phototransduction
initiation on panel B,

b0deb124-7761-4610-a8ba-ac36c8a2af59-1
00:10:40.234 --> 00:10:44.160
I'm showing an example of
phototransduction termination.

12c7558e-f834-48f9-8603-17351599b20a-0
00:10:44.400 --> 00:10:49.872
So what we would see here is that alpha
subunit that was originally from this RH

12c7558e-f834-48f9-8603-17351599b20a-1
00:10:49.872 --> 00:10:53.520
one light sensing protein disassociates
from north A.

326660e0-cc3b-45dd-86be-ba2882e79c77-0
00:10:54.440 --> 00:10:56.560
And then we have a new player come in.

4227c70d-bafb-4008-82c3-7d870bf49efc-0
00:10:56.560 --> 00:11:02.800
This is a protein kinase C or PKC for
short, known as Ena C in Drosophila.

d5f66372-d39d-4f7c-bb3f-b8b8e4117c4f-0
00:11:03.160 --> 00:11:07.389
Ena C needs both DAG,
which can be produced by Norp A,

d5f66372-d39d-4f7c-bb3f-b8b8e4117c4f-1
00:11:07.389 --> 00:11:11.464
and calcium,
which is coming from the influx through

d5f66372-d39d-4f7c-bb3f-b8b8e4117c4f-2
00:11:11.464 --> 00:11:17.462
the Chip channel to phosphorylate other
protein targets such as Ena D and the

d5f66372-d39d-4f7c-bb3f-b8b8e4117c4f-3
00:11:17.462 --> 00:11:19.000
trip channel itself.

4b453681-f6f7-47c1-ad37-415f48280a9b-0
00:11:19.360 --> 00:11:23.853
Now there's a lot of open-ended questions
when it comes to Drosophila termination

4b453681-f6f7-47c1-ad37-415f48280a9b-1
00:11:23.853 --> 00:11:26.320
as this hasn't been fully characterized
yet.

94fa2781-1b23-4218-9c01-c118ab230047-0
00:11:26.600 --> 00:11:31.738
But we do know that Ena C is an important
part of the shutting down of the

94fa2781-1b23-4218-9c01-c118ab230047-1
00:11:31.738 --> 00:11:33.520
phototransduction pathway.

6506eaf9-7f6e-4f5b-92db-a8f4266cd229-0
00:11:35.040 --> 00:11:41.520
So the model that I've been working with
in my lab is a prolonged blue light model.

0da4fb42-3ae2-4c1d-84ce-4bd119ceac41-0
00:11:41.760 --> 00:11:47.819
And that's because we know that that blue
light wavelength is what activates RH one

0da4fb42-3ae2-4c1d-84ce-4bd119ceac41-1
00:11:47.819 --> 00:11:52.291
into its active form,
which can either lead to light response

0da4fb42-3ae2-4c1d-84ce-4bd119ceac41-2
00:11:52.291 --> 00:11:54.600
or endocytosing out of the cell.

d6d8e302-2e3b-4a90-ab1f-c0ca498646b3-0
00:11:55.000 --> 00:11:59.031
And we also know that if the orange
wavelength is shown,

d6d8e302-2e3b-4a90-ab1f-c0ca498646b3-1
00:11:59.031 --> 00:12:03.840
that the active form of RH one will then
go back to being inactive.

6f96aa84-f068-4c59-9660-7b5cd8e7dc64-0
00:12:04.080 --> 00:12:09.215
So we know that using prolonged blue
light as a model can induce an activation

6f96aa84-f068-4c59-9660-7b5cd8e7dc64-1
00:12:09.215 --> 00:12:11.360
of the phototransduction cascade.

f1539ad6-1028-4249-8a5f-033fce1b604a-0
00:12:11.600 --> 00:12:15.520
And this is also important for a lot of
our future studies going forward.

ab5e1884-bf86-472f-8869-a2ded081cb0b-0
00:12:15.720 --> 00:12:20.540
But how our blue light machine works in
our lab is we would take a vial that has

ab5e1884-bf86-472f-8869-a2ded081cb0b-1
00:12:20.540 --> 00:12:25.480
a number of flies in it and you can put
it into these light chambers that we have.

41bcff84-4bce-4da9-b465-3fb3e27272fa-0
00:12:25.760 --> 00:12:29.866
They we can either express,
expose the flies to red light or blue

41bcff84-4bce-4da9-b465-3fb3e27272fa-1
00:12:29.866 --> 00:12:30.240
light.

53a74b45-2427-4831-8fab-206d3cefe871-0
00:12:30.480 --> 00:12:35.549
And then we're able to image the fly eye
to see if there's any loss of those

53a74b45-2427-4831-8fab-206d3cefe871-1
00:12:35.549 --> 00:12:39.960
Omotidium or Rabdimirs,
which would indicate retinal degeneration.

1faf6def-0ba8-4706-b85b-58c3e180a756-0
00:12:41.000 --> 00:12:45.783
So I'm going to be showing you all a few
images today of flies exposed to

1faf6def-0ba8-4706-b85b-58c3e180a756-1
00:12:45.783 --> 00:12:47.400
different types of light.

d18b084e-e20c-45e2-b109-dd628f941af9-0
00:12:47.640 --> 00:12:50.428
But just to remind you of that eye
structure,

d18b084e-e20c-45e2-b109-dd628f941af9-1
00:12:50.428 --> 00:12:54.611
we have the outer photoreceptor,
the outer raptomirs, and the inner,

d18b084e-e20c-45e2-b109-dd628f941af9-2
00:12:54.611 --> 00:12:59.400
and you're able to visualize 7 dots
through imaging and that is the Amitidium.

48a7c635-e69f-4211-ad88-802b3ea4deb1-0
00:13:00.120 --> 00:13:03.862
As a control for these experiments,
we typically use flies that have never

48a7c635-e69f-4211-ad88-802b3ea4deb1-1
00:13:03.862 --> 00:13:04.960
been exposed to light.

55750fd1-9710-4d0c-ad75-aae442f94cbc-0
00:13:04.960 --> 00:13:06.800
This is known as a dark control.

2c5dce6e-08d0-498c-8720-bdfb2624b048-0
00:13:07.120 --> 00:13:11.563
And what you're able to see here is that
these armitidium look very healthy with

2c5dce6e-08d0-498c-8720-bdfb2624b048-1
00:13:11.563 --> 00:13:12.880
all 7 rabdomirs present.

a4131a27-2404-4d07-8545-4fc80eedee1d-0
00:13:13.440 --> 00:13:16.760
But what if we show them a different type
of light other than white?

24213efe-5a03-4935-9db2-29b57a557883-0
00:13:17.360 --> 00:13:18.680
What about red light?

922f77e4-064c-4d6e-84ba-ebfe0d61269c-0
00:13:19.160 --> 00:13:24.040
When we show for 8 hours of red light,
we also see a very healthy looking eye

922f77e4-064c-4d6e-84ba-ebfe0d61269c-1
00:13:24.040 --> 00:13:26.480
structure with no retinal degeneration.

e8609ea3-a906-42ae-9e0b-84d0beaf9eef-0
00:13:27.040 --> 00:13:28.600
But what about our blue light?

493f26d3-df7c-49b1-9fbc-355015f00cfc-0
00:13:29.600 --> 00:13:32.945
When we show these flies to 8 hours of
blue light,

493f26d3-df7c-49b1-9fbc-355015f00cfc-1
00:13:32.945 --> 00:13:38.258
we see mass retinal degeneration with
Sometimes when looking for that amitidium,

493f26d3-df7c-49b1-9fbc-355015f00cfc-2
00:13:38.258 --> 00:13:41.800
you're only able to visualize 1 or maybe
2 Rabdimirs.

38d0b3ea-e84d-4a47-9649-1a04e8215f3f-0
00:13:42.080 --> 00:13:47.217
So we know that in young flies,
prolonged blue light will result in

38d0b3ea-e84d-4a47-9649-1a04e8215f3f-1
00:13:47.217 --> 00:13:49.560
premature retinal degeneration.

e1c912d4-2549-43fd-991b-38ca3cada359-0
00:13:49.960 --> 00:13:54.211
Now when I came to summarize some other
things we know about a blue light model

e1c912d4-2549-43fd-991b-38ca3cada359-1
00:13:54.211 --> 00:13:58.356
from several different papers that have
been published by my lab is that blue

e1c912d4-2549-43fd-991b-38ca3cada359-2
00:13:58.356 --> 00:14:01.120
light would lead to premature retinal
degeneration.

0867446b-4083-42ec-acbb-ecad8b9dd11c-0
00:14:01.440 --> 00:14:04.614
We also know that it leads to increased
hydrogen peroxide,

0867446b-4083-42ec-acbb-ecad8b9dd11c-1
00:14:04.614 --> 00:14:07.520
which is one of those common reactive
oxygen species.

b968414a-19eb-4687-8ec9-3d59a4ce3d09-0
00:14:07.800 --> 00:14:10.880
And we also know that it leads to lipid
peroxidation.

e0472a94-9fca-4b9b-9fed-be48eda69f2f-0
00:14:11.120 --> 00:14:15.222
And if you're unfamiliar with that term,
if you remember back to some of the

e0472a94-9fca-4b9b-9fed-be48eda69f2f-1
00:14:15.222 --> 00:14:18.259
courses you may have taken about a
phospholipid bilayer,

e0472a94-9fca-4b9b-9fed-be48eda69f2f-2
00:14:18.259 --> 00:14:20.763
if you have increased reactive oxygen
species,

e0472a94-9fca-4b9b-9fed-be48eda69f2f-3
00:14:20.763 --> 00:14:23.960
it can actually break down that lipid
bilayer, damaging it.

09f919ee-0ac2-49c0-a477-3d39b6151688-0
00:14:24.800 --> 00:14:29.666
And we also know that blue light leads to
increased oxidative stress because we

09f919ee-0ac2-49c0-a477-3d39b6151688-1
00:14:29.666 --> 00:14:32.707
were able to measure glutathione within
the cell,

09f919ee-0ac2-49c0-a477-3d39b6151688-2
00:14:32.707 --> 00:14:35.080
which is one of our major antioxidants.

4bebdd83-712a-432f-9525-1ca8f16a15e6-0
00:14:35.320 --> 00:14:37.945
And we know that there's decreased
glutathione,

4bebdd83-712a-432f-9525-1ca8f16a15e6-1
00:14:37.945 --> 00:14:40.680
which we can tell is indicating oxidative
stress.

360e1b4c-07ca-4a4a-97be-b539a466fba7-0
00:14:41.160 --> 00:14:45.911
So when I came into the lab,
we were very focused on different types

360e1b4c-07ca-4a4a-97be-b539a466fba7-1
00:14:45.911 --> 00:14:49.699
of oxidative stress,
and particularly my protein fell,

360e1b4c-07ca-4a4a-97be-b539a466fba7-2
00:14:49.699 --> 00:14:53.280
My project fell within the topic of
protein damage.

e93fda8e-4a5a-4495-8836-29d9d99ce977-0
00:14:53.640 --> 00:14:57.920
And when we say that oxidative stress can
cause protein damage, it's not as if,

e93fda8e-4a5a-4495-8836-29d9d99ce977-1
00:14:57.920 --> 00:15:00.648
you know,
holes are appearing in these proteins or

e93fda8e-4a5a-4495-8836-29d9d99ce977-2
00:15:00.648 --> 00:15:02.200
they're all being smashed up.

e6c5cadd-4f00-4102-a1d6-bd0f3daa4fb6-0
00:15:02.680 --> 00:15:07.068
A lot of times we are actually talking
about different types of modifications

e6c5cadd-4f00-4102-a1d6-bd0f3daa4fb6-1
00:15:07.068 --> 00:15:11.120
that we can be made on these proteins,
such as oxidative modifications.

973fad42-1fd8-4532-9e6b-cb5f744b623d-0
00:15:11.720 --> 00:15:14.585
Now,
these oxidative modifications can happen

973fad42-1fd8-4532-9e6b-cb5f744b623d-1
00:15:14.585 --> 00:15:17.513
on a variety of different amino acid
residues,

973fad42-1fd8-4532-9e6b-cb5f744b623d-2
00:15:17.513 --> 00:15:21.999
but one of the ones that we are
specifically interested in are cysteine

973fad42-1fd8-4532-9e6b-cb5f744b623d-3
00:15:21.999 --> 00:15:22.560
residues.

1a37d1cd-81f8-433b-9d5e-821e1068efb4-0
00:15:22.840 --> 00:15:28.000
Cysteine residues are important because
on their side chain they have a thiol

1a37d1cd-81f8-433b-9d5e-821e1068efb4-1
00:15:28.000 --> 00:15:31.440
group which is composed of a sulfur and a
hydrogen.

a09108d9-b1b9-4285-bb23-06e6394cb2a2-0
00:15:31.960 --> 00:15:37.327
These style groups are quite important
because reactive oxygen species are able

a09108d9-b1b9-4285-bb23-06e6394cb2a2-1
00:15:37.327 --> 00:15:42.560
to either readily oxidize this style
group into another type of modification.

c96fe535-803a-4ed8-baa2-5606e62af6d3-0
00:15:42.720 --> 00:15:47.404
Some some of the ones you might you might
be most familiar with are intermolecular

c96fe535-803a-4ed8-baa2-5606e62af6d3-1
00:15:47.404 --> 00:15:50.000
disulfides or intramolecular disulfide
bonds.

4881a5ee-e111-4df7-9c65-187c813494c0-0
00:15:50.440 --> 00:15:55.086
But these modifications can also be
readily reversed back to the original

4881a5ee-e111-4df7-9c65-187c813494c0-1
00:15:55.086 --> 00:15:55.400
file.

adb39b86-ca58-4672-bca3-872f9924cb0b-0
00:15:55.640 --> 00:15:59.690
So you can think of a cysteine as like a
molecular switch where it's responsible

adb39b86-ca58-4672-bca3-872f9924cb0b-1
00:15:59.690 --> 00:16:03.240
for sensing the environment and changing
to different types of states.

5c34d813-d30a-4f51-a710-ab712d155ce4-0
00:16:03.560 --> 00:16:06.200
A lot of this is known as redox signaling.

38c70eb1-ff98-44f4-adf5-f3542027bb3b-0
00:16:06.880 --> 00:16:11.764
But what can happen is if you have that
imbalance of your active oxygen species,

38c70eb1-ff98-44f4-adf5-f3542027bb3b-1
00:16:11.764 --> 00:16:16.348
these cysteine modifications can be
further oxidized and you can get what's

38c70eb1-ff98-44f4-adf5-f3542027bb3b-2
00:16:16.348 --> 00:16:18.640
known as an irreversible modification.

