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- My name is Dr. Ethel Gordon.

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I'm a member of Salem State
University's Biology Faculty

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and Treasurer of the
Darwin Festival Committee.

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It is my pleasure to welcome

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Salem State University community,

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and our guests from local area and beyond.

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We're happy to welcome some of
our area high school students

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as well as STEM students of all ages.

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It is my honor today to introduce

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our Salem State University

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alumnus speaker Mr. Jesse Moreira.

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Jesse is an undergraduate
with a BS in Biology

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from Salem State.

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Following his graduation in 2017,

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he joined Boston University's
Department of Health Sciences

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where he completed a Master
of Science and Physiology

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for Research in Cardiac and
Skeletal Muscle Biology.

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Jesse applied for a pre-doctoral program

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in the same department

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and was awarded the
Dean's Teaching Fellowship

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to study the Neural and
Neuro-immuno Mechanisms Contributing

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to the Development of Neurogenic
Salts Sensing Hypertension.

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Because of his research,

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Jesse was invited to
give oral presentations

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at the International
Experimental Biology Conference

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in both 2019 and 2020

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and was invited to give oral presentations

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at three consecutive Annual

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International American Heart Association

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Hypertension Scientific
Sessions from 2018 to 2020.

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In 2020, he was given the Top
Trainee Investigator Award

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by the American Heart Association's
Council on Hypertension.

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When not conducting research
or writing scientific papers,

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he serves as the primary lab instructor

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for the Gross Human Anatomy Cadaver Lab,

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and is co-instructor

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for Advanced Cardiac Physiology
Pathophysiology course.

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Jesse Moreira's talk
entitled, "Salty Microglia:

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Gaining Mechanistic Insights

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into Sodium-dependent Hypertension"

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is sponsored by the Department of Biology

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and the College of Arts and Science.

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Please give a warm Salem
State welcome to Jesse Moreira

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- Thank you so much

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for that wonderful
introduction, Dr. Gordon.

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Let me go ahead now and share my screen

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so I can bring up my
presentation for today.

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So yes, thank you so much.

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And thank you to the entire
Darwin Festival Committee

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for inviting me here to talk about

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my dissertation work that I'm conducting

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at Boston University.

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As Dr. Gordon said,

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I am in the Department of Health Sciences

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and I work in the Laboratory
of Dr. Richard Wainford

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in collaboration with the
Department of Pharmacology.

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And without further ado,
let me talk to you about

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microglia and salt sensitive hypertension.

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So to provide some background,

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I'll begin with blood pressure.

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What exactly is blood pressure?

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Well, blood pressure is the pressure

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inside your closed cardiovascular system

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that is required to
facilitate tissue gas exchange

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at the level of different
organs and drive blood

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against gravity of the
carotid arteries in your neck.

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So your brain gets perfused

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and it's a very critical
component of physiology,

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is blood pressure and with that,

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the maintenance of normal blood pressure.

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But what exactly
determines blood pressure?

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Well, the mean arteriole
pressure or the average

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between the systolic
pressure that you receive

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at the doctor's office,

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that top number and
the diastolic pressure,

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the bottom number, is really determined

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by two critical factors.

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Primarily, we see cardiac output.

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So you can see that
here, and cardiac output

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is just how much blood is being pumped

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by your heart in a given amount of time.

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And so that's determined principally

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by how often your heart is
beating, your heart rate,

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and how much blood is getting pumped out

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with every beat, your stroke volume.

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Now, arteriole pressure is also determined

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by peripheral resistance.

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So how resistant is the
cardiovascular system as a whole

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to the flow of blood from
the heart into the vessels?

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And really impacting that heavily

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are things like arteriole or radius.

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So how constricted or
dilated are these arterioles,

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these very small little
arteries that start to feed

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into the organs as well
as how thick is the blood?

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How viscous is it?

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Because a more viscous fluid,

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one for example, with less water volume,

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less plasma is more resistant to flow.

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When we think about

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what actually controls
blood pressure, though,

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there are many systems,
both hormonal and structural

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like the nervous system
that impact blood pressure.

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But for the purpose of my research

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that I do with Dr. Wainford,

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what I want to share with you has to do

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with the autonomic division
of the nervous system.

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Now, the autonomic division
or the nervous system

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is a part of the nervous system at large

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which controls all of your function.

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But unlike things like the
voluntary motor control

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of skeletal muscle that
allows us to do things

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like lift our arm,

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this operates below the level of awareness

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and it's constantly helping us do things

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like breathe or maintain
a normal heart rate.

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And functionally, it's divided
into both the sympathetic

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and parasympathetic divisions.

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With the sympathetic division,

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you may have heard of as
your fight or flight response

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actually activated far more frequently

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than when you're doing something
like running from a tiger.

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The sympathetic nervous
system has tonic activity

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at a certain level that helps integrate

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nervous system function and
control of blood pressure

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through its interactions
with many target organs

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like the adrenal gland at
the medulla, the middle,

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the arterioles directly being
innervated by these nerves

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as well as the kidney and the heart.

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And really importantly,
through its impacts

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on all of these different organs,

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it can control the various factors

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that we were just discussing
like cardiac output

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by stimulating the heart directly

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or peripheral resistance
through inducing constriction

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of those arterioles or
sodium and water retention

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at the level of the kidney
to impact blood volume.

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And with respect to the autonomic
nervous system, of course,

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we're going to zoom in a little more

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and see that there are these
cardiovascular control centers,

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many of which integrate functions together

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to ultimately affect responses,

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but critically involving
this one right here

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in the middle, the PVN

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which stands for paraventricular nucleus

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which takes the signals from
all of these different regions

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that are receiving input
about blood pressure

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and fluid and electrolyte balance

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and composes a unified response
through the spinal cord's

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sympathetic nervous system control center

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which it's connected to

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and out to the organs via the nerves.

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To give you a little closer
insight into the PVN itself,

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this is actually a
stain that was performed

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by a colleague of mine who
has since left our laboratory,

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Dr. Parul Chaudhary, and she stained

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for a G-protein coupled
receptor alpha sub units RNA

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using in C2 hybridization.

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And what you can see is the distribution

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throughout the bilateral
paraventricular nucleus here

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of this red stain for
this protein highlighting,

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in particular, of the various sub regions,

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this middle region known

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as the medial parvocellular sub nucleus

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and the dorsal region known
as the dorsal parvocellular.

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And all of the various sub
regions that you can see

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that anatomically makeup the PVN,

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the dorsal and medial
parvocellular regions contain

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those neurons which actually affect

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the biological responses intended
to control blood pressure

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via their connections

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to those other cardiovascular
control centers

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we were just speaking about
and that sympathetic nucleus,

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the IML and the spinal cord.

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So I'll get in a minute
why we're interested

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in the GNAI2 RNA and resultant
G alpha I two subunit protein

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but we'll first talk a bit about

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G-protein coupled receptors as a whole.

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You may or may not have been exposed

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to G-protein coupled receptors before,

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but if not, that's all right.

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These are seven pass transmembrane
domain proteins embedded

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in the plasma membranes of cells

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that are then coupled intracellularly

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to a variety of subunit proteins.

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You have this G alpha subunit

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and then there's this beta gamma sub unit.

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But really the important part

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is that upon ligand binding
at the extracellular domain,

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what you're going to get
is an effect activated

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by the G alpha sub unit.

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So it's going to swap out its inactive GDP

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for a more high energy GTP.

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And then according to the subtype
of alpha subunit you have,

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whether that's a G alpha sub S,

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sub I slash O sub Q or sub Z,

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you're going to get a variety of effects.

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And with respect to the PVN
and blood pressure control,

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what we're really interested in

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are these G alpha I slash O sub units

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and their ability to inhibit
adenylyl cyclase activity

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which reduces intracellular
cyclic AMP production.

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Because this signal pathway,

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as I'll get to, has been
demonstrated to be relevant

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in blood pressure control
through knockout studies.

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Now giving you some clinical background

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for all of this, why is this important?

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Why, is the PVN important?

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Why is blood pressure control important?

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Well, we're going to step and
look at sodium for a second.

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You can see in the CDC infographic

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that the average American is consuming

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three times the daily recommended sodium

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by the CDC at about 3,400 milligrams a day

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in comparison to 1500 recommended.

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And this is problematic when we consider

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that a variety of people
exhibit different phenotypes.

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So if you are this blue line here,

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salt resistant, might
not be such a big deal

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that you eat so much salt because
salt resistant individuals

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possess mechanisms that enable them

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to offload excess dietary
sodium intake into the urine.

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So no big deal for you, but
a significant proportion

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of individuals, I'll get to
how many, are salt sensitive

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which means that they do not
possess these mechanisms,

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rendering them unable
to offload that sodium

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and driving their blood pressure up

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both in the acute and the chronic setting

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such that with an elevated
dietary sodium intake over time,

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they develop salt sensitive hypertension.

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And our laboratory is
particularly interested

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in what the mechanisms are that account

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for the phenotypic difference observed

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between salt resistant and
salt sensitive individuals.

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And so at this point, and
you might be asking this

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and I said, I'd get to this.

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How common is hypertension, right?

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Well, according to the recent

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American Heart association

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Heart disease and Stroke Statistics,

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you can see that approximately

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one in two adults in the United States

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over the age of 18 are
categorized as having hypertension

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which is particularly
problematic because hypertension,

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as you can see in the bottom, contributes

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to about 10 and a 1/2 million
deaths worldwide per year.

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And it's in addition,
the leading risk factor

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for stroke, heart attacks,

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as well as chronic kidney disease.

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And with respect to how
salt sensitivity plays in,

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it's currently estimated

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that 1/2 of hypertensive
individuals, and even a 1/4

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of people who don't have
hypertension are salt sensitive.

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So salt sensitivity of blood pressure

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may actually be driving
a significant component

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of hypertension and its
overall disease burden

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in the United States and in the world.

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Now to kind of bring
these multiple factors

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I've been discussing together

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and investigate the actual
mechanisms that, as I said,

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account for the difference
between salt sensitivity

261
00:12:13,470 --> 00:12:15,720
and salt resistance in hypertension,

262
00:12:15,720 --> 00:12:18,700
our laboratory has
previously used rat models

263
00:12:19,640 --> 00:12:21,270
such as the Dahl salt-resistant rat

264
00:12:21,270 --> 00:12:22,620
or the Sprague-Dawley rat

265
00:12:22,620 --> 00:12:25,650
which I'll talk more
about later to study this.

266
00:12:25,650 --> 00:12:27,520
And so what happens is you take these rats

267
00:12:27,520 --> 00:12:29,750
and you give them a high salt intake.