82a8d309-cf22-4800-bf2d-b3455a5dc767-0
00:16:18.880 --> 00:16:21.760
This can either be sulfonic or sulfonic
acid.

2e111fb6-f3ec-42ff-bc5b-cf218c5bacf7-0
00:16:22.000 --> 00:16:26.464
And these types are what lead to cellular
damage because they can no longer be

2e111fb6-f3ec-42ff-bc5b-cf218c5bacf7-1
00:16:26.464 --> 00:16:29.120
reverted back to the cysteine's original
file.

c6e865d4-89f5-4cb2-84f5-1745a7fe8e3e-0
00:16:29.720 --> 00:16:34.125
So one of the major questions that we
asked upon joining the lab is what

c6e865d4-89f5-4cb2-84f5-1745a7fe8e3e-1
00:16:34.125 --> 00:16:38.953
proteins specifically are susceptible to
this type of oxidative damage at their

c6e865d4-89f5-4cb2-84f5-1745a7fe8e3e-2
00:16:38.953 --> 00:16:40.040
cysteine residues.

2fb7b898-2d07-4708-985d-11a8510648d3-0
00:16:40.560 --> 00:16:45.011
And how we did that is through a
technique called redox profiling of the

2fb7b898-2d07-4708-985d-11a8510648d3-1
00:16:45.011 --> 00:16:45.560
proteome.

af3e370d-0d2e-4873-a0f8-4c1a45148b49-0
00:16:46.120 --> 00:16:49.376
So what we did here is we exposed flies
to blue light,

af3e370d-0d2e-4873-a0f8-4c1a45148b49-1
00:16:49.376 --> 00:16:53.048
we manually dissected their eyes,
extracted the proteins out,

af3e370d-0d2e-4873-a0f8-4c1a45148b49-2
00:16:53.048 --> 00:16:56.720
and then performed a little bit of some
complicated labeling.

3318aa3e-a9fc-4d36-8357-273e619a4f45-0
00:16:57.200 --> 00:17:00.168
So I'm going to just briefly touch on how
this labeling works,

3318aa3e-a9fc-4d36-8357-273e619a4f45-1
00:17:00.168 --> 00:17:04.080
but if you have more specific questions
about it later, I'd be happy to take them.

d53f5506-b7db-4ec2-9631-303be6a885f0-0
00:17:04.080 --> 00:17:07.040
Or if you have one that you'd like to
e-mail, that's fine as well.

90d85656-4b47-4181-98a2-2fcf6bc5fd86-0
00:17:07.560 --> 00:17:11.416
But how this labeling works is once we
extract the proteins,

90d85656-4b47-4181-98a2-2fcf6bc5fd86-1
00:17:11.416 --> 00:17:15.716
if a cysteine is reduced,
meaning it has that original thiol group,

90d85656-4b47-4181-98a2-2fcf6bc5fd86-2
00:17:15.716 --> 00:17:17.360
then A tag can bind to it.

4732d171-0cb8-4481-b27b-4dbc8c943cc6-0
00:17:18.000 --> 00:17:23.820
But if a cysteine is in a different type
of oxidative modification that meaning

4732d171-0cb8-4481-b27b-4dbc8c943cc6-1
00:17:23.820 --> 00:17:28.840
that it's no longer in that original
thiol state, A tag cannot bind.

df0bbc8e-0988-4fad-b80e-83d2c98400db-0
00:17:29.240 --> 00:17:34.272
So we do this type of labeling with all
of the proteins we extracted from the eye,

df0bbc8e-0988-4fad-b80e-83d2c98400db-1
00:17:34.272 --> 00:17:38.638
and then we're able to identify which
proteins we isolated using liquid

df0bbc8e-0988-4fad-b80e-83d2c98400db-2
00:17:38.638 --> 00:17:40.639
chromatography mass spectrometry.

7555f2f7-0704-42b7-ad05-6841b9fdb0e6-0
00:17:40.640 --> 00:17:45.773
And we do this with an incredible group
at the Indiana School of Medicine in

7555f2f7-0704-42b7-ad05-6841b9fdb0e6-1
00:17:45.773 --> 00:17:46.640
Indianapolis.

7d0e0844-060a-45a0-b91e-c3f33fda6506-0
00:17:47.120 --> 00:17:52.813
And essentially what this data is looking
at is changes in cysteine availability

7d0e0844-060a-45a0-b91e-c3f33fda6506-1
00:17:52.813 --> 00:17:54.360
through tag abundance.

17f6cc23-f9fb-4522-951b-5d7e81a0fcc8-0
00:17:54.840 --> 00:18:00.132
So how this is going to look is if you
have a decreased amount of these tags,

17f6cc23-f9fb-4522-951b-5d7e81a0fcc8-1
00:18:00.132 --> 00:18:05.696
this is going to correlate with potential
oxidation because that means a cysteine

17f6cc23-f9fb-4522-951b-5d7e81a0fcc8-2
00:18:05.696 --> 00:18:09.359
was not reduced or wasn't available to
bind that tag.

637ae2b1-af7f-4a87-95ac-21837776e520-0
00:18:09.360 --> 00:18:10.840
So you're going to see less of it.

29991e80-eeb1-4574-9131-e7d935ccd7dd-0
00:18:11.440 --> 00:18:14.418
Whereas if you see an increase in the
number of tags,

29991e80-eeb1-4574-9131-e7d935ccd7dd-1
00:18:14.418 --> 00:18:18.223
this could correlate with potential
reduction because that means the

29991e80-eeb1-4574-9131-e7d935ccd7dd-2
00:18:18.223 --> 00:18:22.359
cysteines were in their original thiol
state and the tag was able to bind.

081c35ef-7ac1-4025-8268-b38493dffbbd-0
00:18:23.000 --> 00:18:27.960
So we're able to summarize all of this
proteomic data in a volcano plot.

c9e15048-965c-401c-a427-86901ad2af79-0
00:18:28.240 --> 00:18:32.170
If you're unfamiliar with looking at
volcano plots, to simplify it here,

c9e15048-965c-401c-a427-86901ad2af79-1
00:18:32.170 --> 00:18:36.478
anything that is shown on the left side
is going to be considered significantly

c9e15048-965c-401c-a427-86901ad2af79-2
00:18:36.478 --> 00:18:39.009
oxidized,
whereas anything that's shown on the

c9e15048-965c-401c-a427-86901ad2af79-3
00:18:39.009 --> 00:18:42.239
right side is going to be considered
significantly reduced.

bd7b75d7-56f9-49cd-8698-e46f651ed566-0
00:18:43.600 --> 00:18:48.675
So when we were looking at this data,
we were able to identify that blue light

bd7b75d7-56f9-49cd-8698-e46f651ed566-1
00:18:48.675 --> 00:18:53.430
resulted in significant oxidation of
several phototransduction machinery,

bd7b75d7-56f9-49cd-8698-e46f651ed566-2
00:18:53.430 --> 00:18:58.570
including some of those important key
players that I mentioned earlier like Ena

bd7b75d7-56f9-49cd-8698-e46f651ed566-3
00:18:58.570 --> 00:19:01.589
C and Ena D,
which are also shown here in this

bd7b75d7-56f9-49cd-8698-e46f651ed566-4
00:19:01.589 --> 00:19:06.280
repeated schematic that I showed before
of Drosophila phototransduction.

86c190df-b592-4727-8582-78907346e9d3-0
00:19:06.600 --> 00:19:11.683
So just to show you again Ena C,
which is that protein kinase C we

86c190df-b592-4727-8582-78907346e9d3-1
00:19:11.683 --> 00:19:17.828
identified as having several oxidative
modifications and again Ena CS role is to

86c190df-b592-4727-8582-78907346e9d3-2
00:19:17.828 --> 00:19:22.760
use both DAG and calcium to phosphorylate
other protein targets.

a0b8598d-7ff7-4461-89a4-92baf64851c1-0
00:19:23.120 --> 00:19:27.634
We also identified oxidation of that
important scaffolding protein Ena D,

a0b8598d-7ff7-4461-89a4-92baf64851c1-1
00:19:27.634 --> 00:19:31.477
which is that protein responsible for
holding all of the other

a0b8598d-7ff7-4461-89a4-92baf64851c1-2
00:19:31.477 --> 00:19:35.320
phototransduction machinery in close
proximity to one another.

7554bf59-1937-427c-8f1b-37ced5737636-0
00:19:36.120 --> 00:19:40.194
Now it's one thing to identify these
oxidative modifications,

7554bf59-1937-427c-8f1b-37ced5737636-1
00:19:40.194 --> 00:19:44.400
but we don't really know what that means
in a functional sense.

552ddacc-a247-4ff5-86d6-74d79f0dbe8f-0
00:19:44.800 --> 00:19:49.339
So one of the follow up questions that we
had from this study is if cysteine

552ddacc-a247-4ff5-86d6-74d79f0dbe8f-1
00:19:49.339 --> 00:19:52.699
availability changes in these
phototransduction protein,

552ddacc-a247-4ff5-86d6-74d79f0dbe8f-2
00:19:52.699 --> 00:19:57.062
then what is happening to the efficiency
of phototransduction under these

552ddacc-a247-4ff5-86d6-74d79f0dbe8f-3
00:19:57.062 --> 00:19:59.715
conditions,
meaning the blue light exposure,

552ddacc-a247-4ff5-86d6-74d79f0dbe8f-4
00:19:59.715 --> 00:20:03.960
Is there a functional consequence for
this oxidation that we're seeing?

eeaa3f19-2b0c-4973-8fff-8aa13232a21c-0
00:20:05.320 --> 00:20:11.335
And one way that we're able to do this is
or evaluate these functional consequences

eeaa3f19-2b0c-4973-8fff-8aa13232a21c-1
00:20:11.335 --> 00:20:15.560
is through a technique called Electro
retinograms or Ergs.

076a7801-86e2-44c6-bf00-bf04fbe4004e-0
00:20:15.800 --> 00:20:20.800
Now Ergs measure the electrical activity
of the retina in response to light.

ed5d1ea7-2922-46eb-8431-684b17ecefaa-0
00:20:21.080 --> 00:20:23.680
So this can actually be done in live
flies.

2040765c-d6e3-4342-b477-e3b4f356ae2e-0
00:20:23.800 --> 00:20:27.704
So over here on the left,
I'm showing a family that has been

2040765c-d6e3-4342-b477-e3b4f356ae2e-1
00:20:27.704 --> 00:20:32.440
mounted to a slide and inserted is a
metal electrode into the fly retina.

81b84f08-1f36-423f-b89b-6f1b38023094-0
00:20:32.600 --> 00:20:36.000
And that's how you're able to measure
those electrical responses.

a4bd59e4-f2a8-4ff2-a9d0-255a1a36b7f3-0
00:20:36.440 --> 00:20:38.937
So before I go into what this data looked
like,

a4bd59e4-f2a8-4ff2-a9d0-255a1a36b7f3-1
00:20:38.937 --> 00:20:41.800
I just want to prepare you for what it
will look like.

a1448e99-783b-4008-8fd3-1f22387fe03a-0
00:20:42.000 --> 00:20:45.926
So when we're looking at ER GS,
there are three main steps that we're

a1448e99-783b-4008-8fd3-1f22387fe03a-1
00:20:45.926 --> 00:20:46.600
looking for.

2f9c10d8-973a-4479-ad26-a1f076f57b88-0
00:20:46.840 --> 00:20:52.168
We're looking for this on transient,
which is called the hyperpolarization of

2f9c10d8-973a-4479-ad26-a1f076f57b88-1
00:20:52.168 --> 00:20:52.920
the neuron.

facfbd3e-1eaa-4af1-a6bc-bba5345ab101-0
00:20:53.400 --> 00:20:56.160
We're also looking at receptor potential.

29dab1d1-2777-46ec-a9d5-d5d5cb27b7a0-0
00:20:56.160 --> 00:20:58.160
So this is depolarization.

57878f10-a1ba-485a-a15e-721fd1c7fdf3-0
00:20:58.520 --> 00:21:01.235
And then we're looking for the sharp peak
at the end,

57878f10-a1ba-485a-a15e-721fd1c7fdf3-1
00:21:01.235 --> 00:21:03.800
which is known as the off term,
the off transient.

ec5a3d7e-5b96-4f3f-b94e-a78a4692ad8b-0
00:21:04.040 --> 00:21:06.560
This is the termination response.

36172e10-7758-446f-9a6f-3b7ea894678d-0
00:21:06.760 --> 00:21:12.378
You will then see this return back to
baseline and it will repeat again when

36172e10-7758-446f-9a6f-3b7ea894678d-1
00:21:12.378 --> 00:21:13.400
showing light.

4f97ebda-3bd9-4c43-9e11-09cf376cbc3b-0
00:21:14.120 --> 00:21:19.924
So one of the questions that we had is
for flies that were exposed to blue light,

4f97ebda-3bd9-4c43-9e11-09cf376cbc3b-1
00:21:19.924 --> 00:21:23.960
how does this erg change with increased
light intensity?

22a48cbc-e7aa-4183-8bb6-445400409560-0
00:21:24.880 --> 00:21:28.400
So this data can be a lot to look at
upfront.

b850d02e-32d4-41f3-a985-a2ca5e485ed1-0
00:21:28.400 --> 00:21:31.150
So I'm going to walk you through it quite
slowly,

b850d02e-32d4-41f3-a985-a2ca5e485ed1-1
00:21:31.150 --> 00:21:35.000
and I'd like to start by noting that this
is in a negative log scale.

75214684-d5a1-4f71-9541-f8b34ca0ec2d-0
00:21:35.160 --> 00:21:39.098
What this means for the data is that as
you read left to right,

75214684-d5a1-4f71-9541-f8b34ca0ec2d-1
00:21:39.098 --> 00:21:43.406
the light intensity increases,
which means that these flies are being

75214684-d5a1-4f71-9541-f8b34ca0ec2d-2
00:21:43.406 --> 00:21:46.360
shown brighter and brighter and brighter
light.

5228772f-ebf4-4e7d-9cfa-39614028d538-0
00:21:46.680 --> 00:21:50.896
And as I mentioned before,
Drosophila phototransduction is a much

5228772f-ebf4-4e7d-9cfa-39614028d538-1
00:21:50.896 --> 00:21:55.560
faster process than humans are,
and they can sense even a single photon.