268
00:12:29,750 --> 00:12:32,780
And what you actually see
is that in these naive rats

269
00:12:32,780 --> 00:12:35,480
on high salt, there's upregulation

270
00:12:35,480 --> 00:12:37,240
in the paraventricular nucleus

271
00:12:37,240 --> 00:12:39,640
that critical cardiovascular
control center

272
00:12:39,640 --> 00:12:42,270
of the G alpha I two class protein.

273
00:12:42,270 --> 00:12:44,823
So a G alpha I subunit
protein version two,

274
00:12:45,660 --> 00:12:48,010
and this ultimately leads to decrease

275
00:12:48,010 --> 00:12:50,500
in sympathetic nervous system activity.

276
00:12:50,500 --> 00:12:52,870
So you can turn down that tonic activity

277
00:12:52,870 --> 00:12:55,330
of the fight or flight
response so that the kidney

278
00:12:55,330 --> 00:12:58,440
is not constantly being
stimulated to retain sodium.

279
00:12:58,440 --> 00:13:00,570
That sodium is offloaded into the urine

280
00:13:01,610 --> 00:13:04,350
and you maintain a
nice, steep relationship

281
00:13:04,350 --> 00:13:06,620
here in the pressure and nature Risa slope

282
00:13:06,620 --> 00:13:10,150
such that very small changes in X,

283
00:13:10,150 --> 00:13:11,930
the mean arteriole pressure are required

284
00:13:11,930 --> 00:13:13,980
to facilitate large changes

285
00:13:13,980 --> 00:13:16,300
in sodium offloading into the urine,

286
00:13:16,300 --> 00:13:18,973
leaving the animal with
normal blood pressure.

287
00:13:19,830 --> 00:13:22,940
Now in contrast, in a
salt sensitive phenotype,

288
00:13:22,940 --> 00:13:27,940
our laboratory has identified
that there is no upregulation

289
00:13:28,190 --> 00:13:30,710
of PVN G alpha I two proteins

290
00:13:30,710 --> 00:13:32,640
in response to a high salt diet.

291
00:13:32,640 --> 00:13:35,210
So then not only does the animal fail

292
00:13:35,210 --> 00:13:38,330
to do the things that previously
did that we just discussed

293
00:13:38,330 --> 00:13:42,500
but there's also renal nerve
dependent simpatico excitation.

294
00:13:42,500 --> 00:13:43,870
So on its head,

295
00:13:43,870 --> 00:13:47,160
excessive sympathetic
nervous system activity

296
00:13:47,160 --> 00:13:49,220
which induces massive sodium retention

297
00:13:49,220 --> 00:13:50,500
at the level of the kidney,

298
00:13:50,500 --> 00:13:52,300
instead of telling the
kidney to disperse it

299
00:13:52,300 --> 00:13:54,250
into the urine and this shifts

300
00:13:54,250 --> 00:13:57,060
this pressure nature
recess relationship right

301
00:13:57,060 --> 00:14:00,460
and down so that now very large changes

302
00:14:00,460 --> 00:14:02,490
in blood pressure are going to be required

303
00:14:02,490 --> 00:14:05,790
to offload that excess of
sodium into the urine resulting

304
00:14:05,790 --> 00:14:09,283
in the development of robust
salt sensitive hypertension.

305
00:14:10,750 --> 00:14:13,260
And in addition to this
work that we've done,

306
00:14:13,260 --> 00:14:16,720
I've got for just two
examples citations here,

307
00:14:16,720 --> 00:14:18,140
there has been literature

308
00:14:18,140 --> 00:14:20,080
in models of hypertension in animals

309
00:14:20,080 --> 00:14:24,950
that has shown a really
critical role of inflammation

310
00:14:24,950 --> 00:14:28,640
in addition to all of the
other physiological mechanisms

311
00:14:28,640 --> 00:14:31,660
going on that drives salt
sensitive hypertension,

312
00:14:31,660 --> 00:14:33,910
as well as other models like angiotensin

313
00:14:33,910 --> 00:14:36,130
two dependent hypertension.

314
00:14:36,130 --> 00:14:40,520
And so this data being new in addition

315
00:14:40,520 --> 00:14:42,770
to all the things we've
previously done led us

316
00:14:42,770 --> 00:14:43,900
to our beginning hypothesis.

317
00:14:43,900 --> 00:14:46,430
And here's where I'll begin
talking about what I've done.

318
00:14:46,430 --> 00:14:49,270
And so my dissertation
began with the thought

319
00:14:49,270 --> 00:14:52,210
that maybe PVN inflammation
would be correlated

320
00:14:52,210 --> 00:14:54,170
with the development of our model,

321
00:14:54,170 --> 00:14:57,600
G alpha I two protein dependent
salt sensitive hypertension

322
00:14:57,600 --> 00:14:59,920
in addition to all these others.

323
00:14:59,920 --> 00:15:04,420
So to provide you some
understanding of what we've done,

324
00:15:04,420 --> 00:15:06,900
I'm going to walk you
through my methods first

325
00:15:06,900 --> 00:15:09,560
and you can see here, I've
got Sprague-Dawley rat,

326
00:15:09,560 --> 00:15:12,000
a classically salt resistant rat

327
00:15:12,000 --> 00:15:13,960
and we put a radio telemetry in it.

328
00:15:13,960 --> 00:15:16,900
So this is an acute surgery we do

329
00:15:16,900 --> 00:15:19,020
where we instrument the femoral artery up

330
00:15:19,020 --> 00:15:22,720
into the aorta with an indwelling cannula

331
00:15:22,720 --> 00:15:25,420
that's connected to a telemetry probe

332
00:15:25,420 --> 00:15:27,080
so that when the cage is near the probe,

333
00:15:27,080 --> 00:15:29,640
I can chronically measure
this rat's blood pressure.

334
00:15:29,640 --> 00:15:32,870
And we leave that in there
for the duration of the study.

335
00:15:32,870 --> 00:15:35,933
Then to achieve knockdown
of the protein of interest,

336
00:15:35,933 --> 00:15:38,150
the G alpha I two protein,

337
00:15:38,150 --> 00:15:40,820
what we're going to do is put
an ICV cannula into the brain.

338
00:15:40,820 --> 00:15:43,810
So we do stereotactic
neurosurgery on these rats

339
00:15:43,810 --> 00:15:46,630
and into the right lateral
cerebral ventricle,

340
00:15:46,630 --> 00:15:48,530
we insert a stainless steel cannula

341
00:15:48,530 --> 00:15:52,070
that's connected to a mini
pump that's on the back,

342
00:15:52,070 --> 00:15:54,723
filled with antisense
oligodeoxynucleotide,

343
00:15:54,723 --> 00:15:57,810
a variety of flavors.

344
00:15:57,810 --> 00:16:00,860
So as a control, a sham surgery,

345
00:16:00,860 --> 00:16:03,610
we do a scrambled sequence
antisense nucleotide

346
00:16:03,610 --> 00:16:06,840
that we validated to not be complimentary

347
00:16:06,840 --> 00:16:10,150
to any known mRNAs in the rat brain

348
00:16:10,150 --> 00:16:13,063
such that it doesn't render
any proteins knocked down.

349
00:16:13,980 --> 00:16:16,136
And then we also use a
targeted G alpha I two

350
00:16:16,136 --> 00:16:18,640
oligodeoxynucleotide,
another subset of rats,

351
00:16:18,640 --> 00:16:21,900
so that they do get
specific sequence binding

352
00:16:21,900 --> 00:16:24,850
of this antisense ODN to the mRNA

353
00:16:24,850 --> 00:16:28,150
for the G alpha I two protein
rendering it untranslatable.

354
00:16:28,150 --> 00:16:30,590
And then to study the
impact of inflammation,

355
00:16:30,590 --> 00:16:32,560
if there is one, we use a combination

356
00:16:32,560 --> 00:16:35,200
of either of these ODNs with minocycline,

357
00:16:35,200 --> 00:16:37,940
which is an anti-inflammatory drug,

358
00:16:37,940 --> 00:16:41,770
also a tetracycline class
antibiotic that has been shown

359
00:16:41,770 --> 00:16:43,020
in other hypertension studies

360
00:16:43,020 --> 00:16:45,420
to reduce inflammation in the brain.

361
00:16:45,420 --> 00:16:48,590
We randomized these rats to
receive either a normal diet

362
00:16:48,590 --> 00:16:52,800
for control or a high salt
diet with 4% sodium chloride.

363
00:16:52,800 --> 00:16:53,870
And at the end of the study,

364
00:16:53,870 --> 00:16:56,580
we're looking at things like
mean arteriole pressure,

365
00:16:56,580 --> 00:16:59,090
western blotting for the
G alpha I two protein

366
00:16:59,090 --> 00:17:01,150
to validate the knockdown,

367
00:17:01,150 --> 00:17:03,840
plasma neuro epinephrin levels as an index

368
00:17:03,840 --> 00:17:06,450
at how much sympathetic outflow there is.

369
00:17:06,450 --> 00:17:08,800
Because if there's excessive
sympathetic outflow,

370
00:17:08,800 --> 00:17:11,583
there will be a lot of plasma
and epinephrin in the blood.

371
00:17:12,579 --> 00:17:15,050
Cytokine mRNA, looking at whether or not

372
00:17:15,050 --> 00:17:20,050
there are transcripts occurring
that suggests inflammation.

373
00:17:20,350 --> 00:17:22,820
And then lastly, immunohistochemistry.

374
00:17:22,820 --> 00:17:26,580
So antibody-based tissue staining
to look at the morphology,

375
00:17:26,580 --> 00:17:29,680
the structure of cells like microglia

376
00:17:29,680 --> 00:17:32,730
as well as astrocytes respectively stained

377
00:17:32,730 --> 00:17:35,960
by these two proteins, CD11b/c and GFAP,

378
00:17:35,960 --> 00:17:38,860
and pro-inflammatory
anti-inflammatory cytokines

379
00:17:38,860 --> 00:17:40,150
as they've actually been expressed

380
00:17:40,150 --> 00:17:41,623
and secreted into the tissue.

381
00:17:43,270 --> 00:17:46,330
Just to give you one more concept

382
00:17:46,330 --> 00:17:48,970
of how these ODNs work,
the current hypothesis

383
00:17:48,970 --> 00:17:52,440
for how an antisense
complimentary oligodeoxynucleotide

384
00:17:52,440 --> 00:17:55,870
induces protein knock
down is this top one.

385
00:17:55,870 --> 00:17:57,500
This is the leading theory

386
00:17:57,500 --> 00:18:00,930
that there's ribonuclease
H-mediated degradation

387
00:18:00,930 --> 00:18:03,450
of this DNA RNA hybrid.

388
00:18:03,450 --> 00:18:05,920
So the antisense oligodeoxynucleotide

389
00:18:05,920 --> 00:18:10,140
is a single-stranded DNA
complimentary to the mRNA,

390
00:18:10,140 --> 00:18:12,070
you get cleavage and then degradation

391
00:18:12,070 --> 00:18:14,620
of both of the fragments
and so no translation,

392
00:18:14,620 --> 00:18:17,730
and that can happen in the
nucleus or in the cytoplasm.

393
00:18:17,730 --> 00:18:19,100
And should this not be the case,

394
00:18:19,100 --> 00:18:20,270
the next theory that we have

395
00:18:20,270 --> 00:18:23,110
is that there's actually
translation inhibition directly.

396
00:18:23,110 --> 00:18:25,800
So the ribosome's not able
to complete translation

397
00:18:25,800 --> 00:18:28,290
into a functional, mature peptide

398
00:18:28,290 --> 00:18:30,480
because of the blockage that's occurring

399
00:18:30,480 --> 00:18:32,053
from the complimentary binding.

400
00:18:33,010 --> 00:18:37,830
So the first thing we see,
and this is a validation here,

401
00:18:37,830 --> 00:18:42,290
was that with targeted G
alpha I two ODN and fusion,

402
00:18:42,290 --> 00:18:43,860
but not scrambled ODN infusion,

403
00:18:43,860 --> 00:18:46,160
we get downregulation of
G alpha I two proteins.

404
00:18:46,160 --> 00:18:47,840
So western blot here, you can see

405
00:18:47,840 --> 00:18:51,480
this is two lanes of ladder
so that we could know exactly

406
00:18:51,480 --> 00:18:53,180
where we were on the block.

407
00:18:53,180 --> 00:18:56,470
And then S1G1, S2G2 represents animals

408
00:18:56,470 --> 00:18:59,550
that were sham scrambled
control ODN infused

409
00:18:59,550 --> 00:19:02,960
versus receiving a
targeted G alpha I two ODN.

410
00:19:02,960 --> 00:19:06,750
And this here is a
representative loading blot

411
00:19:06,750 --> 00:19:09,280
that's been stained with Coomassie

412
00:19:09,280 --> 00:19:11,610
for total protein loading normalization

413
00:19:11,610 --> 00:19:13,900
as the field is actually
moving a little bit away

414
00:19:13,900 --> 00:19:17,644
from using stains for
beta acting or GAPDH,

415
00:19:17,644 --> 00:19:21,210
these housekeeping proteins,
since they can saturate out

416
00:19:21,210 --> 00:19:24,980
quite easily on a western blot imaging.

417
00:19:24,980 --> 00:19:28,060
And so you get the
normalization of protein levels

418
00:19:28,060 --> 00:19:30,450
in your blot to total protein loaded

419
00:19:30,450 --> 00:19:33,990
across all samples in
several different proteins.

420
00:19:33,990 --> 00:19:37,110
And what we see is that as
previously demonstrated,

421
00:19:37,110 --> 00:19:38,880
our G alpha I two ODN does result

422
00:19:38,880 --> 00:19:42,570
in efficacious downregulation
of the G alpha I two protein.

423
00:19:42,570 --> 00:19:45,223
So we're sure this is working this time.

424
00:19:46,370 --> 00:19:50,170
When we look at the blood
pressure phenotype that we see,

425
00:19:50,170 --> 00:19:51,850
you can see first to have a graph here

426
00:19:51,850 --> 00:19:55,530
for the scrambled ODN and fused rats.

427
00:19:55,530 --> 00:19:58,520
And you can see that when
we switch those control rats

428
00:19:58,520 --> 00:20:01,340
from a normal to their experimental diet

429
00:20:01,340 --> 00:20:02,910
whether that's normal or high salt, right,

430
00:20:02,910 --> 00:20:06,470
the high salt does not evoke
an increase in blood pressure.

431
00:20:06,470 --> 00:20:08,680
So they do maintain salt resistance

432
00:20:08,680 --> 00:20:11,850
for the entirety of the duration
of their high salt diet.

433
00:20:11,850 --> 00:20:14,560
And moreover the minocycline co-infusion

434
00:20:14,560 --> 00:20:16,850
had no baseline impact in these animals,

435
00:20:16,850 --> 00:20:19,360
suggesting there's a lack
of inflammation going on

436
00:20:19,360 --> 00:20:21,340
in a salt resistant rat.

437
00:20:21,340 --> 00:20:24,270
Now in contrast, things start
to get interesting here.

438
00:20:24,270 --> 00:20:26,320
We see as we've previously seen

439
00:20:26,320 --> 00:20:28,950
that immediately following
the switch from a normal

440
00:20:28,950 --> 00:20:31,590
to high salt diet,
animals lacking essential

441
00:20:31,590 --> 00:20:35,310
G alpha I two protein
actually have robust increases

442
00:20:35,310 --> 00:20:37,740
in blood pressure that go up

443
00:20:37,740 --> 00:20:39,160
and up and up over the first few days,

444
00:20:39,160 --> 00:20:42,450
and then plateau out on a high salt diet.

445
00:20:42,450 --> 00:20:44,390
And very interestingly, and our first key

446
00:20:44,390 --> 00:20:46,860
that there might be something going on

447
00:20:46,860 --> 00:20:49,940
with inflammation was the
minocycline significantly reduced

448
00:20:49,940 --> 00:20:51,970
the magnitude of the hypertension here.

449
00:20:51,970 --> 00:20:53,730
So the blood pressure comes back down

450
00:20:53,730 --> 00:20:55,780
when you take away inflammation

451
00:20:55,780 --> 00:20:58,010
or whatever we think the
minocycline at this point

452
00:20:58,010 --> 00:20:58,843
might be doing.

453
00:21:00,060 --> 00:21:03,590
Now to get an understanding
of a few different indices

454
00:21:03,590 --> 00:21:06,040
of blood pressure control systems,

455
00:21:06,040 --> 00:21:09,370
we began, as I mentioned,
with plasma norepinephrine.

456
00:21:09,370 --> 00:21:10,970
So a good index of how much

457
00:21:10,970 --> 00:21:13,380
sympathetic nervous
system activity there is.

458
00:21:13,380 --> 00:21:14,950
And on the left here, you can see

459
00:21:14,950 --> 00:21:19,330
that with a scrambled ODN
infusion, a high salt intake

460
00:21:19,330 --> 00:21:22,267
actually suppresses sympathetic
nervous system activity.

461
00:21:22,267 --> 00:21:23,960
And so this goes back to that figure

462
00:21:23,960 --> 00:21:26,800
I showed you before of our
previous demonstrations.

463
00:21:26,800 --> 00:21:29,320
It goes down with high
salt in a normal rat,

464
00:21:29,320 --> 00:21:30,820
meaning that they've turned off

465
00:21:30,820 --> 00:21:32,030
their sympathetic nervous system

466
00:21:32,030 --> 00:21:34,210
to maintain normal blood pressure.

467
00:21:34,210 --> 00:21:37,430
And importantly, minocycline
just as it didn't impact

468
00:21:37,430 --> 00:21:39,560
the blood pressure in
these rats, does not impact

469
00:21:39,560 --> 00:21:41,710
their sympathetic nervous
system at baseline.

470
00:21:41,710 --> 00:21:43,760
So no impact going on.

471
00:21:43,760 --> 00:21:46,700
And in contrast now, when we
look at the knockout rats,

472
00:21:46,700 --> 00:21:49,860
we see the animals that
lack G alpha I two proteins

473
00:21:50,750 --> 00:21:52,020
actually have an increase

474
00:21:52,020 --> 00:21:54,500
in sympathetic nervous
system activity as measured

475
00:21:54,500 --> 00:21:59,150
by epinephrin, so a very
robust sympathoexcitation

476
00:21:59,150 --> 00:22:02,060
which was normalized by minocycline.

477
00:22:02,060 --> 00:22:04,620
So that was really cool
when we did these studies

478
00:22:04,620 --> 00:22:05,480
and we actually found

479
00:22:05,480 --> 00:22:09,520
that you could turn off
excessive sympathetic activity

480
00:22:09,520 --> 00:22:11,230
through this minocycline co-infusion.

481
00:22:11,230 --> 00:22:13,100
And that was the first time
we thought to ourselves

482
00:22:13,100 --> 00:22:14,390
in addition to the blood pressure,

483
00:22:14,390 --> 00:22:15,913
we might have something here.

484
00:22:16,880 --> 00:22:19,513
Now we have two more indices here.

485
00:22:20,720 --> 00:22:23,040
These are almost like
controls that we performed.

486
00:22:23,040 --> 00:22:25,160
There's another pathway that
regulates blood pressure

487
00:22:25,160 --> 00:22:27,820
that's not really something we look at

488
00:22:27,820 --> 00:22:30,770
because as I'll show you,
it's not particularly active.

489
00:22:30,770 --> 00:22:33,700
And it's known as the
renin-angiotensin-aldosterone system.

490
00:22:33,700 --> 00:22:35,820
And this is another kidney-based system

491
00:22:35,820 --> 00:22:38,150
of hormonal blood pressure regulation

492
00:22:38,150 --> 00:22:41,110
that we can actually see
when we look is suppressed.

493
00:22:41,110 --> 00:22:45,500
So plasma renin activity is a good index

494
00:22:45,500 --> 00:22:47,900
of how much renin enzyme activity

495
00:22:47,900 --> 00:22:51,370
generating angiotensin one
there is in these rats.

496
00:22:51,370 --> 00:22:54,720
And what you can see is that
regardless of ODN infusion,

497
00:22:54,720 --> 00:22:57,810
so whether they have G
alpha I two proteins or not,

498
00:22:57,810 --> 00:23:01,250
high salt diet evokes
significant suppression of this

499
00:23:01,250 --> 00:23:05,710
as well as suppression of
angiotensinogen in the urine.

500
00:23:05,710 --> 00:23:10,060
So what you see is that with regards

501
00:23:10,060 --> 00:23:11,520
to both of these assessments

502
00:23:11,520 --> 00:23:14,110
of renin-angiotensin-aldosterone
system activity,

503
00:23:14,110 --> 00:23:16,530
that system appears to be A, suppress

504
00:23:16,530 --> 00:23:20,150
and B, not related to
G alpha I two proteins.

505
00:23:20,150 --> 00:23:22,380
So what we're thinking is we mostly have

506
00:23:22,380 --> 00:23:25,210
a sympathetic nervous system model

507
00:23:25,210 --> 00:23:27,010
of blood pressure regulation here,

508
00:23:27,010 --> 00:23:30,193
independent from rat's pathway activity.