62b061c7-604b-4c2e-80cd-17af195d9de4-0
00:21:55.800 --> 00:21:59.151
So all though we're seeing an increase in
the light intensity,

62b061c7-604b-4c2e-80cd-17af195d9de4-1
00:21:59.151 --> 00:22:02.823
if I were to show this to you,
it would almost be as if there was no

62b061c7-604b-4c2e-80cd-17af195d9de4-2
00:22:02.823 --> 00:22:06.920
change in the light because we can't
respond as fast as the fruit flies can.

e40aacc7-0275-474b-8ea1-16e984a37ad3-0
00:22:07.640 --> 00:22:12.040
So we're going to start here along the
bottom with this white light illumination.

69cc7ff1-8b7b-4145-9d1e-0c60bbde849b-0
00:22:12.320 --> 00:22:17.120
So each of this,
each of these ER GS is an individual fly.

337049b1-a8a4-4974-9cbb-bdd2c5f5a07a-0
00:22:17.360 --> 00:22:23.174
This is done on live flies and that were
either exposed to white light or to blue

337049b1-a8a4-4974-9cbb-bdd2c5f5a07a-1
00:22:23.174 --> 00:22:26.436
light,
which is our experimental prior to the

337049b1-a8a4-4974-9cbb-bdd2c5f5a07a-2
00:22:26.436 --> 00:22:26.720
ERG.

2bdd3733-5959-4dd2-999e-2d7ebc46c0a3-0
00:22:27.160 --> 00:22:31.040
Flies are dark adapted so they're kept in
a space with no light.

88c3e17a-e2d9-4269-b351-e310cdd374c7-0
00:22:31.280 --> 00:22:35.343
That way when you're doing this assay,
they are reporting from a baseline and

88c3e17a-e2d9-4269-b351-e310cdd374c7-1
00:22:35.343 --> 00:22:39.511
it's not like they're already responding
to light prior to the beginning of the

88c3e17a-e2d9-4269-b351-e310cdd374c7-2
00:22:39.511 --> 00:22:39.720
erg.

14b7cd6a-f2b4-493d-ad85-150690a811f6-0
00:22:40.560 --> 00:22:45.623
But what we're looking for in this data
is those 3 distinct portions again the on

14b7cd6a-f2b4-493d-ad85-150690a811f6-1
00:22:45.623 --> 00:22:48.896
transient,
the receptor potential in the termination

14b7cd6a-f2b4-493d-ad85-150690a811f6-2
00:22:48.896 --> 00:22:53.157
and for flies that are our control,
which means just the white light

14b7cd6a-f2b4-493d-ad85-150690a811f6-3
00:22:53.157 --> 00:22:53.960
illumination.

737ab36d-73cf-4de9-883a-ccdb2828da2c-0
00:22:54.320 --> 00:22:59.455
We start to see that response around
negative log 5 where you can see the peak

737ab36d-73cf-4de9-883a-ccdb2828da2c-1
00:22:59.455 --> 00:23:04.330
at the top, this little drop off,
which is the receptor potential that off

737ab36d-73cf-4de9-883a-ccdb2828da2c-2
00:23:04.330 --> 00:23:06.800
transient and then return to baseline.

9bd1e87b-f087-4c29-a176-f2ad5538ff10-0
00:23:07.440 --> 00:23:12.160
And as you if you notice as the light
intensity increases,

9bd1e87b-f087-4c29-a176-f2ad5538ff10-1
00:23:12.160 --> 00:23:18.800
meaning as we go more towards the right,
this drop down which we know is amplitude

9bd1e87b-f087-4c29-a176-f2ad5538ff10-2
00:23:18.800 --> 00:23:23.280
keeps going further down,
meaning that the amplitude is

9bd1e87b-f087-4c29-a176-f2ad5538ff10-3
00:23:23.280 --> 00:23:26.160
proportional to the light intensity.

50d98ab2-67b4-493a-9d0b-6fc91c99270e-0
00:23:26.160 --> 00:23:33.497
So we expect this to continuously the we
expect the amplitude to go down further

50d98ab2-67b4-493a-9d0b-6fc91c99270e-1
00:23:33.497 --> 00:23:35.400
as we read the graph.

96912a27-1eef-44af-befc-fb8250696be1-0
00:23:36.040 --> 00:23:37.720
Now what about blue light?

add0d9ee-69bf-45d2-86ec-040d03840c49-0
00:23:37.720 --> 00:23:39.960
So this is testing that functional
consequence.

6cff261b-8928-453c-ad63-ab63a70efd52-0
00:23:39.960 --> 00:23:42.760
Does blue light change how the flies
respond to light?

06624ae5-d8f7-4102-a81d-e32b2d4943ed-0
00:23:43.360 --> 00:23:47.787
So if we look at negative log flat 5
where we saw the white light illumination

06624ae5-d8f7-4102-a81d-e32b2d4943ed-1
00:23:47.787 --> 00:23:51.206
response starting,
we don't see anything for flies that were

06624ae5-d8f7-4102-a81d-e32b2d4943ed-2
00:23:51.206 --> 00:23:52.439
exposed to blue light.

db3e1334-cf2f-4c58-ba27-938afcd20597-0
00:23:52.720 --> 00:23:58.915
And in fact we don't start to see a full
response until we get to about negative

db3e1334-cf2f-4c58-ba27-938afcd20597-1
00:23:58.915 --> 00:23:59.680
log 3 1/2.

71dd5649-989b-4d54-9c68-0eb14b0fbc7b-0
00:23:59.960 --> 00:24:02.200
So their response is starting much later.

7ad7d1d5-f3f2-4da5-83a2-ce02baf85853-0
00:24:02.680 --> 00:24:07.859
And another important fact that we see
when looking at this data is that their

7ad7d1d5-f3f2-4da5-83a2-ce02baf85853-1
00:24:07.859 --> 00:24:11.269
amplitude,
this down portion is not proportional to

7ad7d1d5-f3f2-4da5-83a2-ce02baf85853-2
00:24:11.269 --> 00:24:15.399
the light free intensity as it was in our
white light control.

871235bd-e343-4488-bc6a-fa2df42287e8-0
00:24:15.960 --> 00:24:21.660
So this graph down the bottom where we're
showing the same negative log scale as we

871235bd-e343-4488-bc6a-fa2df42287e8-1
00:24:21.660 --> 00:24:24.781
are up here and we're showing that
amplitude,

871235bd-e343-4488-bc6a-fa2df42287e8-2
00:24:24.781 --> 00:24:27.360
which is this drop down on our X axis.

d698ca7c-7d46-46de-aae7-5fe486ae8ad8-0
00:24:27.960 --> 00:24:33.734
What we can see in the summarized data is
that we have a shift in response time,

d698ca7c-7d46-46de-aae7-5fe486ae8ad8-1
00:24:33.734 --> 00:24:35.160
so delayed response.

96221418-98e1-4891-b949-ce784d8da6fa-0
00:24:35.440 --> 00:24:39.487
And also if you notice that this blue
line runs below the white,

96221418-98e1-4891-b949-ce784d8da6fa-1
00:24:39.487 --> 00:24:42.040
that is showing that decreased amplitude.

65d698fe-6df8-434b-85d3-e2ec580d15a2-0
00:24:43.240 --> 00:24:46.520
So to summarize this data or like what
does this data mean?

7303759a-fda6-43a1-8fe6-6bb2bec88fc8-0
00:24:46.760 --> 00:24:51.320
The ERG is showing decreased sensitivity
and dampened amplitude.

727a2b19-047d-4f3b-b004-04d2098eeb2c-0
00:24:51.640 --> 00:24:56.533
This means that blue light in fact
dampens the flies electrical response to

727a2b19-047d-4f3b-b004-04d2098eeb2c-1
00:24:56.533 --> 00:24:56.920
light.

03ce6825-20f3-4356-a345-17bb404c5ea5-0
00:24:57.280 --> 00:25:01.560
And what we're seeing is something that's
very similar to light adaptation.

da233648-d336-4842-b593-c8f7923bc84f-0
00:25:01.840 --> 00:25:06.659
This means that although the flies were
kept in the dark prior to the erg,

da233648-d336-4842-b593-c8f7923bc84f-1
00:25:06.659 --> 00:25:11.800
they respond to the first point of light
as if they've been responding to light

da233648-d336-4842-b593-c8f7923bc84f-2
00:25:11.800 --> 00:25:13.600
constantly without shopping.

ecc7706e-eccd-43a0-ac35-40251bf8d01c-0
00:25:14.600 --> 00:25:17.120
The next question might be,
why is this important?

85f34af8-a545-4237-99e5-3f92da109ef2-0
00:25:17.600 --> 00:25:21.842
So having the ability to shut the pathway
down or turn it off,

85f34af8-a545-4237-99e5-3f92da109ef2-1
00:25:21.842 --> 00:25:27.026
it's just as important as being able to
turn one on or initiate one to begin

85f34af8-a545-4237-99e5-3f92da109ef2-2
00:25:27.026 --> 00:25:29.720
without terminating the signaling event.

e2c5201e-efd8-4edb-81ee-d5762385323c-0
00:25:30.120 --> 00:25:33.771
The eye is not able to adapt to different
intensities of light,

e2c5201e-efd8-4edb-81ee-d5762385323c-1
00:25:33.771 --> 00:25:37.309
and this means that your
phototransduction pathway can become

e2c5201e-efd8-4edb-81ee-d5762385323c-2
00:25:37.309 --> 00:25:37.880
saturated.

476e7de5-b5dd-4813-965f-e18def811d74-0
00:25:38.200 --> 00:25:42.077
So although there's nothing physically
wrong with the eye,

476e7de5-b5dd-4813-965f-e18def811d74-1
00:25:42.077 --> 00:25:43.720
it is functionally blind.

1316f1b2-9f5b-4c4a-b23f-5dd6e03f125d-0
00:25:45.160 --> 00:25:50.740
So when we were thinking about this data
and knowing that we saw oxidation of

1316f1b2-9f5b-4c4a-b23f-5dd6e03f125d-1
00:25:50.740 --> 00:25:54.030
several different phototransduction
proteins,

1316f1b2-9f5b-4c4a-b23f-5dd6e03f125d-2
00:25:54.030 --> 00:25:59.825
we wanted to know which of these proteins
might contribute to this ERG phenotype

1316f1b2-9f5b-4c4a-b23f-5dd6e03f125d-3
00:25:59.825 --> 00:26:03.760
that we're seeing,
which protein might be responsible.

29643035-296c-495c-a26c-402469ff3c13-0
00:26:04.320 --> 00:26:09.249
And when reading about these proteins,
we started to really focus on Ena C or

29643035-296c-495c-a26c-402469ff3c13-1
00:26:09.249 --> 00:26:10.640
that protein kinase C.

6076877e-2dd0-4d5f-8d2b-0bd707788712-0
00:26:11.240 --> 00:26:14.608
So again,
that's responsible for phosphorylating

6076877e-2dd0-4d5f-8d2b-0bd707788712-1
00:26:14.608 --> 00:26:19.626
one of the substrates is a known
substrate for Ena D and potentially the

6076877e-2dd0-4d5f-8d2b-0bd707788712-2
00:26:19.626 --> 00:26:20.520
trip channel.

e6b1eaca-6906-40ff-8b6a-cf6295293437-0
00:26:20.520 --> 00:26:22.680
But there are also substrates that we
don't know.

1ebbdc4a-a7f0-48b6-8d3d-0bca2885389a-0
00:26:23.280 --> 00:26:28.163
And here on the right,
I'm showing a predicted Drosophila alpha

1ebbdc4a-a7f0-48b6-8d3d-0bca2885389a-1
00:26:28.163 --> 00:26:32.818
fold structure of Ena C And through our
proteomic profiling,

1ebbdc4a-a7f0-48b6-8d3d-0bca2885389a-2
00:26:32.818 --> 00:26:38.160
we found that three individual cysteines
had oxidative modifications.

8f12335f-1537-4559-be7f-0142603ee9a4-0
00:26:38.520 --> 00:26:43.532
And what's interesting is that each of
these oxidative modifications are

8f12335f-1537-4559-be7f-0142603ee9a4-1
00:26:43.532 --> 00:26:49.095
clustered together within a single domain
of Ena C and this happens to be in the

8f12335f-1537-4559-be7f-0142603ee9a4-2
00:26:49.095 --> 00:26:50.400
DAG binding domain.

c8b904d5-460e-430b-8e9a-b8aa28198f27-0
00:26:50.560 --> 00:26:56.556
So Ena C needs to bind DAG and calcium in
order to perform its phosphorylation

c8b904d5-460e-430b-8e9a-b8aa28198f27-1
00:26:56.556 --> 00:26:57.240
activity.

164177f1-4e1a-4426-8256-b42756abd5bd-0
00:26:57.800 --> 00:27:01.900
So we had a question of if the cysteines
are oxidized,

164177f1-4e1a-4426-8256-b42756abd5bd-1
00:27:01.900 --> 00:27:05.480
how might that change ENAC activity in
the eye.

98ab3ba1-e8f0-4f62-9eee-39d81dc7609f-0
00:27:06.280 --> 00:27:12.200
And so we started digging in the
literature and we mammalian PKC's.

86a1f030-f556-4546-962e-c25afbf73952-0
00:27:12.320 --> 00:27:18.450
So human PKC's and Drosophila PKC's are
highly conserved and they have the same

86a1f030-f556-4546-962e-c25afbf73952-1
00:27:18.450 --> 00:27:21.822
domain regions such as a regulatory
domain,

86a1f030-f556-4546-962e-c25afbf73952-2
00:27:21.822 --> 00:27:27.110
which includes that DAG binding domain as
well as its kinase domain,

86a1f030-f556-4546-962e-c25afbf73952-3
00:27:27.110 --> 00:27:28.720
its catalytic domain.

5d5438ab-30bf-4346-b158-f8563386030c-0
00:27:29.280 --> 00:27:35.256
And what we were able to find from
previous studies that came out in the 90s

5d5438ab-30bf-4346-b158-f8563386030c-1
00:27:35.256 --> 00:27:41.077
was that oxidation of cysteines in that
DAG binding domain actually led to

5d5438ab-30bf-4346-b158-f8563386030c-2
00:27:41.077 --> 00:27:46.200
activation of the PKC regardless of
whether or not DAG was bound.