509
00:23:32,260 --> 00:23:34,560
When we look at cytokine
expression levels,

510
00:23:34,560 --> 00:23:38,020
which now is gonna be a direct
assessment of inflammation,

511
00:23:38,020 --> 00:23:42,830
what we see is that with
respect to A, B and C

512
00:23:42,830 --> 00:23:45,420
which are IL one beta, IL six

513
00:23:45,420 --> 00:23:48,720
and TNF alpha pro-inflammatory cytokines

514
00:23:48,720 --> 00:23:52,950
and D which IL10, an
anti-inflammatory cytokine,

515
00:23:52,950 --> 00:23:56,000
there's no impact of a high salt diet

516
00:23:56,000 --> 00:23:59,330
in our control scrambled ODN infused rats.

517
00:23:59,330 --> 00:24:02,010
So there's no pro-inflammatory signaling,

518
00:24:02,010 --> 00:24:04,520
there's no anti-inflammatory
signaling changes

519
00:24:04,520 --> 00:24:07,230
and minocycline doesn't impact baseline

520
00:24:07,230 --> 00:24:08,240
in these animals either.

521
00:24:08,240 --> 00:24:10,820
So not really much going on here.

522
00:24:10,820 --> 00:24:11,930
There's some pathway inhibiting,

523
00:24:11,930 --> 00:24:15,270
they've got normal blood
pressure, no inflammation.

524
00:24:15,270 --> 00:24:18,700
When we look in the knockout
rats, we see this seesaw effect

525
00:24:18,700 --> 00:24:22,260
where pro-inflammatory signaling comes up

526
00:24:22,260 --> 00:24:24,490
at the expression level of the transcripts

527
00:24:24,490 --> 00:24:28,040
and anti-inflammatory
signaling is suppressed.

528
00:24:28,040 --> 00:24:30,530
So you actually get an imbalance

529
00:24:30,530 --> 00:24:33,200
of pro and anti inflammatory
signaling favoring

530
00:24:33,200 --> 00:24:36,430
pro-inflammatory
transcripts in these rats.

531
00:24:36,430 --> 00:24:38,740
And all of these data were normalized

532
00:24:38,740 --> 00:24:40,450
by minocycline infusion as well.

533
00:24:40,450 --> 00:24:42,970
So this brought us to
the notion that, okay.

534
00:24:42,970 --> 00:24:45,470
Yep, we're definitely
changing inflammation

535
00:24:45,470 --> 00:24:49,380
when we give this minocycline
on top of the blood pressure

536
00:24:49,380 --> 00:24:52,380
and the sympathetic changes
that are going back to baseline.

537
00:24:53,770 --> 00:24:57,440
And here I have some preliminary
immunofluorescent stains

538
00:24:57,440 --> 00:24:58,273
in the PVN.

539
00:24:58,273 --> 00:24:59,610
So the histological slides

540
00:24:59,610 --> 00:25:03,960
that we've done to show
upregulation of the protein

541
00:25:03,960 --> 00:25:05,680
within the actual tissue

542
00:25:07,478 --> 00:25:10,650
that's happening following
these transcriptional changes.

543
00:25:10,650 --> 00:25:12,220
And we're currently working out ways

544
00:25:12,220 --> 00:25:14,790
to better assess the
protein, but you can see here

545
00:25:14,790 --> 00:25:18,330
between a scrambled rat and
a G alpha I two knockdown rat

546
00:25:18,330 --> 00:25:21,610
on a high salt diet, that
transcriptional change

547
00:25:21,610 --> 00:25:25,320
where there's elevation in IL
six and IL one beta follows

548
00:25:25,320 --> 00:25:27,900
through to the protein level it appears

549
00:25:27,900 --> 00:25:30,683
because there's so much
more robust immunostaining.

550
00:25:32,260 --> 00:25:34,930
And now trying to understand

551
00:25:34,930 --> 00:25:37,610
the cellular localization of these,

552
00:25:37,610 --> 00:25:40,050
we go to a couple of
different cells in the brain

553
00:25:40,050 --> 00:25:41,890
which can contribute to inflammation.

554
00:25:41,890 --> 00:25:42,723
And as I mentioned before,

555
00:25:42,723 --> 00:25:44,790
those are microglia and astrocytes.

556
00:25:44,790 --> 00:25:47,850
Now astrocytes, to start, is
what you're looking at here.

557
00:25:47,850 --> 00:25:52,500
You've got these images,
astrocytes are neural cells.

558
00:25:52,500 --> 00:25:54,710
So they derive from the same stem cells

559
00:25:54,710 --> 00:25:58,140
that neurons do in the developing brain.

560
00:25:58,140 --> 00:26:00,350
And they are a critical component

561
00:26:00,350 --> 00:26:01,500
of the blood brain barrier,

562
00:26:01,500 --> 00:26:04,180
as you can see them
surrounding blood vessels here,

563
00:26:04,180 --> 00:26:05,650
as well as playing a role

564
00:26:05,650 --> 00:26:08,770
in interstitial glutamate homeostasis,

565
00:26:08,770 --> 00:26:13,640
pH regulation of the brain
tissue, and they can contribute

566
00:26:13,640 --> 00:26:16,750
to a cytokine production
when they're inflamed.

567
00:26:16,750 --> 00:26:19,380
But what we see when
we do a quantification

568
00:26:19,380 --> 00:26:21,800
across all these groups,

569
00:26:21,800 --> 00:26:24,010
whether they have G alpha
I two proteins or not,

570
00:26:24,010 --> 00:26:27,160
is that a high salt diet
does not evoke an increase

571
00:26:27,160 --> 00:26:29,080
in the density in that tissue

572
00:26:30,690 --> 00:26:33,540
of these astrocytes,
suggesting to us at this point

573
00:26:33,540 --> 00:26:37,100
that there's no change going
on in astrocyte reactivity.

574
00:26:37,100 --> 00:26:38,440
They're probably not contributing

575
00:26:38,440 --> 00:26:41,220
to this inflammatory process
because they would change

576
00:26:41,220 --> 00:26:43,740
on immunostaining and take up more space,

577
00:26:43,740 --> 00:26:46,883
stain more intensely, and
they're not doing that.

578
00:26:47,730 --> 00:26:49,570
But when we look at microglia

579
00:26:49,570 --> 00:26:51,640
which are innate immune cells derived

580
00:26:51,640 --> 00:26:56,150
from precursor immune
cells during development

581
00:26:56,150 --> 00:26:58,140
that migrate to the nervous system

582
00:26:58,140 --> 00:27:02,120
and play immune functions there,
there's a different story.

583
00:27:02,120 --> 00:27:04,480
Now, if we look at the percent

584
00:27:04,480 --> 00:27:07,330
that are categorized as active or reactive

585
00:27:07,330 --> 00:27:11,110
in the PVN in a scrambled control rat,

586
00:27:11,110 --> 00:27:14,440
we see that we don't have any
changes in immunoreactivity

587
00:27:14,440 --> 00:27:18,300
from normal to high salt diet,
no change in the actual shape

588
00:27:18,300 --> 00:27:21,060
and structure of these microglia
and minocycline, moreover,

589
00:27:21,060 --> 00:27:22,880
doesn't really do anything here either.

590
00:27:22,880 --> 00:27:25,760
You see, we have nice,
branched to microglia

591
00:27:25,760 --> 00:27:28,770
which suggests that they
possess an anti-inflammatory

592
00:27:28,770 --> 00:27:31,040
or surveillant phenotype.

593
00:27:31,040 --> 00:27:33,970
And that's what the G alpha
I two knockout rats look like

594
00:27:33,970 --> 00:27:36,310
at rest at normal salt as well.

595
00:27:36,310 --> 00:27:37,540
But as you can see,

596
00:27:37,540 --> 00:27:40,540
there's a robust increase
in the tissue expression

597
00:27:40,540 --> 00:27:43,750
by these microglia of the CD11b/c marker

598
00:27:43,750 --> 00:27:46,320
also known as the
compliment receptor three

599
00:27:46,320 --> 00:27:48,380
following a high salt diet.

600
00:27:48,380 --> 00:27:50,010
So what it might be is that

601
00:27:50,010 --> 00:27:53,320
these microglia are
actually the cellular source

602
00:27:53,320 --> 00:27:55,940
of the cytokines that we
saw being up-regulated

603
00:27:55,940 --> 00:28:00,117
because they possess a much
more inflammatory morphology

604
00:28:01,890 --> 00:28:06,890
at this point, as well as
being upregulated in quantity

605
00:28:07,310 --> 00:28:10,400
and having upregulated
their compliment receptor.

606
00:28:10,400 --> 00:28:14,390
And just like the rest of the
indices we saw in these rats,

607
00:28:14,390 --> 00:28:16,793
they were normalized by
minocycline infusion.

608
00:28:17,970 --> 00:28:20,670
This does go back as well to literature

609
00:28:20,670 --> 00:28:23,100
that suggests minocycline can act

610
00:28:23,100 --> 00:28:24,690
by inhibiting immune cells.

611
00:28:24,690 --> 00:28:26,240
And so it's quite possible here

612
00:28:26,240 --> 00:28:29,640
that the effective minocycline
infused into the brain

613
00:28:29,640 --> 00:28:33,450
was directly to inhibit
microglial reactivity

614
00:28:33,450 --> 00:28:35,150
in response to high salt

615
00:28:35,150 --> 00:28:37,570
to prevent the production of cytokines

616
00:28:37,570 --> 00:28:41,250
and the upregulation of
sympathetic nervous system activity

617
00:28:41,250 --> 00:28:44,300
which is what ultimately
helps bring the blood pressure

618
00:28:44,300 --> 00:28:45,763
partially back down.

619
00:28:47,450 --> 00:28:50,480
Now, when we assess those microglia here,

620
00:28:50,480 --> 00:28:52,790
we used a semi quantitative method

621
00:28:52,790 --> 00:28:56,383
that was somewhat subjective
of a manual cell counting,

622
00:28:57,560 --> 00:28:59,340
but we wanted to really
be sure we were right

623
00:28:59,340 --> 00:29:01,460
about the morphologic
changes that we were seeing

624
00:29:01,460 --> 00:29:02,830
in these microglia from being nice

625
00:29:02,830 --> 00:29:05,010
and branched out anti-inflammatory

626
00:29:05,010 --> 00:29:07,010
to having their processes pulled in,

627
00:29:07,010 --> 00:29:10,750
and their somas, what's known
as hypertrophic or amoeboid.

628
00:29:10,750 --> 00:29:13,600
And so we used an index Sholl Analysis.

629
00:29:13,600 --> 00:29:14,720
And with Sholl Analysis,

630
00:29:14,720 --> 00:29:19,720
what we ultimately did
was we took each cell

631
00:29:19,990 --> 00:29:22,940
for many cells from each tissue slice

632
00:29:22,940 --> 00:29:26,700
and cropped images of them
alone down, and subjected them

633
00:29:26,700 --> 00:29:30,840
to a computerized process
where a software program places

634
00:29:30,840 --> 00:29:32,740
a dot in the center of the soma

635
00:29:32,740 --> 00:29:36,500
and a set of equally distant
concentric rings radiating

636
00:29:36,500 --> 00:29:37,820
out from the soma.

637
00:29:37,820 --> 00:29:39,340
And then the software counts

638
00:29:39,340 --> 00:29:42,290
how many times the
processes of these microglia

639
00:29:42,290 --> 00:29:45,290
actually intersect these rings

640
00:29:45,290 --> 00:29:47,350
and it formulates a chart like this.

641
00:29:47,350 --> 00:29:49,010
And what you get is on the X axis,

642
00:29:49,010 --> 00:29:50,810
how far away you're going from that dot

643
00:29:50,810 --> 00:29:53,150
in the center of the soma and the Y axis,

644
00:29:53,150 --> 00:29:55,620
how many intersections are occurring.

645
00:29:55,620 --> 00:29:59,260
And so you get this fun
peak that's occurring here.

646
00:29:59,260 --> 00:30:01,310
And basically, the way you interpret this

647
00:30:01,310 --> 00:30:04,837
is that the higher the peak
is over a given distance,

648
00:30:04,837 --> 00:30:09,837
the more branched and
complexity this microglia has.

649
00:30:09,920 --> 00:30:13,050
And so when you get average
plots for each group,

650
00:30:13,050 --> 00:30:15,420
what you can do is compare them

651
00:30:15,420 --> 00:30:18,060
and see whether or not
average branch in complexity

652
00:30:18,060 --> 00:30:23,060
as an index of microglial
inflammatory status changes.

653
00:30:23,130 --> 00:30:25,120
And so first what we saw

654
00:30:25,120 --> 00:30:27,560
in our scrambled control rats was that

655
00:30:27,560 --> 00:30:30,970
all of the peaks for these
different rats basically happened

656
00:30:30,970 --> 00:30:33,840
at the same point and
their standard deviations

657
00:30:33,840 --> 00:30:36,580
amongst the groups
overlapped significantly,

658
00:30:36,580 --> 00:30:39,300
suggesting to us that
what we had seen first

659
00:30:39,300 --> 00:30:42,720
with no changes in our subjective
morphology were correct.

660
00:30:42,720 --> 00:30:45,050
There was no change in
branch of complexity

661
00:30:45,050 --> 00:30:46,910
of these microglia induced by high salt.

662
00:30:46,910 --> 00:30:50,193
So they're maintaining their
anti-inflammatory phenotype.

663
00:30:51,050 --> 00:30:53,210
And in contrast, what you can see here

664
00:30:53,210 --> 00:30:55,970
is this peak occurring in the normal salt

665
00:30:55,970 --> 00:31:00,180
G alpha I two knockdown rats
is significantly reduced

666
00:31:00,180 --> 00:31:05,080
by about seven intersections
per group overall.

667
00:31:06,670 --> 00:31:11,500
So there's really less branching
going on in this group,

668
00:31:11,500 --> 00:31:14,280
a significant lesser amount of branching,

669
00:31:14,280 --> 00:31:16,740
suggesting that they've
pulled in those processes.

670
00:31:16,740 --> 00:31:19,870
And they do have that
hypertrophic morphology

671
00:31:19,870 --> 00:31:21,330
that we were seeing on histology.

672
00:31:21,330 --> 00:31:24,070
And so this kind of
validated for us, oh yeah,

673
00:31:24,070 --> 00:31:25,230
we were seeing activation.

674
00:31:25,230 --> 00:31:27,650
And what you can see was that

675
00:31:27,650 --> 00:31:29,960
towards the tail end of the branches,

676
00:31:29,960 --> 00:31:33,920
they began to branch back
out and significantly come up

677
00:31:33,920 --> 00:31:36,460
from the high salt group
when you gave minocycline.

678
00:31:36,460 --> 00:31:39,540
So there was restoration
of the branch in complexity

679
00:31:39,540 --> 00:31:42,460
with minocycline infusion,
suggesting that, yep,

680
00:31:42,460 --> 00:31:45,283
the minocycline was acting
through the microglia.

681
00:31:46,440 --> 00:31:49,080
And altogether, these things led us

682
00:31:49,080 --> 00:31:50,906
to our first conclusion
and the first conclusion

683
00:31:50,906 --> 00:31:54,490
of my studies as a PhD student,

684
00:31:54,490 --> 00:31:57,120
which was kind of to
remake that figure about

685
00:31:57,120 --> 00:31:59,570
salt sensitive rats I
showed you at the beginning

686
00:31:59,570 --> 00:32:01,520
that in a salt sensitive rat,

687
00:32:01,520 --> 00:32:04,320
a high sodium intake
does not evoke changes

688
00:32:04,320 --> 00:32:06,173
in PVN G alpha I two protein levels,

689
00:32:08,040 --> 00:32:09,840
and that in a renal nerve dependent way,

690
00:32:09,840 --> 00:32:12,130
you still get some pathogenic citation

691
00:32:12,130 --> 00:32:14,240
and renal sodium resorption

692
00:32:14,240 --> 00:32:17,450
and thus changes in pressure,
nature, recess relationship,

693
00:32:17,450 --> 00:32:19,900
meaning big changes in
blood pressure required

694
00:32:19,900 --> 00:32:22,830
to offset that sodium dysregulation.

695
00:32:22,830 --> 00:32:24,470
But now we've added in a component

696
00:32:24,470 --> 00:32:27,970
where there's a pro-inflammatory
PVN microenvironment

697
00:32:27,970 --> 00:32:30,760
featuring microglia that produce cytokines

698
00:32:30,760 --> 00:32:33,480
like interleukin six
and interleukin one beta

699
00:32:33,480 --> 00:32:36,373
to drive the development of
salt sensitive hypertension.

700
00:32:37,980 --> 00:32:40,700
Now that was a lot, but I've
got a little more for you.

701
00:32:40,700 --> 00:32:44,670
And so these studies led us to think

702
00:32:44,670 --> 00:32:45,830
a couple of different things

703
00:32:45,830 --> 00:32:48,100
that formed the next chapter of my thesis.

704
00:32:48,100 --> 00:32:50,390
And I'm going to go over
the first one with you

705
00:32:50,390 --> 00:32:51,710
because we were thinking to ourselves,

706
00:32:51,710 --> 00:32:52,990
this happened in male rats.

707
00:32:52,990 --> 00:32:56,040
But studies have shown
robust sex differences.

708
00:32:56,040 --> 00:32:58,230
So what about female rats?

709
00:32:58,230 --> 00:33:00,800
And these phenomena are correlated

710
00:33:00,800 --> 00:33:03,420
but asking a chicken and egg question,

711
00:33:03,420 --> 00:33:06,600
which comes first,
inflammation, hypertension,

712
00:33:06,600 --> 00:33:09,123
some currently undiscovered factor.

713
00:33:10,400 --> 00:33:13,620
And so to get into the possibility

714
00:33:13,620 --> 00:33:15,620
that there are gonna be sex differences,

715
00:33:17,030 --> 00:33:19,550
let me just talk about
that for a little bit.

716
00:33:19,550 --> 00:33:22,000
We've got sex differences

717
00:33:22,000 --> 00:33:23,823
in multiple models of hypertension.

718
00:33:24,880 --> 00:33:28,310
In humans and in animals,
there has been a robust role

719
00:33:28,310 --> 00:33:32,210
for sex steroid hormones,
particularly estrogen

720
00:33:32,210 --> 00:33:37,210
like estradiol, estrone in
modulating cardiovascular risk.

721
00:33:38,150 --> 00:33:41,660
And this is evident in females,

722
00:33:41,660 --> 00:33:44,240
human females, people
assigned female at birth

723
00:33:44,240 --> 00:33:47,900
that have female sex steroid hormones

724
00:33:47,900 --> 00:33:50,730
and their cardiovascular
risk pre-menopause

725
00:33:50,730 --> 00:33:55,040
is very, very low versus post-menopause

726
00:33:55,040 --> 00:33:58,060
when it actually shoots up over two fold.

727
00:33:58,060 --> 00:34:01,550
And so to kind of get a
model of this and understand

728
00:34:01,550 --> 00:34:03,670
whether or not this would
hold true with respect

729
00:34:03,670 --> 00:34:06,743
to the expression of G
alpha I two proteins,

730
00:34:07,830 --> 00:34:10,240
we started looking in female rats.

731
00:34:10,240 --> 00:34:12,670
So using a similar paradigm as last time,

732
00:34:12,670 --> 00:34:15,730
what you can see is that with
the female Sprague-Dawley rat,

733
00:34:15,730 --> 00:34:18,920
we inserted the same
intracerebral ventricular cannula

734
00:34:18,920 --> 00:34:21,300
into the right lateral ventricle.

735
00:34:21,300 --> 00:34:24,240
And we infused these rats with
either a controlled scrambled

736
00:34:24,240 --> 00:34:28,550
or targeted G alpha I two ODN,
randomized them to a normal

737
00:34:28,550 --> 00:34:30,340
or elevated dietary salt intake

738
00:34:30,340 --> 00:34:32,220
and looked at things like blood pressure,

739
00:34:32,220 --> 00:34:34,360
G alpha I two protein
expression to validate

740
00:34:34,360 --> 00:34:37,070
whether or not the knockdown
was still occurring.

741
00:34:37,070 --> 00:34:39,870
Sodium balance, plasma norepinephrine

742
00:34:39,870 --> 00:34:44,870
as an index of sympathetic
nervous system activity.

743
00:34:45,230 --> 00:34:49,330
And again, tissue markers
of microglia and astrocytes,

744
00:34:49,330 --> 00:34:51,640
the immune cells that
can possibly contribute

745
00:34:51,640 --> 00:34:56,603
to inflammation if there
should be any in the brain.

746
00:34:58,130 --> 00:35:00,490
What we first saw, and this
is a very similar graph

747
00:35:00,490 --> 00:35:02,000
to what you've seen last time,

748
00:35:02,000 --> 00:35:04,780
was that the scrambled ODM infused rats

749
00:35:04,780 --> 00:35:07,890
have a robust expression of
the G alpha I two protein

750
00:35:07,890 --> 00:35:09,893
at about 41 kilodaltons.