080554b8-38f6-43cc-b4b7-af7665125f3a-0
00:27:46.680 --> 00:27:52.877
So our current working hypothesis for
this the project is that we think that

080554b8-38f6-43cc-b4b7-af7665125f3a-1
00:27:52.877 --> 00:27:57.062
redox signaling or oxidative stress
activates ENAC,

080554b8-38f6-43cc-b4b7-af7665125f3a-2
00:27:57.062 --> 00:28:03.582
that protein kinase C independent of the
PLC beta mediated DAG production or the

080554b8-38f6-43cc-b4b7-af7665125f3a-3
00:28:03.582 --> 00:28:06.560
calcium influx from the trip channel.

aa74ef51-665a-44d4-a7a0-322643da6361-0
00:28:06.800 --> 00:28:11.395
And this might be what's enabling
sustained light up adaptation following

aa74ef51-665a-44d4-a7a0-322643da6361-1
00:28:11.395 --> 00:28:13.320
those bright light intensities.

0dfa3e6e-1200-4cac-9821-2777199dee73-0
00:28:14.040 --> 00:28:19.774
And some new data that we've just
received as well is showing that if you

0dfa3e6e-1200-4cac-9821-2777199dee73-1
00:28:19.774 --> 00:28:26.127
knockout or remove Ena C from the fly eye,
they actually have increased intensity

0dfa3e6e-1200-4cac-9821-2777199dee73-2
00:28:26.127 --> 00:28:28.840
response after blue light exposure.

15d524d0-dad8-45e7-9902-2e4817d4d28c-0
00:28:29.120 --> 00:28:33.960
So we are able to rescue that delayed
response time.

59672921-001e-40b5-bc55-cdf93ddf43d5-0
00:28:34.840 --> 00:28:40.505
Now some of the remaining questions that
we have for this project is how does Ena

59672921-001e-40b5-bc55-cdf93ddf43d5-1
00:28:40.505 --> 00:28:43.960
C contribute to these two intense light
response?

6ff2bf6e-3e73-46c1-8ce0-a665360b1e0f-0
00:28:44.160 --> 00:28:50.181
So we're thinking how could Ena C maybe
change its activity in order to alter the

6ff2bf6e-3e73-46c1-8ce0-a665360b1e0f-1
00:28:50.181 --> 00:28:53.560
termination of our phototransduction
cascade?

86e85d64-6b9c-4372-80be-8d23c175464e-0
00:28:54.120 --> 00:28:57.160
And also how does blue light affect Ena C?

4e8466ac-36d3-49cf-9129-3ef587f42c81-0
00:28:57.400 --> 00:28:59.120
How might this change its structure?

031b0512-f524-4d16-bb25-61a0b314bfe6-0
00:28:59.280 --> 00:29:03.960
We saw that those cysteine modifications
are made within a single domain that DAG

031b0512-f524-4d16-bb25-61a0b314bfe6-1
00:29:03.960 --> 00:29:04.360
domain.

374a3291-8313-4275-8417-eb74e4d54277-0
00:29:04.760 --> 00:29:09.999
Some of the questions that we have are
might these modifications change how that

374a3291-8313-4275-8417-eb74e4d54277-1
00:29:09.999 --> 00:29:10.840
domain looks?

67d416cf-157b-40c0-87c7-f1e6249559ac-0
00:29:10.840 --> 00:29:15.520
Could that domain unfold giving in ACA
different act or a change in activity?

a7de346c-d307-4bc3-8c7e-70da2adafb71-0
00:29:16.080 --> 00:29:21.905
Another question is does blue light make
in AC hyperactive or could blue light

a7de346c-d307-4bc3-8c7e-70da2adafb71-1
00:29:21.905 --> 00:29:24.560
impair protein protein interactions?

29710182-ffea-425f-9f87-b6c71bee1271-0
00:29:24.840 --> 00:29:29.534
As we know Ena D that important
scaffolding protein holds all of the

29710182-ffea-425f-9f87-b6c71bee1271-1
00:29:29.534 --> 00:29:32.800
phototransduction machinery in close
proximity.

0ed0e9fc-abea-4115-b1a3-29f2ba6a44d6-0
00:29:33.040 --> 00:29:35.762
So if we have oxidation of that protein
kinase C,

0ed0e9fc-abea-4115-b1a3-29f2ba6a44d6-1
00:29:35.762 --> 00:29:39.520
could that prevent it from interacting
with the scaffolding protein?

e582220f-6556-4cb5-9cba-c9ae8fd3d929-0
00:29:40.600 --> 00:29:45.560
Another big question are what are Ena CS
phosphorylation substrates?

9662e38e-e2f3-4ce6-be99-83af973372dd-0
00:29:45.800 --> 00:29:47.920
Only some are known, but not all.

eae3a4d0-8b1d-4b18-b464-de57c10b6f8a-0
00:29:48.640 --> 00:29:53.274
And one other big question that we have
is what could initiate this type of redox

eae3a4d0-8b1d-4b18-b464-de57c10b6f8a-1
00:29:53.274 --> 00:29:53.840
signaling.

d4d537a3-aa29-458c-9701-6cb7f0806810-0
00:29:54.160 --> 00:29:59.313
Reactive oxygen species aren't just
floating around in the cell and they have

d4d537a3-aa29-458c-9701-6cb7f0806810-1
00:29:59.313 --> 00:30:03.608
to be produced locally,
which means a protein has to be in close

d4d537a3-aa29-458c-9701-6cb7f0806810-2
00:30:03.608 --> 00:30:07.374
proximity to Ena C,
producing reactive oxygen species in

d4d537a3-aa29-458c-9701-6cb7f0806810-3
00:30:07.374 --> 00:30:12.000
order for Ena C to become oxidized in the
area that we're seeing now.

a4635b2d-64a4-4233-8646-8e94e4576a43-0
00:30:12.000 --> 00:30:17.652
This is currently an open thesis project
for a PhD student at my lab at Purdue

a4635b2d-64a4-4233-8646-8e94e4576a43-1
00:30:17.652 --> 00:30:18.440
University.

60733c80-3b3f-4cfe-b3d6-629b22923251-0
00:30:19.040 --> 00:30:22.805
So if you're interested in grad school or
this project sounds interesting to you,

60733c80-3b3f-4cfe-b3d6-629b22923251-1
00:30:22.805 --> 00:30:26.248
I'd be happy to take questions at the end
or you can e-mail me for further

60733c80-3b3f-4cfe-b3d6-629b22923251-2
00:30:26.248 --> 00:30:26.800
information.

8c458c8a-8de9-4603-ab1c-b145d4db75ec-0
00:30:27.880 --> 00:30:33.043
But for the last portion of my talk,
I would like to touch on what I have been

8c458c8a-8de9-4603-ab1c-b145d4db75ec-1
00:30:33.043 --> 00:30:37.226
studying for my thesis work,
which is characterizing S adenosyl

8c458c8a-8de9-4603-ab1c-b145d4db75ec-2
00:30:37.226 --> 00:30:40.560
homocystinase or AHCY as a redox
regulated enzyme.

da475b15-1135-416c-8d7b-071676f7fc1a-0
00:30:42.200 --> 00:30:47.030
So although blue light heavily affected
phototransduction machinery,

da475b15-1135-416c-8d7b-071676f7fc1a-1
00:30:47.030 --> 00:30:52.700
it was the what was interesting is that
actually our most significantly oxidized

da475b15-1135-416c-8d7b-071676f7fc1a-2
00:30:52.700 --> 00:30:58.090
protein that was identified in Drosophila
eyes and cultured cell lines was a

da475b15-1135-416c-8d7b-071676f7fc1a-3
00:30:58.090 --> 00:31:03.620
metabolic enzyme known as a HCYAHCY is an
important enzyme when we're thinking

da475b15-1135-416c-8d7b-071676f7fc1a-4
00:31:03.620 --> 00:31:06.840
about epigenetic or epigenetic
modifications.

53b615fe-973a-47ea-9608-5be9d2751a50-0
00:31:07.200 --> 00:31:11.289
And for those of you that are unfamiliar
with epigenetics or the modifications

53b615fe-973a-47ea-9608-5be9d2751a50-1
00:31:11.289 --> 00:31:14.861
applications that can take place,
I'm just going to show you a quick

53b615fe-973a-47ea-9608-5be9d2751a50-2
00:31:14.861 --> 00:31:16.880
schematic of what this might look like.

88ed64f2-1aea-4883-8c09-2c43c3ceb8c4-0
00:31:17.200 --> 00:31:22.733
So I'm sure many people know what
chromosomes are, but when you unwind them,

88ed64f2-1aea-4883-8c09-2c43c3ceb8c4-1
00:31:22.733 --> 00:31:26.040
we have another term for it called
chromatin.

5f98aa54-fa9b-4918-a8e5-eb11d9301b1d-0
00:31:26.320 --> 00:31:32.880
And chromatin is made-up of tightly wound
DNA around histone proteins.

be6c7637-6e67-42a2-b6c5-c2cef6be9784-0
00:31:33.160 --> 00:31:38.608
So down here the purple is the tightly
wrapped DNA where the blue is the

be6c7637-6e67-42a2-b6c5-c2cef6be9784-1
00:31:38.608 --> 00:31:39.280
histones.

8064200e-b5fc-4f54-a834-16262671c200-0
00:31:39.720 --> 00:31:41.920
Now histone proteins are quite important.

cb3a30ab-f36d-47de-b322-d6a68441d4e3-0
00:31:42.160 --> 00:31:45.120
They have this feature called a histone
tail.

d43d4b36-ff2a-4bd9-a514-376865922168-0
00:31:45.560 --> 00:31:49.460
Now dependent on what type of
modification is taking place,

d43d4b36-ff2a-4bd9-a514-376865922168-1
00:31:49.460 --> 00:31:53.360
this can have different ramifications for
DNA and histones.

ebb4cca3-8135-436f-a28e-01a751ed41e4-0
00:31:53.760 --> 00:31:58.228
One of the most prevalent modifications
that I'm going to be talking about for

ebb4cca3-8135-436f-a28e-01a751ed41e4-1
00:31:58.228 --> 00:32:01.000
the majority of this talk is called
methylation.

804f2f40-ffd5-46a2-879e-1d70e6399ffa-0
00:32:01.240 --> 00:32:05.680
And for methylation to occur,
you need the addition of a methyl group.

ffdd62d2-ab75-442d-9354-a4a2b2167c45-0
00:32:06.000 --> 00:32:10.242
So what's very interesting about histones
is if they are modified,

ffdd62d2-ab75-442d-9354-a4a2b2167c45-1
00:32:10.242 --> 00:32:14.358
say by a methyl group at a specific spot
in their histone tails,

ffdd62d2-ab75-442d-9354-a4a2b2167c45-2
00:32:14.358 --> 00:32:19.423
it can cause them the they can cause them
to separate and for the DNA to become

ffdd62d2-ab75-442d-9354-a4a2b2167c45-3
00:32:19.423 --> 00:32:20.120
accessible.

9690702b-cc1b-4104-a87e-8adfc82f46d9-0
00:32:20.280 --> 00:32:25.170
Sometimes this will be taught to people
as beads on a string where it can either

9690702b-cc1b-4104-a87e-8adfc82f46d9-1
00:32:25.170 --> 00:32:29.880
be tightly wound or they can separate
where the histones are then spaced out.

22a1369f-8a9b-49b2-8e2f-203f2f1af3a5-0
00:32:30.080 --> 00:32:34.365
Your DNA is accessible in between and
this can lead for active transcription of

22a1369f-8a9b-49b2-8e2f-203f2f1af3a5-1
00:32:34.365 --> 00:32:36.240
the genes and that's located there.

a0c02ffe-233c-40ba-9c18-8929f83d2fa4-0
00:32:36.720 --> 00:32:41.341
But what's important is that if the
modification is made on a different

a0c02ffe-233c-40ba-9c18-8929f83d2fa4-1
00:32:41.341 --> 00:32:45.769
location of the histone tail,
they can actually revert back to being

a0c02ffe-233c-40ba-9c18-8929f83d2fa4-2
00:32:45.769 --> 00:32:50.840
inaccessible or tightly wound DNA where
your gene expression is then inactive.

93618a0d-b656-4ea8-a09b-a99ceab50138-0
00:32:51.320 --> 00:32:54.840
And you might be thinking,
how does this relate to that protein?

32cc9d2f-30a7-4d21-a7a3-f49be6caa95a-0
00:32:54.840 --> 00:32:58.864
I mentioned Hcy,
and that's because Hcy functions in a

32cc9d2f-30a7-4d21-a7a3-f49be6caa95a-1
00:32:58.864 --> 00:33:03.840
very important branch of metabolism known
as one carbon metabolism.

58ec09e2-ec05-4dc5-9d3b-a491bdb32544-0
00:33:04.360 --> 00:33:10.640
One carbon metabolism is what produces
this universal methyl donor known as Sam.

babad07b-bece-4d20-aef0-31cbc01deaad-0
00:33:10.840 --> 00:33:15.040
So how this process works is that you
have your essential amino acid,

babad07b-bece-4d20-aef0-31cbc01deaad-1
00:33:15.040 --> 00:33:18.640
methionine gets broken down to generate
the metabolite Sam.

f265e7ae-5b20-41cf-a324-e794cc8289c4-0
00:33:18.920 --> 00:33:22.920
This M on the end is for the meth,
the methyl group.

c34f81cb-6f57-41fd-a340-6b354962490c-0
00:33:23.400 --> 00:33:28.011
So this is a universal methyl donor for
all methylation reactions in the cell,

c34f81cb-6f57-41fd-a340-6b354962490c-1
00:33:28.011 --> 00:33:32.040
including DNA methylation,
mRNA methylation and histone methylation.

2e3774b0-ce55-40b4-a41c-2d85b0241082-0
00:33:32.320 --> 00:33:37.088
And it's highly abundant within your
cells and it's the second highest used

2e3774b0-ce55-40b4-a41c-2d85b0241082-1
00:33:37.088 --> 00:33:38.280
cofactor after ATP.

53444e81-e249-41d0-a013-204920c05c57-0
00:33:39.320 --> 00:33:44.987
And what's important about Sam is once
that methyl group gets used by proteins

53444e81-e249-41d0-a013-204920c05c57-1
00:33:44.987 --> 00:33:50.151
known as methyl transferases,
it generates A byproduct metabolite known

53444e81-e249-41d0-a013-204920c05c57-2
00:33:50.151 --> 00:33:55.460
as S Adenosyl homocysteine or SAHSAH,
has an opposing role to Sam as it's

53444e81-e249-41d0-a013-204920c05c57-3
00:33:55.460 --> 00:33:58.760
actually an inhibitor of methyl
transferases.