751
00:35:11,280 --> 00:35:15,360
And our G alpha I two ODN
infusion significantly brings down

752
00:35:15,360 --> 00:35:17,700
that expression almost to zero.

753
00:35:17,700 --> 00:35:21,373
So very, very robust decrease
in tissue protein expression.

754
00:35:22,290 --> 00:35:25,610
And that's again, a Coomassie stained blot

755
00:35:25,610 --> 00:35:28,150
that was loaded simultaneously to the gel

756
00:35:28,150 --> 00:35:30,200
from which this was
transferred to a membrane

757
00:35:30,200 --> 00:35:33,030
so that you can validate
normal protein loading

758
00:35:33,030 --> 00:35:34,363
across all lines.

759
00:35:36,260 --> 00:35:38,700
And then we looked at blood pressure,

760
00:35:38,700 --> 00:35:41,300
and really interestingly what we saw was,

761
00:35:41,300 --> 00:35:43,400
again, at a scrambled control rat,

762
00:35:43,400 --> 00:35:47,430
there's no high salt diet evoked
increase in blood pressure.

763
00:35:47,430 --> 00:35:49,170
And in our knockout rat,

764
00:35:49,170 --> 00:35:51,930
there's interestingly
only a mild increase.

765
00:35:51,930 --> 00:35:53,910
And to give you some comparison here,

766
00:35:53,910 --> 00:35:55,870
these are day seven acute blood pressures,

767
00:35:55,870 --> 00:35:58,630
not chronic blood pressures
over time like I showed you,

768
00:35:58,630 --> 00:36:00,393
but just one day blood pressures.

769
00:36:01,539 --> 00:36:03,330
And at the same time and point,

770
00:36:03,330 --> 00:36:06,210
these female rats increase
their average blood pressure

771
00:36:06,210 --> 00:36:08,330
by about seven or eight
millimeters of mercury,

772
00:36:08,330 --> 00:36:13,330
whereas the males increased
from about 125 to almost 150.

773
00:36:13,970 --> 00:36:17,530
So there's a significant
difference in the magnitude

774
00:36:17,530 --> 00:36:19,000
of hypertension evoked

775
00:36:19,000 --> 00:36:22,480
following G alpha I two
protein downregulation.

776
00:36:22,480 --> 00:36:26,570
So we're already seeing sex
differences occurring here.

777
00:36:26,570 --> 00:36:28,320
Now, that's interesting.

778
00:36:28,320 --> 00:36:31,400
When we look at plasma
norepinephrine as a measure

779
00:36:31,400 --> 00:36:33,423
of sympathetic nervous system activity,

780
00:36:34,270 --> 00:36:37,650
we first see that there's
actually a significant reduction

781
00:36:37,650 --> 00:36:39,420
in plasma norepinephrine levels

782
00:36:39,420 --> 00:36:41,923
in the scrambled control
rats, these female rats,

783
00:36:43,070 --> 00:36:47,030
but what was really kind of a brain teaser

784
00:36:47,030 --> 00:36:49,850
was that they still sympathoinhibited

785
00:36:49,850 --> 00:36:52,840
even after you'd taken out
essential G alpha I two protein.

786
00:36:52,840 --> 00:36:56,480
So they actually don't
develop sympathoexcitation

787
00:36:56,480 --> 00:36:57,870
the way a male rat does.

788
00:36:57,870 --> 00:36:59,820
And this was interesting because

789
00:36:59,820 --> 00:37:01,900
when we sat down to think about it,

790
00:37:01,900 --> 00:37:05,810
we thought, well, maybe these
rats won't sympathoexcite.

791
00:37:05,810 --> 00:37:08,450
Maybe there'll be protected
from going up with respect

792
00:37:08,450 --> 00:37:12,130
to neuro epinephrin, but
they shouldn't still go down.

793
00:37:12,130 --> 00:37:14,890
They've lost their regulatory
mechanism responsible

794
00:37:14,890 --> 00:37:16,063
for facilitating that.

795
00:37:17,410 --> 00:37:20,510
And sometimes science
is a mystery. (chuckles)

796
00:37:20,510 --> 00:37:23,970
When we look at sodium balance
now, because we want to see

797
00:37:23,970 --> 00:37:25,950
if they even developed salt sensitivity,

798
00:37:25,950 --> 00:37:27,640
do they retain sodium?

799
00:37:27,640 --> 00:37:30,230
What you can see is we've got
these nice baseline values

800
00:37:30,230 --> 00:37:32,570
here on this longitudinal time graph

801
00:37:32,570 --> 00:37:34,550
where they're on normal salt.

802
00:37:34,550 --> 00:37:37,340
And when you switch these
rats to a high salt diet,

803
00:37:37,340 --> 00:37:40,450
there's only ever a single
significant increase

804
00:37:40,450 --> 00:37:42,910
in sodium retention calculated

805
00:37:42,910 --> 00:37:45,690
as the difference between
the sodium consumed

806
00:37:45,690 --> 00:37:48,250
and the sodium excrete it in the urine.

807
00:37:48,250 --> 00:37:50,600
And then you returned to
non-significant difference

808
00:37:50,600 --> 00:37:54,610
from baseline and the scrambled
rats of course never deviate

809
00:37:54,610 --> 00:37:57,690
from baseline in a
statistically significant way.

810
00:37:57,690 --> 00:38:00,620
So they only develop mild hypertension.

811
00:38:00,620 --> 00:38:02,993
They maintain their
ability to sympathoinhibit,

812
00:38:02,993 --> 00:38:05,690
and their sodium retention
is not very bad at all

813
00:38:05,690 --> 00:38:08,870
especially when you consider
it in comparison to the males

814
00:38:08,870 --> 00:38:12,580
which display a greater salt
sensitivity of blood pressure,

815
00:38:12,580 --> 00:38:15,010
such that even the scrambled control rats

816
00:38:15,010 --> 00:38:17,670
significantly retain sodium for some time

817
00:38:17,670 --> 00:38:21,350
before coming to baseline,
although not nearly as bad

818
00:38:21,350 --> 00:38:23,110
as the G alpha I two knockdown rats

819
00:38:23,110 --> 00:38:25,680
which have a very
exacerbated sodium retention

820
00:38:25,680 --> 00:38:28,010
over several days that doesn't return

821
00:38:28,010 --> 00:38:31,210
to baseline values until
the very end of the study,

822
00:38:31,210 --> 00:38:32,763
seven days of high salt diet.

823
00:38:33,630 --> 00:38:37,510
So they're protected from
many things here, it appears.

824
00:38:37,510 --> 00:38:38,740
When we looked in the PVN

825
00:38:38,740 --> 00:38:41,710
at indices of inflammation,
you can see up here,

826
00:38:41,710 --> 00:38:44,070
these scrambled control rats,

827
00:38:44,070 --> 00:38:46,660
no change in microglial cell count,

828
00:38:46,660 --> 00:38:50,870
no change on average in
the number or percent

829
00:38:50,870 --> 00:38:53,170
that are active amongst these animals,

830
00:38:53,170 --> 00:38:54,970
and the same holds true

831
00:38:54,970 --> 00:38:57,100
following G alpha I two
protein knocked down

832
00:38:57,100 --> 00:38:58,040
in high salt diet.

833
00:38:58,040 --> 00:39:00,820
There's a failure to evoke an increase

834
00:39:00,820 --> 00:39:02,160
in microglial reactivity.

835
00:39:02,160 --> 00:39:04,740
So the next big difference, obviously,

836
00:39:04,740 --> 00:39:07,080
as I'm showing you here is a complete lack

837
00:39:07,080 --> 00:39:09,680
of inflammatory cell activation

838
00:39:09,680 --> 00:39:11,610
which may partially be responsible

839
00:39:11,610 --> 00:39:14,980
for the lack of
sympathoexcitation we see here

840
00:39:14,980 --> 00:39:17,460
because as we saw with the male rats,

841
00:39:17,460 --> 00:39:21,930
when you take down the
inflammation, you completely remove

842
00:39:21,930 --> 00:39:25,390
the sympathetic nervous
system excessive activity.

843
00:39:25,390 --> 00:39:28,310
So without it ever happening at baseline,

844
00:39:28,310 --> 00:39:31,540
it may be that they are not developing

845
00:39:31,540 --> 00:39:33,290
robust salt sensitive hypertension.

846
00:39:34,160 --> 00:39:36,220
And lastly, we look at these astrocytes

847
00:39:36,220 --> 00:39:37,670
and we can see the same thing.

848
00:39:37,670 --> 00:39:39,930
There's a complete lack of an impact

849
00:39:39,930 --> 00:39:43,130
of high salt diet on changing
the immunostaining intensity

850
00:39:43,130 --> 00:39:45,480
or reactivity of astrocytes, suggesting

851
00:39:45,480 --> 00:39:48,470
that female rats are not
going to develop inflammation

852
00:39:48,470 --> 00:39:49,303
in the brain.

853
00:39:50,640 --> 00:39:52,690
And this led us to an initial conclusion

854
00:39:52,690 --> 00:39:54,860
for these studies that
they develop blunted

855
00:39:54,860 --> 00:39:57,500
salt sensitive hypertension, female rats,

856
00:39:57,500 --> 00:40:01,390
which lack inflammation and
sympathoexcitation overall.

857
00:40:01,390 --> 00:40:03,200
So there are robust sex differences

858
00:40:03,200 --> 00:40:07,240
which we may be able to attribute
to sex steroid hormones.

859
00:40:07,240 --> 00:40:09,070
Now, just to give you some
quick future directions

860
00:40:09,070 --> 00:40:11,710
for these studies, we
want to investigate things

861
00:40:11,710 --> 00:40:14,010
like the electrophysiological impacts

862
00:40:14,010 --> 00:40:15,720
of G alpha I two proteins.

863
00:40:15,720 --> 00:40:18,340
So how do they impact neuronal firing?

864
00:40:18,340 --> 00:40:21,700
How do they impact neuronal
resting membrane potentials

865
00:40:21,700 --> 00:40:23,620
as well as what is the impact of these

866
00:40:23,620 --> 00:40:27,010
on single cell parvocellular
neuron transcriptomics?

867
00:40:27,010 --> 00:40:29,930
How is the phenotype of a
neuron, for example, altered

868
00:40:29,930 --> 00:40:31,770
when these proteins are lost

869
00:40:31,770 --> 00:40:35,380
and further studies impact
investigating the impact of,

870
00:40:35,380 --> 00:40:37,760
as I just mentioned,
the sex steroid hormones

871
00:40:37,760 --> 00:40:40,490
on this model of
hypertension in the absence

872
00:40:40,490 --> 00:40:42,830
and presence of G alpha I two proteins?