5d9bb43a-6932-4f7c-9689-b2d64222f7cb-0
00:33:59.040 --> 00:34:04.586
So methyl transferases can either bind
Sam to use its methyl group to methylate

5d9bb43a-6932-4f7c-9689-b2d64222f7cb-1
00:34:04.586 --> 00:34:08.400
another substrate or methyl transferases
can bind SAH.

e1ba828c-cc62-47e0-86af-51f007850502-0
00:34:08.400 --> 00:34:12.645
And if they find SAH,
then they are no longer able to methylate

e1ba828c-cc62-47e0-86af-51f007850502-1
00:34:12.645 --> 00:34:13.640
other proteins.

af2d3eed-a868-49f2-85ee-f9bc48a7b4a6-0
00:34:14.080 --> 00:34:19.052
And when we talk about Sam and SCH,
we normally refer to them as a ratio,

af2d3eed-a868-49f2-85ee-f9bc48a7b4a6-1
00:34:19.052 --> 00:34:22.480
and this can also be termed the
methylation index.

45fcf334-411c-462f-9c08-875aa93579f5-0
00:34:22.840 --> 00:34:27.465
And if this ratio is disrupted,
it can actually prevent all methylation

45fcf334-411c-462f-9c08-875aa93579f5-1
00:34:27.465 --> 00:34:29.200
reactions within your cell.

92da5179-4a92-4f2a-be37-0b44a28793af-0
00:34:30.200 --> 00:34:36.400
And HCY has a very important role in this
process by its interaction with SAH.

7c1c8c64-6325-41e4-a1fd-e0d65290702b-0
00:34:37.640 --> 00:34:43.040
Now HCY is one of the most conserved
enzymes across all living species.

5c487db6-9b3c-48ec-ac2c-c10f6b7aef15-0
00:34:43.320 --> 00:34:49.065
And here I'm showing just a rough domain
map of HCY where you have it substrate

5c487db6-9b3c-48ec-ac2c-c10f6b7aef15-1
00:34:49.065 --> 00:34:53.230
binding domain,
it's hinge region which allows it to move

5c487db6-9b3c-48ec-ac2c-c10f6b7aef15-2
00:34:53.230 --> 00:34:55.600
it's and it's NAD binding domain.

6a25a162-54e3-430b-9f68-e9e397835047-0
00:34:55.880 --> 00:34:58.920
So HCY functions as a **** tetramer.

fe60b8e2-7442-4c44-a2ce-07d40d42fb21-0
00:34:59.160 --> 00:35:03.077
So here in panel D I'm showing a
predicted structure of what a monomer

fe60b8e2-7442-4c44-a2ce-07d40d42fb21-1
00:35:03.077 --> 00:35:03.960
would look like.

29b3b132-1f1c-4e04-ae27-b994a3c7ff9f-0
00:35:03.960 --> 00:35:09.694
So one of its subunits and four of these
monomer subunits will come together to

29b3b132-1f1c-4e04-ae27-b994a3c7ff9f-1
00:35:09.694 --> 00:35:14.067
form the **** tetramer,
which is then it's able to be in its

29b3b132-1f1c-4e04-ae27-b994a3c7ff9f-2
00:35:14.067 --> 00:35:18.440
active form and facilitate local
transmethylation reactions.

74c6ddaf-61de-432b-a35c-ff0cc12d4490-0
00:35:19.280 --> 00:35:24.645
And one of the most important things
about HCY is that across all living

74c6ddaf-61de-432b-a35c-ff0cc12d4490-1
00:35:24.645 --> 00:35:28.100
species,
it is the only enzyme to hydrolyze or

74c6ddaf-61de-432b-a35c-ff0cc12d4490-2
00:35:28.100 --> 00:35:31.040
breakdown that inhibitor metabolite SAH.

a7a389da-87d8-436a-9072-304a4d46b462-0
00:35:31.760 --> 00:35:33.680
So this is just a structure of SAH.

032cfa0a-446d-469e-bcea-fa1e7472d471-0
00:35:33.680 --> 00:35:38.803
And when HCY breaks it down,
it breaks it down into two components

032cfa0a-446d-469e-bcea-fa1e7472d471-1
00:35:38.803 --> 00:35:41.480
known as homocysteine or adenosine.

f82a8eef-7df9-4002-a732-3be0e3b2996b-0
00:35:41.720 --> 00:35:44.720
Adenosine is very important for nucleic
acids,

f82a8eef-7df9-4002-a732-3be0e3b2996b-1
00:35:44.720 --> 00:35:49.700
but homocysteine is actually of hot
commodity within your cell and it can get

f82a8eef-7df9-4002-a732-3be0e3b2996b-2
00:35:49.700 --> 00:35:51.359
used in a variety of ways.

f3f418e1-b346-47b8-a911-e6da47a526c4-0
00:35:51.760 --> 00:35:56.305
Two of the most common ways that
homocysteine is needed in the cell is

f3f418e1-b346-47b8-a911-e6da47a526c4-1
00:35:56.305 --> 00:35:59.123
either through the transulfuration
pathway,

f3f418e1-b346-47b8-a911-e6da47a526c4-2
00:35:59.123 --> 00:36:03.349
which this pathway uses homocysteine to
convert it into cysteine,

f3f418e1-b346-47b8-a911-e6da47a526c4-3
00:36:03.349 --> 00:36:07.318
that amino acid residue,
and this can then be used for either

f3f418e1-b346-47b8-a911-e6da47a526c4-4
00:36:07.318 --> 00:36:10.199
protein synthesis or antioxidant
production.

e9b6e69a-06d3-46c3-8947-9bdc0ed838d4-0
00:36:10.400 --> 00:36:13.318
Like glutathione,
which I mentioned earlier,

e9b6e69a-06d3-46c3-8947-9bdc0ed838d4-1
00:36:13.318 --> 00:36:17.080
Homocysteine can also be used in the
remethylation cycle.

337499f1-1923-4856-a508-eb1a2a517030-0
00:36:17.360 --> 00:36:22.119
This means that homocysteine would get
further broken down and it's used to

337499f1-1923-4856-a508-eb1a2a517030-1
00:36:22.119 --> 00:36:26.440
regenerate methionine to restart that one
carbon metabolism pathway.

08f8ce55-8cb1-4434-8b68-b565d2bdad48-0
00:36:29.120 --> 00:36:31.922
Now,
although I'm very interested in HCY in

08f8ce55-8cb1-4434-8b68-b565d2bdad48-1
00:36:31.922 --> 00:36:36.571
the context of ocular disease,
there are also a variety of diseases that

08f8ce55-8cb1-4434-8b68-b565d2bdad48-2
00:36:36.571 --> 00:36:40.520
are associated with HCY,
including several different types of

08f8ce55-8cb1-4434-8b68-b565d2bdad48-3
00:36:40.520 --> 00:36:43.960
cardio, cardiovascular diseases,
metabolic disorders.

b2aa2f0a-144c-413b-bcd2-86bd17dc2874-0
00:36:44.600 --> 00:36:51.273
HCY has also been studied as an antiviral
target for virus infections as well as a

b2aa2f0a-144c-413b-bcd2-86bd17dc2874-1
00:36:51.273 --> 00:36:52.640
tumor suppressor.

b0eea562-b6d9-4768-b0c2-27027ad76fca-0
00:36:53.800 --> 00:36:56.548
Now for us,
when we're thinking about a HCY,

b0eea562-b6d9-4768-b0c2-27027ad76fca-1
00:36:56.548 --> 00:37:00.639
we know that it was the most
significantly oxidized protein in our

b0eea562-b6d9-4768-b0c2-27027ad76fca-2
00:37:00.639 --> 00:37:01.800
proteomic data set.

e6689d61-1202-484b-a2cf-519e32c16b63-0
00:37:02.200 --> 00:37:07.707
And we also identified a single oxidative
modification at a residue known as

e6689d61-1202-484b-a2cf-519e32c16b63-1
00:37:07.707 --> 00:37:11.498
cysteine 195,
which I'll be referring to as C195 for

e6689d61-1202-484b-a2cf-519e32c16b63-2
00:37:11.498 --> 00:37:13.000
the rest of the talk.

9d499837-f93f-4fb2-9f51-d3953b302f68-0
00:37:13.920 --> 00:37:19.435
Cysteine 195 is in a very important
location for AHCY and it's located near

9d499837-f93f-4fb2-9f51-d3953b302f68-1
00:37:19.435 --> 00:37:20.960
its catalytic center.

1894b090-befc-402f-bc6a-7b0413f47b04-0
00:37:21.440 --> 00:37:25.528
And a study that was done in the 90s,
which is an in vitro study,

1894b090-befc-402f-bc6a-7b0413f47b04-1
00:37:25.528 --> 00:37:30.360
meaning that this protein wasn't taken
from a whole Organism like Drosophila.

2a1ea9a9-8cf8-4f6d-8b91-91037fd4c42b-0
00:37:30.360 --> 00:37:33.240
It was produced recombinantly using
bacteria.

d9329bb7-162b-4c04-b809-713e802a0707-0
00:37:33.520 --> 00:37:39.093
They found that if you modified that
recombinant protein at location C195 to a

d9329bb7-162b-4c04-b809-713e802a0707-1
00:37:39.093 --> 00:37:44.807
different amino acid such as a serine,
that this revealed a significant decrease

d9329bb7-162b-4c04-b809-713e802a0707-2
00:37:44.807 --> 00:37:46.360
in catalytic turnover.

b03f27b9-ae32-4eb9-a086-11ec4be128ad-0
00:37:46.640 --> 00:37:51.664
And catalytic turnover is a fancy word
for saying that if you mutate the

b03f27b9-ae32-4eb9-a086-11ec4be128ad-1
00:37:51.664 --> 00:37:54.968
cysteine,
HCY as a protein has a very difficult

b03f27b9-ae32-4eb9-a086-11ec4be128ad-2
00:37:54.968 --> 00:38:00.199
time releasing its substrate SAH,
meaning that it's catalytic efficiency is

b03f27b9-ae32-4eb9-a086-11ec4be128ad-3
00:38:00.199 --> 00:38:03.915
much lower because instead of being able
to bind SAH,

b03f27b9-ae32-4eb9-a086-11ec4be128ad-4
00:38:03.915 --> 00:38:06.600
break it down and release the products.

f5aaaf97-4f46-40fb-b9f6-a74715a1ea70-0
00:38:06.800 --> 00:38:11.101
If you mutate this residue,
SCH almost gets stuck and it's not able

f5aaaf97-4f46-40fb-b9f6-a74715a1ea70-1
00:38:11.101 --> 00:38:12.240
to be broken down.

fc941779-a4cb-4334-a301-514fbb181b13-0
00:38:13.040 --> 00:38:19.021
So what we wanted to know in vivo for the
Drosophila eye is does this modification

fc941779-a4cb-4334-a301-514fbb181b13-1
00:38:19.021 --> 00:38:21.400
have that functional consequence?

e3d6fa57-3d4d-41d2-a6b0-41e8c99ca283-0
00:38:21.560 --> 00:38:25.840
What might this oxidative modification be
doing to the protein activity?

82c7d333-734a-4c33-b018-1d684eaddc0d-0
00:38:26.800 --> 00:38:32.707
Although we don't have an activity assay
yet for Hcy, we have the next best thing,

82c7d333-734a-4c33-b018-1d684eaddc0d-1
00:38:32.707 --> 00:38:37.760
which is being able to measure its
substrate abundance within the eye.

31c706a6-6e01-4b5f-a66c-572f5e23a751-0
00:38:38.320 --> 00:38:41.057
So just to remind you again of that
pathway,

31c706a6-6e01-4b5f-a66c-572f5e23a751-1
00:38:41.057 --> 00:38:45.680
we have methionine being used to generate
Sam, that universal methyl donor.

366764ef-012d-42ca-b238-2a27dd8eef1f-0
00:38:45.680 --> 00:38:48.891
So it will get taken up by a methyl
transferase,

366764ef-012d-42ca-b238-2a27dd8eef1f-1
00:38:48.891 --> 00:38:51.840
which then is used to methylate a
substrate.

f38ee293-8bcc-432d-9cde-fa3b883f5289-0
00:38:52.120 --> 00:38:56.898
When this occurs,
it generates SAH which can inhibit methyl

f38ee293-8bcc-432d-9cde-fa3b883f5289-1
00:38:56.898 --> 00:39:00.960
transferases,
and SAH can be broken down by a HCY.

c2ea23be-e3af-4ad5-8257-a7b40e85e724-0
00:39:01.440 --> 00:39:04.840
So what I'm showing you here is targeted
metabolite analysis.

a9341aa0-29d9-4bf9-a328-d7c5f8adceb6-0
00:39:05.480 --> 00:39:08.286
This means that we had two subsets of
flies,

a9341aa0-29d9-4bf9-a328-d7c5f8adceb6-1
00:39:08.286 --> 00:39:13.463
either flies that were exposed to normal
white light or flies that were exposed to

a9341aa0-29d9-4bf9-a328-d7c5f8adceb6-2
00:39:13.463 --> 00:39:17.205
8 hours of blue light,
which we then dissected their heads,

a9341aa0-29d9-4bf9-a328-d7c5f8adceb6-3
00:39:17.205 --> 00:39:22.320
extracted the metabolites and quantified
how much of each metabolite was present.

a265f9bc-010a-4dce-a0a7-43ce311577fc-0
00:39:23.360 --> 00:39:27.387
So when we look at Sam,
there is no significant change between

a265f9bc-010a-4dce-a0a7-43ce311577fc-1
00:39:27.387 --> 00:39:31.160
flies that were showing or shown to 8
hours of blue light.

eb7fb0a3-093e-438d-942e-0287c6797932-0
00:39:31.400 --> 00:39:35.200
Their Sam abundance,
which again is here in the pathway,

eb7fb0a3-093e-438d-942e-0287c6797932-1
00:39:35.200 --> 00:39:36.200
is not changed.

b8987b3b-a322-4737-a14d-efdaba4dd7b9-0
00:39:36.520 --> 00:39:39.667
However,
what we see with eight hours of blue

b8987b3b-a322-4737-a14d-efdaba4dd7b9-1
00:39:39.667 --> 00:39:43.429
light is a significant increase in the
metabolite SAH,

b8987b3b-a322-4737-a14d-efdaba4dd7b9-2
00:39:43.429 --> 00:39:48.424
which indicates that Hcy may not be
functioning properly upon blue light

b8987b3b-a322-4737-a14d-efdaba4dd7b9-3
00:39:48.424 --> 00:39:49.039
exposure.