873
00:40:42,830 --> 00:40:46,040
With that, I'd like to
think these are just

874
00:40:46,040 --> 00:40:47,630
some of the people that are listed here

875
00:40:47,630 --> 00:40:50,290
but several of these wonderful individuals

876
00:40:50,290 --> 00:40:53,290
as well as my laboratory and
my mentor, Dr. Richard Winford,

877
00:40:54,690 --> 00:40:56,100
some previous members are here.

878
00:40:56,100 --> 00:40:58,820
Current members are here, but
my dissertation committee,

879
00:40:58,820 --> 00:40:59,810
they've all been fabulous

880
00:40:59,810 --> 00:41:01,980
in helping me refine this project

881
00:41:01,980 --> 00:41:05,020
as well as my department and our funders.

882
00:41:05,020 --> 00:41:07,923
And with that, I'm very
happy to take your questions.

883
00:41:09,630 --> 00:41:11,820
- Thank you so much for such a great talk.

884
00:41:11,820 --> 00:41:13,670
I really appreciate your time

885
00:41:13,670 --> 00:41:15,700
and I appreciate you sharing with us

886
00:41:15,700 --> 00:41:17,750
all this good stuff you've been up to.

887
00:41:17,750 --> 00:41:19,230
So thank you.

888
00:41:19,230 --> 00:41:21,600
All right, so we have a few
questions here for you already.

889
00:41:21,600 --> 00:41:26,540
So the first one is what
tests can be done to find out

890
00:41:26,540 --> 00:41:29,890
if you are salt sensitive
or if you possess mechanisms

891
00:41:29,890 --> 00:41:31,563
for salt resistance?

892
00:41:32,530 --> 00:41:34,820
- So that's a really great question.

893
00:41:34,820 --> 00:41:36,930
In humans, we're currently studying

894
00:41:36,930 --> 00:41:40,750
the G alpha I two protein to
look for easier biomarkers

895
00:41:40,750 --> 00:41:43,550
for the identification
of salt sensitivity.

896
00:41:43,550 --> 00:41:44,950
We've done a couple of studies

897
00:41:44,950 --> 00:41:47,980
with respect to a single
nucleotide polymorphisms

898
00:41:47,980 --> 00:41:50,900
in the GNAI2 gene as well
as some other groups have,

899
00:41:50,900 --> 00:41:53,020
showing its significant correlations

900
00:41:53,020 --> 00:41:54,380
with essential hypertension.

901
00:41:54,380 --> 00:41:57,350
And that goes back to
looking for easier biomarkers

902
00:41:57,350 --> 00:41:59,940
because it's a very robust study

903
00:41:59,940 --> 00:42:02,070
that you have to do with
humans over several weeks

904
00:42:02,070 --> 00:42:05,450
where you sodium deplete
them, feed them high sodium,

905
00:42:05,450 --> 00:42:08,620
sodium deplete them and take
ambulatory blood pressures

906
00:42:08,620 --> 00:42:09,453
all throughout the way.

907
00:42:09,453 --> 00:42:12,360
So it's very involved to
determine at this point

908
00:42:12,360 --> 00:42:13,960
if you're salt sensitive or not.

909
00:42:17,480 --> 00:42:19,040
- We have another question.

910
00:42:19,040 --> 00:42:22,440
What was the motivation for you to move

911
00:42:22,440 --> 00:42:26,003
from muscle physiology to neuro
immunology and neuroscience?

912
00:42:27,720 --> 00:42:32,090
- Oh, I want to be as honest as possible.

913
00:42:32,090 --> 00:42:35,930
It wasn't as direct of a
motivation as it may seem.

914
00:42:35,930 --> 00:42:39,130
I just love everything in physiology.

915
00:42:39,130 --> 00:42:42,500
And I spent my fair
share of time doing this

916
00:42:42,500 --> 00:42:44,240
and then I wanted to do something else,

917
00:42:44,240 --> 00:42:46,400
and then I did that and I
wanted it to do something else.

918
00:42:46,400 --> 00:42:48,850
And some of you who
know me from Salem State

919
00:42:48,850 --> 00:42:51,180
know that I was interested in microbiology

920
00:42:51,180 --> 00:42:53,010
and pharmacology when I was there.

921
00:42:53,010 --> 00:42:56,870
And so I've been very fortunate
and had the opportunity

922
00:42:56,870 --> 00:43:00,000
to just kind of pursue
my interests on a whim.

923
00:43:00,000 --> 00:43:05,000
And this was the most recent
of those whims. (laughing)

924
00:43:05,090 --> 00:43:06,990
- Honest is good, I like that.

925
00:43:06,990 --> 00:43:08,840
So another question we have here

926
00:43:08,840 --> 00:43:11,293
is how is this connected to evolution?

927
00:43:13,150 --> 00:43:15,900
- So when I think about
cardiovascular disease

928
00:43:15,900 --> 00:43:19,870
and evolution, really
interesting concepts come up,

929
00:43:19,870 --> 00:43:24,870
like the notion that usually
with respect to evolution,

930
00:43:25,280 --> 00:43:28,440
traits that are higher

931
00:43:28,440 --> 00:43:31,130
in inducing morbidity
are not selected for.

932
00:43:31,130 --> 00:43:33,500
You usually don't get
selection for those things,

933
00:43:33,500 --> 00:43:37,880
but that has to do mostly
with its impact on fitness.

934
00:43:37,880 --> 00:43:39,190
So what's really interesting about

935
00:43:39,190 --> 00:43:40,780
things like cardiovascular disease

936
00:43:40,780 --> 00:43:43,470
is that they don't necessarily
have a direct impact

937
00:43:43,470 --> 00:43:45,910
on the life span per se.

938
00:43:45,910 --> 00:43:49,130
Most individuals make it
well beyond reproductive age.

939
00:43:49,130 --> 00:43:53,600
And so we kind of have
to fight that evolution

940
00:43:53,600 --> 00:43:56,190
isn't selecting against
these negative traits

941
00:43:56,190 --> 00:44:00,570
like salt sensitivity by coming
up with our own solutions

942
00:44:01,460 --> 00:44:04,210
through understanding the
mechanisms that mediate them.

943
00:44:07,620 --> 00:44:09,220
- We have another question.

944
00:44:09,220 --> 00:44:10,500
What would be your advice

945
00:44:10,500 --> 00:44:12,700
to any undergraduate
students wishing to go on

946
00:44:12,700 --> 00:44:14,973
to conduct a graduate level research?

947
00:44:15,940 --> 00:44:17,640
- Oh, that's such a great question.

948
00:44:17,640 --> 00:44:21,340
I didn't know I wanted
to do research, honestly.

949
00:44:21,340 --> 00:44:23,760
I got involved in research in undergrad,

950
00:44:23,760 --> 00:44:25,220
but not super heavily.

951
00:44:25,220 --> 00:44:27,980
And in pursuing a master's degree first,

952
00:44:27,980 --> 00:44:30,070
as Dr. Gordon said before,

953
00:44:30,070 --> 00:44:32,970
that was to enhance my
qualifications for Medical School,

954
00:44:32,970 --> 00:44:35,170
but I ended up incidentally
falling in love

955
00:44:35,170 --> 00:44:36,540
with the research process

956
00:44:36,540 --> 00:44:38,890
and sticking with it along the way.

957
00:44:38,890 --> 00:44:42,180
So I guess the advice in that
is just to be open-minded

958
00:44:42,180 --> 00:44:44,950
and you don't know where your
passion may end up lying.

959
00:44:44,950 --> 00:44:49,300
So try everything, dip
your toes in the water.

960
00:44:49,300 --> 00:44:50,543
You might like it.

961
00:44:53,700 --> 00:44:54,720
- That's a great advice.

962
00:44:54,720 --> 00:44:57,110
We have a question here
from one of our colleagues

963
00:44:57,110 --> 00:44:59,010
in the Biology Department is asking,

964
00:44:59,010 --> 00:45:03,440
your female rats had less
inflammation in response to salt,

965
00:45:03,440 --> 00:45:06,250
but in humans, we know that
females are more likely

966
00:45:06,250 --> 00:45:09,040
than males to get inflammatory
autoimmune diseases

967
00:45:09,040 --> 00:45:11,110
that include inflammation.

968
00:45:11,110 --> 00:45:12,193
Any thoughts?

969
00:45:13,487 --> 00:45:16,650
- Oh, that is such a fabulous question.

970
00:45:16,650 --> 00:45:19,580
Honestly, I don't know.

971
00:45:19,580 --> 00:45:22,110
And that's a really
powerful answer, I think,

972
00:45:22,110 --> 00:45:23,450
even though it's a cop out

973
00:45:23,450 --> 00:45:27,930
because the role of sex steroid
hormones really evades us.

974
00:45:27,930 --> 00:45:31,370
In some instances, we see
that sex steroid hormones

975
00:45:31,370 --> 00:45:33,540
directly exert like estrogens

976
00:45:33,540 --> 00:45:36,520
on anti-inflammatory
polarization on immune cells.

977
00:45:36,520 --> 00:45:39,478
But then we see that
as it was pointed out,

978
00:45:39,478 --> 00:45:42,980
it's likely that females
have more autoimmune disease

979
00:45:42,980 --> 00:45:45,270
and dysregulation of the
immune system featuring

980
00:45:45,270 --> 00:45:47,740
these hyper inflammatory responses.

981
00:45:47,740 --> 00:45:50,990
I think that that mechanism
slightly remains a mystery

982
00:45:50,990 --> 00:45:52,830
or at least to me at this point.

983
00:45:52,830 --> 00:45:53,783
I'm not sure.

984
00:45:58,270 --> 00:46:02,630
- We have another question
that's sort of related

985
00:46:02,630 --> 00:46:06,570
to a previous question, but
what inspired your research?

986
00:46:06,570 --> 00:46:09,940
Do you have advice for those
who are looking to do research

987
00:46:09,940 --> 00:46:12,833
and how do they go about
looking for research topics?

988
00:46:14,810 --> 00:46:17,320
- That's really a great set of questions

989
00:46:17,320 --> 00:46:18,630
when we tackle them one at a time.

990
00:46:18,630 --> 00:46:20,880
My research was inspired largely

991
00:46:20,880 --> 00:46:24,050
by my personal interests in neuroscience

992
00:46:24,050 --> 00:46:26,450
and my mentor, Dr. Wainford.