42551280-cffa-4b82-ae53-f864b9f8b34e-0
00:39:49.680 --> 00:39:54.294
And as I mentioned earlier,
normally we talk about Sam and SAH as a

42551280-cffa-4b82-ae53-f864b9f8b34e-1
00:39:54.294 --> 00:39:55.720
ratio to one another.

d52f1db7-f3f4-4a96-9b25-80ff510ced52-0
00:39:56.040 --> 00:40:02.194
And what we can tell through this also is
that blue light significantly reduces the

d52f1db7-f3f4-4a96-9b25-80ff510ced52-1
00:40:02.194 --> 00:40:03.440
Sam to SAH ratio.

d0f667bc-644d-4b82-ab8b-e1067287d526-0
00:40:03.800 --> 00:40:08.546
And this disruption of the methylation
index can prevent the methylation

d0f667bc-644d-4b82-ab8b-e1067287d526-1
00:40:08.546 --> 00:40:12.707
reactions in the cell,
because too much accumulation of SAH can

d0f667bc-644d-4b82-ab8b-e1067287d526-2
00:40:12.707 --> 00:40:15.763
lead to the inhibition of methyl
transferases,

d0f667bc-644d-4b82-ab8b-e1067287d526-3
00:40:15.763 --> 00:40:19.600
which could subsequently alter your
epigenetic expression.

6463b4d6-7ac2-462e-a91d-673fa5c86a7d-0
00:40:19.800 --> 00:40:22.000
Or changes within your gene expression.

526148fc-8813-408b-92d6-5021644e36c0-0
00:40:22.960 --> 00:40:28.698
So our current working model for this is
that a HCY is a redox regulated enzyme,

526148fc-8813-408b-92d6-5021644e36c0-1
00:40:28.698 --> 00:40:33.870
meaning that under normal physiological
conditions Sam is used by methyl

526148fc-8813-408b-92d6-5021644e36c0-2
00:40:33.870 --> 00:40:39.254
transferases to produce SAH which can
then be broken down into two separate

526148fc-8813-408b-92d6-5021644e36c0-3
00:40:39.254 --> 00:40:43.080
products such as adenosine and
homocysteine by a HCY.

be6b2d85-d094-486c-a6b5-02def33b8cb7-0
00:40:43.520 --> 00:40:47.322
However,
under stress conditions like blue light,

be6b2d85-d094-486c-a6b5-02def33b8cb7-1
00:40:47.322 --> 00:40:53.559
these reactive oxygen species make that
oxidative modification at C195 leading to

be6b2d85-d094-486c-a6b5-02def33b8cb7-2
00:40:53.559 --> 00:40:59.416
possibly inactivation of AHCY mean,
which means you don't get the production

be6b2d85-d094-486c-a6b5-02def33b8cb7-3
00:40:59.416 --> 00:41:04.816
of adenosine and homocysteine,
but instead increased abundance of SAH,

be6b2d85-d094-486c-a6b5-02def33b8cb7-4
00:41:04.816 --> 00:41:09.000
which we then think is inhibiting methyl
transferases.

399ceca2-223e-408e-b06f-a61be687cc4a-0
00:41:09.800 --> 00:41:14.319
Now one thing to keep in mind when we do
redox proteomic profiling,

399ceca2-223e-408e-b06f-a61be687cc4a-1
00:41:14.319 --> 00:41:18.240
we are identifying 10s of thousands of
proteins at a time.

495ffffa-00f2-4756-9917-6380323dd3c3-0
00:41:18.720 --> 00:41:21.960
And we,
when you're analyzing these data sets

495ffffa-00f2-4756-9917-6380323dd3c3-1
00:41:21.960 --> 00:41:26.045
using bioinformatics,
we try our best to identify true or

495ffffa-00f2-4756-9917-6380323dd3c3-2
00:41:26.045 --> 00:41:31.117
significant hits while eliminating
anything that might be considered an

495ffffa-00f2-4756-9917-6380323dd3c3-3
00:41:31.117 --> 00:41:33.160
artifact or a false positive.

9823d768-eea0-4866-af18-c318381048d6-0
00:41:33.520 --> 00:41:38.480
So it's very important that when you do
an omics data set that you have a second

9823d768-eea0-4866-af18-c318381048d6-1
00:41:38.480 --> 00:41:40.440
way of validating your findings.

b95fffcf-1993-4e2f-87ff-ad29512367e9-0
00:41:40.800 --> 00:41:45.311
And one way that we've done this in our
lab is through a different type of

b95fffcf-1993-4e2f-87ff-ad29512367e9-1
00:41:45.311 --> 00:41:48.560
labeling that can then be visualized by
Western blot.

a0dffc42-eda0-4df1-90bf-1d350287c50a-0
00:41:49.160 --> 00:41:52.920
So here I'm going to walk you through how
this type of labeling works.

cc363e24-9069-4f3e-a335-ab8477a74681-0
00:41:53.240 --> 00:42:00.160
And here's a schematic of HCY with its
cysteine residue at position C195.

8dd0ffa2-6b01-407c-abab-bb7c0541228b-0
00:42:00.560 --> 00:42:02.440
And this is in a reduced state.

4ad3fe40-bb89-46ad-a507-a6e7018cacb6-0
00:42:02.600 --> 00:42:07.080
If your cysteine is in a reduced state,
it can bind to A tag.

dd2eab1a-77cb-4181-a275-9e27ef5c1601-0
00:42:07.280 --> 00:42:10.600
This tag is known as MMM PEG 24.

d4adfbdb-2860-4b22-9885-c7b63bcdd1ba-0
00:42:10.880 --> 00:42:16.585
This is a compound that can increase
proteins molecular weight by 1.

d4adfbdb-2860-4b22-9885-c7b63bcdd1ba-1
00:42:16.585 --> 00:42:19.480
24 kilodolins per cysteine residue.

1af6ddd8-0378-495e-9341-cbacc4577bd3-0
00:42:20.120 --> 00:42:24.996
But if you have a cysteine that's in an
oxidative modification like I showed

1af6ddd8-0378-495e-9341-cbacc4577bd3-1
00:42:24.996 --> 00:42:29.303
earlier, your tag won't bind,
meaning that your molecular weight of

1af6ddd8-0378-495e-9341-cbacc4577bd3-2
00:42:29.303 --> 00:42:31.520
your protein will remain unchanged.

8307273f-a128-4aa6-bb5e-22d58bf925f1-0
00:42:32.240 --> 00:42:35.628
Also,
if you mutate that cysteine residue to

8307273f-a128-4aa6-bb5e-22d58bf925f1-1
00:42:35.628 --> 00:42:39.844
say a serine,
where the serine no longer has that thiol

8307273f-a128-4aa6-bb5e-22d58bf925f1-2
00:42:39.844 --> 00:42:45.040
side chain and said it has an 0,
the tag also won't be able to bind.

1e68b5ca-ef18-47f0-97b5-4615ff5d27d9-0
00:42:45.240 --> 00:42:48.360
So you will not have a change in the
protein molecular weight.

e476b39f-481e-4f9d-ab54-23676ac61af4-0
00:42:48.800 --> 00:42:50.320
So what does this look like on a block?

825bf90c-1c08-4e4d-8287-2738289bc7c8-0
00:42:51.000 --> 00:42:53.920
So I'm going to walk you through slowly
through each lane.

e873438a-c52d-4faf-ba46-220644bd9003-0
00:42:54.280 --> 00:42:59.280
Here we're focusing on sizes between 75K
daltons and 63K daltons.

8aaaaa0e-a207-4cf9-bb79-2900a355b5c8-0
00:42:59.760 --> 00:43:07.040
I'm showing a wild type HCY,
an HCY with at that cysteine 195.

36c05a4a-77f0-483b-88fe-8d5574f735a0-0
00:43:07.040 --> 00:43:11.840
We've mutated it to an alanine or we've
mutated it to a serine.

0267a830-b194-4c33-b788-af4d3f35f2ec-0
00:43:12.160 --> 00:43:19.280
So this first 3 where it says HCY ox C
195 ox or C195 S ox.

849f94e3-d91c-4d74-86fa-db929ed23697-0
00:43:19.640 --> 00:43:24.696
This is proteins that have been extracted
from Drosophila cells that have been

849f94e3-d91c-4d74-86fa-db929ed23697-1
00:43:24.696 --> 00:43:29.368
exposed to oxidizing reagents,
which means that we are forcing available

849f94e3-d91c-4d74-86fa-db929ed23697-2
00:43:29.368 --> 00:43:32.440
cysteines to take on an oxidative
modification.

8f71c73b-553c-419b-9b50-d5adcd5193c7-0
00:43:32.920 --> 00:43:36.723
Or this next three,
we're looking at the wild type or either

8f71c73b-553c-419b-9b50-d5adcd5193c7-1
00:43:36.723 --> 00:43:41.274
of the mutants that have been reduced,
meaning they have been exposed to

8f71c73b-553c-419b-9b50-d5adcd5193c7-2
00:43:41.274 --> 00:43:45.825
reducing agent that forces those
cysteines to be in their original thiol

8f71c73b-553c-419b-9b50-d5adcd5193c7-3
00:43:45.825 --> 00:43:46.200
state.

6220cc2d-ded1-4c76-b763-23a5edd5ed06-0
00:43:47.000 --> 00:43:52.045
And one thing that we noticed so down
these last three are true experimental

6220cc2d-ded1-4c76-b763-23a5edd5ed06-1
00:43:52.045 --> 00:43:56.893
samples where we're looking for that
weight increase with the MMM PEG tag

6220cc2d-ded1-4c76-b763-23a5edd5ed06-2
00:43:56.893 --> 00:43:57.680
being bound.

1212d732-bc96-4478-8aff-bb005bd7c772-0
00:43:58.000 --> 00:44:04.128
And what we can see here is that this
wild type AHCY meaning that it has

1212d732-bc96-4478-8aff-bb005bd7c772-1
00:44:04.128 --> 00:44:11.179
cysteine 195 when incubated with that MMM
PEG tag runs at a higher molecular weight

1212d732-bc96-4478-8aff-bb005bd7c772-2
00:44:11.179 --> 00:44:17.979
just slightly by what looks to be around
1 kilodol and higher than if you mutate

1212d732-bc96-4478-8aff-bb005bd7c772-3
00:44:17.979 --> 00:44:22.260
the cysteine 195 to either an alanine or
a serine,

1212d732-bc96-4478-8aff-bb005bd7c772-4
00:44:22.260 --> 00:44:28.892
indicating that a HCY at position C 195
is the major oxidized cysteine residue

1212d732-bc96-4478-8aff-bb005bd7c772-5
00:44:28.892 --> 00:44:30.320
for this protein.

4d9f0ab5-41b3-4a04-b33f-fe8c7d4ffd9b-0
00:44:30.960 --> 00:44:33.821
Now we still,
I still have a lot of work to do on this

4d9f0ab5-41b3-4a04-b33f-fe8c7d4ffd9b-1
00:44:33.821 --> 00:44:37.723
project and we have several open
questions that are currently being worked

4d9f0ab5-41b3-4a04-b33f-fe8c7d4ffd9b-2
00:44:37.723 --> 00:44:37.879
on.

7f943c86-6b73-43c3-9d8d-5f750b086b5c-0
00:44:38.240 --> 00:44:45.336
And the first part of the question would
be how would an oxidative modification or

7f943c86-6b73-43c3-9d8d-5f750b086b5c-1
00:44:45.336 --> 00:44:49.440
a mutation at cysteine 195 affect HCI
activity.

c62a64d2-7e12-4a79-b28f-2add02db0d72-0
00:44:49.720 --> 00:44:53.388
So as I mentioned earlier,
we didn't have an activity assay at the

c62a64d2-7e12-4a79-b28f-2add02db0d72-1
00:44:53.388 --> 00:44:55.852
time,
which is why we measured its substrate

c62a64d2-7e12-4a79-b28f-2add02db0d72-2
00:44:55.852 --> 00:44:56.400
abundance.

fed77409-6ea2-461f-8943-25ada7545123-0
00:44:56.720 --> 00:45:03.465
So I'm working to characterize HCI
activity using deabsorption electrospray

fed77409-6ea2-461f-8943-25ada7545123-1
00:45:03.465 --> 00:45:06.040
ionization mass spectrometry.

d0bec9b6-1825-405f-978f-97b1b382538e-0
00:45:06.040 --> 00:45:08.080
This is abbreviated as DESI MISS.

54032255-3987-4cb9-a1b8-3ef432281b7f-0
00:45:08.360 --> 00:45:11.255
This is a newer form of enzyme activity
assay,

54032255-3987-4cb9-a1b8-3ef432281b7f-1
00:45:11.255 --> 00:45:16.245
which is known as a high throughput and
highly sensitive way of measuring enzyme

54032255-3987-4cb9-a1b8-3ef432281b7f-2
00:45:16.245 --> 00:45:16.800
activity.

6f131f06-44d2-47c7-99f7-08187fb3fb36-0
00:45:17.520 --> 00:45:22.480
Our second question is what genes are
directly regulated by HCY in the eye?

9b98ce57-13d9-41c7-9c2f-62ed1fc71d7f-0
00:45:22.720 --> 00:45:27.180
This is currently in progress and we're
doing a large scale sequencing experiment

9b98ce57-13d9-41c7-9c2f-62ed1fc71d7f-1
00:45:27.180 --> 00:45:28.160
using cut and run.

54d9a9dd-4fda-4aa0-976e-987b5c4ea7a7-0
00:45:28.360 --> 00:45:34.511
If you're not familiar with cut and run,
this is a way to pull down DNA that is in

54d9a9dd-4fda-4aa0-976e-987b5c4ea7a7-1
00:45:34.511 --> 00:45:37.920
either in close proximity to or bound by
HCY.

d611be23-06fc-425d-aa53-eaaba809f952-0
00:45:38.760 --> 00:45:44.252
A third question is if HTI is depleted,
meaning that you either knock it down or

d611be23-06fc-425d-aa53-eaaba809f952-1
00:45:44.252 --> 00:45:45.880
knock it out in the eye.

11eee75d-5bd3-47d5-8fce-bc186819208f-0
00:45:46.200 --> 00:45:49.745
We think that this would correlate with
increased SAH abundance,

11eee75d-5bd3-47d5-8fce-bc186819208f-1
00:45:49.745 --> 00:45:53.890
but we want to know how would that change
gene expression in the eye 'cause

11eee75d-5bd3-47d5-8fce-bc186819208f-2
00:45:53.890 --> 00:45:58.309
remember, if you have an increase in SAH,
you can potentially be inhibiting your

11eee75d-5bd3-47d5-8fce-bc186819208f-3
00:45:58.309 --> 00:45:59.399
methyl transferases.

442dd708-5edb-4db9-88f6-ac0c8e583808-0
00:45:59.720 --> 00:46:04.268
So how we're answering this question is
doing another type of large sequencing

442dd708-5edb-4db9-88f6-ac0c8e583808-1
00:46:04.268 --> 00:46:05.880
experiment known as RNA seq.

1ee54383-ba99-4de1-8ca2-169568d86085-0
00:46:06.080 --> 00:46:10.908
And this is when you sequence all of the
RNA with and in the photoreceptor cells

1ee54383-ba99-4de1-8ca2-169568d86085-1
00:46:10.908 --> 00:46:12.280
that do not have a HCY.

bd7a0e68-ec80-4de2-817b-22eba922f953-0
00:46:12.880 --> 00:46:15.280
And these are things that I'm currently
doing in my lab.

862167de-8c67-4d34-8112-cea370dc142d-0
00:46:16.280 --> 00:46:20.154
So before I end my talk today,
I would like to just show a little bit

862167de-8c67-4d34-8112-cea370dc142d-1
00:46:20.154 --> 00:46:24.637
about the Department of Biochemistry at
Purdue in case there are any students in

862167de-8c67-4d34-8112-cea370dc142d-2
00:46:24.637 --> 00:46:29.119
their upper level of their undergraduate
that are interested in Graduate School.

d099f468-ee2b-42c0-a5d8-e3847e2e9861-0
00:46:29.360 --> 00:46:33.444
So our biochemistry department has many
different focuses of research,

d099f468-ee2b-42c0-a5d8-e3847e2e9861-1
00:46:33.444 --> 00:46:36.953
including metabolic and natural product
biochemistry, omics,

d099f468-ee2b-42c0-a5d8-e3847e2e9861-2
00:46:36.953 --> 00:46:41.037
so that some of the things that I've
shown you today like genomics and

d099f468-ee2b-42c0-a5d8-e3847e2e9861-3
00:46:41.037 --> 00:46:43.798
proteomic data,
as well as cancer biochemistry,

d099f468-ee2b-42c0-a5d8-e3847e2e9861-4
00:46:43.798 --> 00:46:47.480
epigenetics and gene expression,
as well as structural biology.

a0b3bdc3-91d5-4c12-9b8b-c11a3e3d2ea1-0
00:46:47.960 --> 00:46:52.880
And if you're interested in applying to
Graduate School, Purdue does offer a PhD.

e5cf2f1b-0db3-4c68-a630-8f5e70d8443f-0
00:46:53.200 --> 00:46:55.560
We have 35 week rotations.

dff05679-524b-48c3-ad43-feef21750e4f-0
00:46:55.680 --> 00:46:58.440
The application deadline would be
November 15th.

e01025da-ef46-4256-bca3-1ce34b2077fa-0
00:46:58.600 --> 00:47:03.290
We do not require a GRE and PhD programs
do offer a stipend,

e01025da-ef46-4256-bca3-1ce34b2077fa-1
00:47:03.290 --> 00:47:05.520
which is currently at 33,000.

18ea6ff4-436d-4864-8788-2ae384f835d4-0
00:47:05.720 --> 00:47:09.235
And if you are interested,
I've also provided a link to our

18ea6ff4-436d-4864-8788-2ae384f835d4-1
00:47:09.235 --> 00:47:09.880
department.

e69dac8e-1c6e-4415-8f20-2a091d38edb5-0
00:47:10.880 --> 00:47:13.490
And with that,
I would like to thank the organizers of

e69dac8e-1c6e-4415-8f20-2a091d38edb5-1
00:47:13.490 --> 00:47:15.200
the Darwin Festival for inviting me.

0dfef620-bf14-4219-97ae-39b8ee8e4b65-0
00:47:15.480 --> 00:47:18.024
I would like to also thank my thesis
committee,

0dfef620-bf14-4219-97ae-39b8ee8e4b65-1
00:47:18.024 --> 00:47:21.152
members of the current graduate students
that I work with,

0dfef620-bf14-4219-97ae-39b8ee8e4b65-2
00:47:21.152 --> 00:47:25.128
which are shown here in this photo,
as well as graduate students that have

0dfef620-bf14-4219-97ae-39b8ee8e4b65-3
00:47:25.128 --> 00:47:28.627
graduated, and of course,
the funding that is provided through my

0dfef620-bf14-4219-97ae-39b8ee8e4b65-4
00:47:28.627 --> 00:47:28.840
lab.

6a57be03-f4f1-4823-a9f3-0803d802418c-0
00:47:29.080 --> 00:47:31.520
And with that,
I'd be happy to take any questions.

81c028ea-dbd5-448a-8b43-74565c413377-0
00:47:38.120 --> 00:47:40.840
Thank you Sarah, great talk.

07279111-7f55-4046-9d0e-c8dd325e5f4a-0
00:47:41.000 --> 00:47:42.000
Nice to see you again.

65005a90-1839-479a-aad2-8254945bdfbc-0
00:47:42.920 --> 00:47:43.560
You as well.

4afa0275-7b24-409d-957b-642f540cafd6-0
00:47:46.320 --> 00:47:52.541
We come from a line that is the mammals
where regeneration and nerve tissue is

4afa0275-7b24-409d-957b-642f540cafd6-1
00:47:52.541 --> 00:47:53.880
very seldom seen.

78e3a45b-761e-44c5-be25-27595250fcc3-0
00:47:54.320 --> 00:47:59.967
Yes,
in in Drosophila can retinal cells

78e3a45b-761e-44c5-be25-27595250fcc3-1
00:47:59.967 --> 00:48:01.520
regenerate?

476b7b54-20f7-4479-81fb-751ccd6bcd36-0
00:48:02.320 --> 00:48:03.640
So they can't regenerate.

bea9f56d-8110-4de0-8e04-b1f9450084b3-0
00:48:03.640 --> 00:48:05.960
So these are still post mitotic cells.

a6aa2cf8-be49-4f51-8545-501b567488b6-0
00:48:06.160 --> 00:48:10.485
But what we have seen through
transcriptomic data that we've done in

a6aa2cf8-be49-4f51-8545-501b567488b6-1
00:48:10.485 --> 00:48:14.622
our lab is that when the cells are,
when the photoreceptor cells,

a6aa2cf8-be49-4f51-8545-501b567488b6-2
00:48:14.622 --> 00:48:19.449
which are post mitotic cells are under
stress with either with aging or blue

a6aa2cf8-be49-4f51-8545-501b567488b6-3
00:48:19.449 --> 00:48:22.960
light before they die,
they do re enter the cell cycle.

61a781e1-cd9d-4580-916f-a0d1e204667c-0
00:48:22.960 --> 00:48:25.886
And it's almost as if those neurons are
trying to save themselves,

61a781e1-cd9d-4580-916f-a0d1e204667c-1
00:48:25.886 --> 00:48:27.240
but they never quite get there.

92cae4b0-8c49-44d6-a1fd-b115a1637a07-0
00:48:30.240 --> 00:48:30.720
Great talk.

7f627ce9-a475-4446-9bab-560b98eaadcf-0
00:48:30.720 --> 00:48:33.702
Sarah,
there's actually a question from one of

7f627ce9-a475-4446-9bab-560b98eaadcf-1
00:48:33.702 --> 00:48:37.320
my students about your final slide and I
cheated, Sarah.

5496d3a2-b803-4cc9-b7df-7a49c1f61412-0
00:48:37.320 --> 00:48:38.480
I took a screenshot.

96711175-6954-4df0-87af-465a42399406-0
00:48:38.480 --> 00:48:45.327
So I'll e-mail that to you,
the student who just asked that question

96711175-6954-4df0-87af-465a42399406-1
00:48:45.327 --> 00:48:49.000
and a question for our undergraduate.

413c34b9-32cc-47d9-93af-de5a202d12b6-0
00:48:49.000 --> 00:48:54.536
Sarah, when you were here at Salem State,
how did you look into which graduate

413c34b9-32cc-47d9-93af-de5a202d12b6-1
00:48:54.536 --> 00:48:56.920
programs might be be good for you?

0b2bf227-a2d5-4893-b823-7a7e2d5ec6e6-0
00:48:56.920 --> 00:48:58.400
And how did you decide on Purdue?

d0ba74cd-6772-4af6-9027-48c1bc50e420-0
00:48:59.160 --> 00:49:04.008
Yeah, so when I was at Salem State,
I did multiple semesters of research with

d0ba74cd-6772-4af6-9027-48c1bc50e420-1
00:49:04.008 --> 00:49:07.676
Doctor Jason Brown,
which I know unfortunately couldn't be

d0ba74cd-6772-4af6-9027-48c1bc50e420-2
00:49:07.676 --> 00:49:08.360
here today.

2af68066-b35a-41c0-88d2-dc36bda345be-0
00:49:08.640 --> 00:49:14.165
But he was a great help when it came to
looking at what was available to me for

2af68066-b35a-41c0-88d2-dc36bda345be-1
00:49:14.165 --> 00:49:19.000
Graduate School and different types of
programs and things like that.

2555a56c-2241-4af2-8437-9815e432cac9-0
00:49:19.240 --> 00:49:21.933
But when I was looking for graduate
programs,

2555a56c-2241-4af2-8437-9815e432cac9-1
00:49:21.933 --> 00:49:26.500
there were things that were important to
me like the ability to do rotations,

2555a56c-2241-4af2-8437-9815e432cac9-2
00:49:26.500 --> 00:49:31.067
which means that you basically get a
trial run for whatever or like a several

2555a56c-2241-4af2-8437-9815e432cac9-3
00:49:31.067 --> 00:49:31.359
week.

cb4dfa87-75cc-429b-94f2-33b4f5420f4a-0
00:49:31.640 --> 00:49:35.040
In a different labs and you can see what
is the right fit for you.

285fd3dc-ae76-4033-910b-03190752f969-0
00:49:35.320 --> 00:49:40.094
So I knew I wanted to be in a program
that offered that and I also wanted to be

285fd3dc-ae76-4033-910b-03190752f969-1
00:49:40.094 --> 00:49:42.720
in an area that had a lower cost of
living.

aaff8144-2437-48a4-8001-28da7f3b007c-0
00:49:42.960 --> 00:49:46.440
That way you can make your grad stipend
work no matter what.

028e9ade-a355-4954-a7b1-cfa89f51a41f-0
00:49:46.840 --> 00:49:51.426
And one other important thing is when
you're looking at programs,

028e9ade-a355-4954-a7b1-cfa89f51a41f-1
00:49:51.426 --> 00:49:56.360
make sure that you are interested in more
than one faculties research.

51bb29e9-2bce-41c3-a46f-692ebc949d60-0
00:49:57.040 --> 00:50:00.160
It's important that you have a variety of
options.

2ff72a91-0477-4fd3-801e-98bfebc6ba09-0
00:50:00.160 --> 00:50:02.273
That way,
if you get into a lab and on paper,

2ff72a91-0477-4fd3-801e-98bfebc6ba09-1
00:50:02.273 --> 00:50:05.306
it seemed perfect for you,
but maybe it wasn't the right fit once

2ff72a91-0477-4fd3-801e-98bfebc6ba09-2
00:50:05.306 --> 00:50:07.880
you got there,
you weren't stuck in a program where you

2ff72a91-0477-4fd3-801e-98bfebc6ba09-3
00:50:07.880 --> 00:50:10.040
didn't think anything else was as
interesting.

e53eb775-1638-461e-9199-00eaf5bf4983-0
00:50:11.880 --> 00:50:12.160
Thank you.

187bedb7-acdf-4469-be2a-852f6bd9d654-0
00:50:13.040 --> 00:50:14.000
I have a question for you.

51e7f549-3155-42a6-8fd1-c371330b76ec-0
00:50:14.000 --> 00:50:19.280
The next question is Sarah, for us,
where is blue light exposure most common?

e78fedcd-5b2a-4965-9559-03de08de31dd-0
00:50:19.360 --> 00:50:22.640
So for humans,
when are we going to encounter blue light?

74fa89cc-968d-4aab-9e0a-51dc0e47deb3-0
00:50:23.400 --> 00:50:27.914
Any time that you are under LED light,
there are blue light wavelengths within

74fa89cc-968d-4aab-9e0a-51dc0e47deb3-1
00:50:27.914 --> 00:50:28.200
that.

b027fdaf-c8f4-4710-8c0e-d9bc090ca9c3-0
00:50:28.200 --> 00:50:31.480
Of course,
our blue light model is an extreme model.

c0b3e531-adbf-496a-8515-cd611d570ac0-0
00:50:31.760 --> 00:50:35.080
That way we can see the direct effects of
blue light.

42525938-b2e2-4ed9-adf5-8930ab75ac40-0
00:50:35.320 --> 00:50:38.405
But anytime you're looking at your
computer screen, your phone,

42525938-b2e2-4ed9-adf5-8930ab75ac40-1
00:50:38.405 --> 00:50:42.261
or even the lights in your classrooms,
those all have blue light wavelengths in

42525938-b2e2-4ed9-adf5-8930ab75ac40-2
00:50:42.261 --> 00:50:42.840
them, right?

862b61ca-087a-4a79-a432-72dbca062be9-0
00:50:48.440 --> 00:50:48.840
Thanks, Paul.

ec813f63-8cfa-44f0-9cc1-cb6df554054b-0
00:50:48.840 --> 00:50:49.800
Are you going to do the next one?

cd00fc2d-5421-4205-a22e-381ab49b798e-0
00:50:52.160 --> 00:50:53.440
I don't have an X1, do you?

8c6e8df7-4a44-46b0-9fa3-a561acf06a35-0
00:50:54.080 --> 00:50:57.800
Oh, I All right,
so I have an extra somehow.

ef7e2cd4-5f30-40c5-b64b-e5a31bb07e59-0
00:50:58.640 --> 00:51:01.200
So a question from one of our upper level
students.

e080b8f7-cf8d-47cf-b6ef-0074ffd6ab4e-0
00:51:01.200 --> 00:51:04.166
Sarah,
thanking you for a very interesting and

e080b8f7-cf8d-47cf-b6ef-0074ffd6ab4e-1
00:51:04.166 --> 00:51:05.240
informative talk.

79a401cc-2f9c-48af-94fd-0bd790d85c70-0
00:51:06.840 --> 00:51:11.440
Some general questions and comments from
you about being a PhD student.

fde42c98-d915-4699-9756-e4c1e7dfae3c-0
00:51:11.440 --> 00:51:12.400
What's it like?

60761ab9-46ed-472d-8dbd-2714e900d6e6-0
00:51:13.160 --> 00:51:20.760
And to answer the students interest
because she's interested in a PhD program.

f578cc8e-b629-4bc5-9422-e6252faa0689-0
00:51:20.800 --> 00:51:23.200
So yeah, what's it like?

11556339-69d9-48eb-8c02-d475eb498e80-0
00:51:23.440 --> 00:51:28.374
Well, it's very, it's busy,
but every day is so interesting because I

11556339-69d9-48eb-8c02-d475eb498e80-1
00:51:28.374 --> 00:51:33.731
get to learn something new constantly,
whether that's new techniques or new

11556339-69d9-48eb-8c02-d475eb498e80-2
00:51:33.731 --> 00:51:35.000
areas of research.

949c027a-e242-4411-a9ae-cfe4dad95a5b-0
00:51:35.520 --> 00:51:40.181
Sometimes the best part of my day is if
when new data comes in and you are at

949c027a-e242-4411-a9ae-cfe4dad95a5b-1
00:51:40.181 --> 00:51:43.348
that moment,
that first person or the only person to

949c027a-e242-4411-a9ae-cfe4dad95a5b-2
00:51:43.348 --> 00:51:47.531
know this type of information before
you're able to put it out there,

949c027a-e242-4411-a9ae-cfe4dad95a5b-3
00:51:47.531 --> 00:51:50.520
and then sometimes the best feeling in
the world.

02351dd5-6213-4666-907f-af2667139f0d-0
00:51:51.400 --> 00:51:54.640
It's pretty different, I would imagine.

f08284da-3931-47c7-a325-2e5fabfa8745-0
00:51:55.840 --> 00:51:59.618
Yes, Sarah,
are humans damaged by blue light and

f08284da-3931-47c7-a325-2e5fabfa8745-1
00:51:59.618 --> 00:52:03.320
should we try to avoid it or filter it
somehow?

0d71d94e-0999-44b1-b2f3-7a3548423672-0
00:52:04.240 --> 00:52:09.765
So there it is known that prolonged
exposure to a lot of blue light is

0d71d94e-0999-44b1-b2f3-7a3548423672-1
00:52:09.765 --> 00:52:11.400
damaging to your eye.

22e40c52-2025-47c8-9d45-239a7cd32b22-0
00:52:12.560 --> 00:52:15.240
I don't know if there's any way to really
avoid it.

97c371fe-2dfe-4f3b-8431-ec4c476aa9e9-0
00:52:15.240 --> 00:52:18.888
They do sell those blue light filtered
glasses that you can buy,

97c371fe-2dfe-4f3b-8431-ec4c476aa9e9-1
00:52:18.888 --> 00:52:22.480
but those haven't really been shown to
have any type of effect.

efb0a76a-4cd4-4a22-b0e7-51900d2be976-0
00:52:22.800 --> 00:52:26.379
But I think just monitoring how much
screen time you're doing throughout your

efb0a76a-4cd4-4a22-b0e7-51900d2be976-1
00:52:26.379 --> 00:52:27.160
day is important.

be1b9efb-d822-4b4b-b624-c2b735d9435a-0
00:52:28.200 --> 00:52:29.920
Thanks, Sarah.

0ce7f157-fa68-49a4-9d9d-f9cfc7cad777-0
00:52:29.920 --> 00:52:33.120
Your last slide,
you showed us how a student might find

0ce7f157-fa68-49a4-9d9d-f9cfc7cad777-1
00:52:33.120 --> 00:52:37.520
out more information about a PhD program
and I'll, I'll make that available.

0778ffc8-2646-4ec8-baf8-446758eaa6d6-0
00:52:37.520 --> 00:52:40.040
As I, as I said,
I took a screenshot of that.

94530e47-5aba-4a1f-b370-385c1d358433-0
00:52:40.960 --> 00:52:44.254
But if a student,
and there are a few of them,

94530e47-5aba-4a1f-b370-385c1d358433-1
00:52:44.254 --> 00:52:49.160
judging by the questions of thinking
about your type of PhD research,

94530e47-5aba-4a1f-b370-385c1d358433-2
00:52:49.160 --> 00:52:53.716
if you cast your mind back to being here
as a junior and senior,

94530e47-5aba-4a1f-b370-385c1d358433-3
00:52:53.716 --> 00:52:56.800
what courses would you recommend they
take?

e64a908f-a93f-4d8e-b6a8-047de5320e79-0
00:52:58.160 --> 00:53:02.360
I So I guess it would depend on what type
of program they're looking into.

8a077482-8cbf-40c6-bf37-67ab452f3ccb-0
00:53:02.400 --> 00:53:06.956
If you're leaning more towards a biology
PhD or a microbiology PhD,

8a077482-8cbf-40c6-bf37-67ab452f3ccb-1
00:53:06.956 --> 00:53:11.914
taking those upper level courses like
microbiology would probably be very

8a077482-8cbf-40c6-bf37-67ab452f3ccb-2
00:53:11.914 --> 00:53:13.120
important for you.

c412fa61-d06a-4e4c-b025-dbda3f5ecf61-0
00:53:13.560 --> 00:53:16.866
If you're interested in something more
similar to what I'm doing with

c412fa61-d06a-4e4c-b025-dbda3f5ecf61-1
00:53:16.866 --> 00:53:17.480
biochemistry.

ee601ac2-ca5f-44cf-a234-9be561552fef-0
00:53:17.720 --> 00:53:21.149
If you're a biology major taking extra
chemistry classes,

ee601ac2-ca5f-44cf-a234-9be561552fef-1
00:53:21.149 --> 00:53:25.701
really paying attention in organic
chemistry and taking biological chemistry

ee601ac2-ca5f-44cf-a234-9be561552fef-2
00:53:25.701 --> 00:53:28.894
is important,
but also if you have the ability to get

ee601ac2-ca5f-44cf-a234-9be561552fef-3
00:53:28.894 --> 00:53:33.447
research experience that also makes a
world of difference when applying to a

ee601ac2-ca5f-44cf-a234-9be561552fef-4
00:53:33.447 --> 00:53:33.919
program.

1282b898-207b-49cc-8451-ad37ef62fe4d-0
00:53:34.800 --> 00:53:35.400
Great thanks.

47e4d094-1f3e-40f5-ac9d-111d50625aa5-0
00:53:39.440 --> 00:53:41.040
Oh there is one more I can see.

95cd4059-48b0-4af1-ba8b-225f0f3f2639-0
00:53:41.600 --> 00:53:42.440
Oh should I read it or?

9d2badde-c3bb-41fd-ade0-9427390e44f9-0
00:53:43.240 --> 00:53:44.320
Oh I had to Scroll down.

7d77d129-f102-4530-8479-fda12b2246eb-0
00:53:44.320 --> 00:53:44.760
OK.

d89983c0-622f-423c-8f82-d40a329b61a0-0
00:53:47.440 --> 00:53:51.565
Is there a cut off for blue light
wavelength beyond which damage does not

d89983c0-622f-423c-8f82-d40a329b61a0-1
00:53:51.565 --> 00:53:52.680
occur or is minimal?

dc76010b-88ee-4633-ba33-71aa5acc1a19-0
00:53:54.120 --> 00:53:59.320
So what we've done in the Drosophila eye
is we've done various time courses.

4f0f5d6b-287c-4046-b259-dc0c9d44bbaa-0
00:53:59.320 --> 00:54:03.232
So for the studies here,
I showed 8 hours and we know that 8 hours

4f0f5d6b-287c-4046-b259-dc0c9d44bbaa-1
00:54:03.232 --> 00:54:05.160
causes mass retinal degeneration.

09404189-e3fb-4e11-aa2c-b94195260b73-0
00:54:05.440 --> 00:54:08.136
But if we do something smaller like 3
hours,

09404189-e3fb-4e11-aa2c-b94195260b73-1
00:54:08.136 --> 00:54:11.072
which is what we consider to be an acute
stress,

09404189-e3fb-4e11-aa2c-b94195260b73-2
00:54:11.072 --> 00:54:15.565
there is some damage to the rabdomers,
meaning that some of those might be

09404189-e3fb-4e11-aa2c-b94195260b73-3
00:54:15.565 --> 00:54:19.640
missing within those images,
but not to the same extent as 8 hours.

e3d466c4-21cc-471d-9228-e043c81886db-0
00:54:19.640 --> 00:54:24.367
So time does matter and is it a single
wavelength or do you use multiple

e3d466c4-21cc-471d-9228-e043c81886db-1
00:54:24.367 --> 00:54:25.080
wavelength?

a5f71a75-7004-4947-8b16-6773e696cbae-0
00:54:25.080 --> 00:54:26.400
This is a single wavelength.

a16c1239-fc60-4210-95ef-336aa896c6f9-0
00:54:28.480 --> 00:54:31.678
Sarah,
a question from I'm sure you took Cell

a16c1239-fc60-4210-95ef-336aa896c6f9-1
00:54:31.678 --> 00:54:34.320
Biology with Doctor Nelson Scott Gale.

d34ca5e0-d1fb-40d0-86aa-8e873fbffd2c-0
00:54:34.560 --> 00:54:34.880
Yes.

be2c8e8b-8d0d-4c09-bdc7-5a61852fae3d-0
00:54:35.600 --> 00:54:40.880
Is macular degeneration correlated with
increased exposure to blue light exposure?

162b72bc-465a-4019-bee7-7ccba1b432fd-0
00:54:41.480 --> 00:54:45.418
So I know that age-related macular
degeneration is more,

162b72bc-465a-4019-bee7-7ccba1b432fd-1
00:54:45.418 --> 00:54:48.320
you have more of a genetic predisposition.

68ca5f73-27b9-4ab9-af2f-3d38bb84f486-0
00:54:48.320 --> 00:54:53.262
I don't know if blue light might
correlate with an increased chance of

68ca5f73-27b9-4ab9-af2f-3d38bb84f486-1
00:54:53.262 --> 00:54:55.560
age-related macular degeneration.

0fe7a998-c515-4825-8ef5-9b49e393c23c-0
00:54:55.560 --> 00:54:56.600
I'm not sure on that one.

667a3442-fee2-4e4d-bd5f-3c4ba38327b7-0
00:54:57.800 --> 00:54:57.920
Right.

b980a4b9-99f1-45dd-9cac-a4dc0cfdac47-0
00:54:59.480 --> 00:55:03.385
And another student question,
how do you think your research with

b980a4b9-99f1-45dd-9cac-a4dc0cfdac47-1
00:55:03.385 --> 00:55:06.640
Drosophila will impact your future
research endeavors?

b87298b8-bf57-4639-9a7d-aab9c7707103-0
00:55:08.480 --> 00:55:11.116
Well,
I think that working with Drosophila

b87298b8-bf57-4639-9a7d-aab9c7707103-1
00:55:11.116 --> 00:55:14.120
means that you have a lot of genetic
background.

00ffd991-d5da-4adc-a126-3fe5dbf5376c-0
00:55:14.120 --> 00:55:19.196
So Drosophila is a incredible model
Organism for a lot of different types of

00ffd991-d5da-4adc-a126-3fe5dbf5376c-1
00:55:19.196 --> 00:55:23.481
genetic based techniques,
but how it will influence or impact my

00ffd991-d5da-4adc-a126-3fe5dbf5376c-2
00:55:23.481 --> 00:55:24.800
future career goals?

030ce38c-7edc-4264-a988-15d4cde89cfd-0
00:55:24.800 --> 00:55:30.151
I think one of the nice things about
doing a PhD is you're not going to get or

030ce38c-7edc-4264-a988-15d4cde89cfd-1
00:55:30.151 --> 00:55:34.080
lose a job based on what model Organism
you did your PhD.

2edcfa0a-fa5e-4de2-9c3c-6bd65181c37d-0
00:55:34.080 --> 00:55:35.920
And I don't think it's a limiting factor.

6d3923f6-2008-4f2a-bdbd-6347da2cc3a1-0
00:55:36.920 --> 00:55:40.440
I think it's based on what you're
interested in at the time.

99ca4aad-b9c6-48f3-a492-32bc7dca4571-0
00:55:41.160 --> 00:55:42.920
Thank you Sarah.

119e839b-cbc5-4be4-aa3b-5089e794abcc-0
00:55:42.920 --> 00:55:47.918
We'll make this the final question given
everybody in the audience that we have

119e839b-cbc5-4be4-aa3b-5089e794abcc-1
00:55:47.918 --> 00:55:52.979
another tool in just over 20 minutes by
whether or not a question Sarah that you

119e839b-cbc5-4be4-aa3b-5089e794abcc-2
00:55:52.979 --> 00:55:58.039
probably had asked of you in one of the
seminars you forces here at Salem State.

0e6170f9-4242-45df-b281-dcedd55429d8-0
00:55:58.400 --> 00:56:00.880
Where do you see yourself in 10 years
time?

551ed472-c9b8-4f2e-b5b9-622d1a6fddd1-0
00:56:01.880 --> 00:56:03.280
In 10 years time?

7151a11c-22d3-4d7e-9fdf-3faa8a2f04e2-0
00:56:03.360 --> 00:56:08.360
I so my career goal after finishing my
PhD is industry work.

18e81f88-30da-449d-aba3-8e8d7f9d8f79-0
00:56:08.880 --> 00:56:15.640
So I hope that I'll be working somewhere
at maybe a proteomic base or protein

18e81f88-30da-449d-aba3-8e8d7f9d8f79-1
00:56:15.640 --> 00:56:18.760
based drug discovery company, Right.

066df479-626a-42b6-a73a-925eb2d89b9f-0
00:56:18.840 --> 00:56:21.000
Well, thank you very,
very much for joining us.

78977aa0-4fda-4993-9dcc-2bf4b1678e53-0
00:56:21.000 --> 00:56:27.725
And you told me earlier before we came
online with everyone hoping to graduate

78977aa0-4fda-4993-9dcc-2bf4b1678e53-1
00:56:27.725 --> 00:56:30.960
in how soon spring 2025 S next spring.

385e0810-fdfe-43b2-9f11-9f4c625b645a-0
00:56:32.200 --> 00:56:35.126
Thanks very much Sarah,
take care and everybody,

385e0810-fdfe-43b2-9f11-9f4c625b645a-1
00:56:35.126 --> 00:56:36.560
thank you for having me.

3c131cbe-4f99-4826-b7a9-eaf4ca4bb087-0
00:56:36.800 --> 00:56:37.560
Thank you, Sarah.

50e60c7d-c240-46a8-99a4-c574d94b021a-0
00:56:38.200 --> 00:56:38.600
Bye.