993
00:46:26,450 --> 00:46:30,420
He had this decades long set of research

994
00:46:30,420 --> 00:46:32,360
investigating the physiological impact

995
00:46:32,360 --> 00:46:34,120
of the G alpha I two protein.

996
00:46:34,120 --> 00:46:37,260
And I took it simply to
what is its role in gating

997
00:46:37,260 --> 00:46:38,890
these inflammatory pathways

998
00:46:38,890 --> 00:46:42,580
in addition to sympathetic
nervous system control?

999
00:46:42,580 --> 00:46:45,820
So very largely inspired
by his previous work

1000
00:46:47,120 --> 00:46:50,513
and his funding, which was to
investigate this work further.

1001
00:46:51,880 --> 00:46:54,050
For those looking to go into research,

1002
00:46:54,050 --> 00:46:57,730
I think a critical component
is following something

1003
00:46:57,730 --> 00:46:58,790
you're passionate about.

1004
00:46:58,790 --> 00:47:01,830
You're not going to be
passionate about research

1005
00:47:01,830 --> 00:47:04,700
that's on a topic you don't care about.

1006
00:47:04,700 --> 00:47:09,520
And then finding a mentor
with whom you really click

1007
00:47:09,520 --> 00:47:12,960
is so important because
it's like a marriage.

1008
00:47:12,960 --> 00:47:15,750
You are gonna have your
fights, but when you're working

1009
00:47:15,750 --> 00:47:20,750
with a research mentor, it's
better when you like them.

1010
00:47:21,840 --> 00:47:24,680
And then looking for research topics,

1011
00:47:24,680 --> 00:47:27,220
that comes with the other
two things I just said,

1012
00:47:27,220 --> 00:47:29,930
your interests and your
mentor's interests aligning.

1013
00:47:29,930 --> 00:47:32,550
And that last part, the research topic

1014
00:47:32,550 --> 00:47:35,360
should just come a little more naturally.

1015
00:47:35,360 --> 00:47:37,183
Do something you're passionate about.

1016
00:47:39,070 --> 00:47:41,670
- Yeah, and speaking
about finding a mentor

1017
00:47:41,670 --> 00:47:46,360
and the doctor path,
one of the questions was

1018
00:47:46,360 --> 00:47:49,570
can you share with us some
that was most challenging about

1019
00:47:49,570 --> 00:47:53,023
either your undergraduate
path or your doctoral path?

1020
00:47:54,100 --> 00:47:58,560
- Yeah, a huge challenge,
especially with respect

1021
00:47:58,560 --> 00:48:01,290
to graduate school is time management.

1022
00:48:01,290 --> 00:48:03,500
As Dr. Gordon mentioned,
I served through a portion

1023
00:48:03,500 --> 00:48:06,030
of graduate school as a teaching fellow.

1024
00:48:06,030 --> 00:48:09,360
So I was teaching gross anatomy
labs, neuro anatomy labs,

1025
00:48:09,360 --> 00:48:11,420
cardiovascular physiology lectures,

1026
00:48:11,420 --> 00:48:15,080
while conducting 40, 50 hours
a week of my own research.

1027
00:48:15,080 --> 00:48:17,770
And then the part we all forget about,

1028
00:48:17,770 --> 00:48:20,106
being a person and having a life

1029
00:48:20,106 --> 00:48:24,100
and an interpersonal
relationship with a partner

1030
00:48:24,100 --> 00:48:26,750
and a family to visit for birthdays.

1031
00:48:26,750 --> 00:48:30,620
And so really getting into a solid swing

1032
00:48:30,620 --> 00:48:34,750
of time management is super helpful.

1033
00:48:34,750 --> 00:48:36,363
That's the most important part,

1034
00:48:38,730 --> 00:48:40,320
and the hardest part. (chuckles).

1035
00:48:42,690 --> 00:48:46,800
- This is sort of out of your area

1036
00:48:46,800 --> 00:48:48,370
but I think it's an interesting question.

1037
00:48:48,370 --> 00:48:52,231
Why are women more prone to
getting auto immune disease?

1038
00:48:52,231 --> 00:48:56,220
- (sighs) It's a fascinating question

1039
00:48:56,220 --> 00:48:57,770
and there is various is research

1040
00:48:57,770 --> 00:48:59,683
currently being done, I'm certain.

1041
00:49:00,870 --> 00:49:04,240
I would say it's got
to have something to do

1042
00:49:04,240 --> 00:49:08,613
with the biological differences
that reside in our genetics.

1043
00:49:10,010 --> 00:49:12,310
Almost everything is rooted there

1044
00:49:12,310 --> 00:49:14,350
in tissue-specific gene expression

1045
00:49:14,350 --> 00:49:16,780
and hormonal impacts on those things.

1046
00:49:16,780 --> 00:49:20,053
But I would encourage you to pub-med it.

1047
00:49:20,957 --> 00:49:23,900
(both laughing)

1048
00:49:23,900 --> 00:49:25,850
- Another question
says, why did you choose

1049
00:49:25,850 --> 00:49:27,000
this animal model?

1050
00:49:27,000 --> 00:49:29,780
And can your new
information be transferred

1051
00:49:29,780 --> 00:49:31,273
to human studies?

1052
00:49:32,330 --> 00:49:33,430
- Fabulous question.

1053
00:49:33,430 --> 00:49:37,570
We chose this animal model
because there is 70 years

1054
00:49:37,570 --> 00:49:39,760
of research characterizing the physiology

1055
00:49:39,760 --> 00:49:43,910
of the Sprague-Dawley rat,
it's extremely widely used

1056
00:49:43,910 --> 00:49:47,580
in studies on salt resistance
and salt sensitivity

1057
00:49:47,580 --> 00:49:51,384
and with respect to
transfer to human studies,

1058
00:49:51,384 --> 00:49:53,900
that's along with what
I brought up earlier,

1059
00:49:53,900 --> 00:49:57,600
having to do with polymorphisms
in the GNAI2 gene in humans.

1060
00:49:57,600 --> 00:50:00,810
So several studies published
by us and other labs

1061
00:50:00,810 --> 00:50:03,670
have shown correlations between snips

1062
00:50:03,670 --> 00:50:07,420
and essential hypertension as
a phenotype, salt sensitivity,

1063
00:50:07,420 --> 00:50:09,510
blood pressures as a
phenotype in multiple cohorts,

1064
00:50:09,510 --> 00:50:12,950
like the GenSalt study
performed in North China

1065
00:50:12,950 --> 00:50:16,700
in the 1900s, as well as
Tissue from the UK Biobank

1066
00:50:16,700 --> 00:50:19,170
in western European individuals performed

1067
00:50:19,170 --> 00:50:21,490
much more recently, these analyses.

1068
00:50:21,490 --> 00:50:24,360
So we think that with the correlation

1069
00:50:24,360 --> 00:50:28,100
between protein expression and snips

1070
00:50:28,100 --> 00:50:31,150
and salt sensitivity
holding true in humans,

1071
00:50:31,150 --> 00:50:35,340
the mechanistic information
may provide useful

1072
00:50:35,340 --> 00:50:37,040
because of the conservation of things

1073
00:50:37,040 --> 00:50:39,250
like fluid and electrolyte
balance pathways

1074
00:50:39,250 --> 00:50:41,293
across different mammalian species.

1075
00:50:46,550 --> 00:50:51,550
- I wanted to ask a question,
why high blood pressure

1076
00:50:54,670 --> 00:50:56,800
would lead to inflammation?

1077
00:50:56,800 --> 00:51:00,570
I assume, partly because of
leakage, I'm not a physiologist

1078
00:51:01,820 --> 00:51:06,603
but what is the possible reasoning

1079
00:51:07,580 --> 00:51:10,790
behind why that's happening?

1080
00:51:10,790 --> 00:51:12,110
Does that make sense?

1081
00:51:12,110 --> 00:51:13,840
- I've got some fun.

1082
00:51:13,840 --> 00:51:18,238
Yeah, I've got some fun insights on that.

1083
00:51:18,238 --> 00:51:20,450
We've recently been doing studies

1084
00:51:20,450 --> 00:51:24,630
along with other labs doing
studies that have shown

1085
00:51:24,630 --> 00:51:26,350
that there's changes that occur

1086
00:51:26,350 --> 00:51:29,330
within the neuronal phenotypes in response

1087
00:51:29,330 --> 00:51:32,050
to inflammation and as
well as just studies

1088
00:51:32,050 --> 00:51:35,620
that baseline show that
cytokine production

1089
00:51:35,620 --> 00:51:37,080
as a predisposing factor

1090
00:51:37,080 --> 00:51:39,750
can then change neuronal firing rates.

1091
00:51:39,750 --> 00:51:43,280
So we think the inflammation
is directly acting locally

1092
00:51:43,280 --> 00:51:46,920
on those projection neurons and
increasing their firing rate

1093
00:51:46,920 --> 00:51:49,330
as well as exacerbating
the number of neurons

1094
00:51:49,330 --> 00:51:51,300
that express excitatory phenotypes

1095
00:51:51,300 --> 00:51:53,770
in comparison to inhibitory phenotypes.

1096
00:51:53,770 --> 00:51:56,540
So you get uncoupling of
excitation in addition

1097
00:51:56,540 --> 00:52:00,100
as well as exacerbated
amount of signaling.

1098
00:52:00,100 --> 00:52:02,677
It's quite fascinating to unravel that.

1099
00:52:02,677 --> 00:52:04,677
That's a great question.

1100
00:52:05,526 --> 00:52:06,510
- I have a question here

1101
00:52:06,510 --> 00:52:08,450
that I wanted to elaborate further too.

1102
00:52:08,450 --> 00:52:12,240
So the question is, do you
feel that SSU prepare you

1103
00:52:12,240 --> 00:52:14,250
to be successful in research?

1104
00:52:14,250 --> 00:52:15,710
So I want to add to that.

1105
00:52:15,710 --> 00:52:19,530
How did you use the tools that SSU has?

1106
00:52:19,530 --> 00:52:21,480
What kind of tools were not working?

1107
00:52:21,480 --> 00:52:25,580
What resources did you use
from SSU to be today successful

1108
00:52:25,580 --> 00:52:28,540
in your research or you
feel used additional tools

1109
00:52:28,540 --> 00:52:29,970
that are connected to SSU,

1110
00:52:29,970 --> 00:52:33,080
share a little bit about
those as well, thank you.

1111
00:52:33,080 --> 00:52:35,430
- Yeah, I think with
one thing with respect

1112
00:52:35,430 --> 00:52:38,080
to your professional career
is you can't really be afraid

1113
00:52:38,080 --> 00:52:41,620
to ask the question and it was really

1114
00:52:41,620 --> 00:52:44,850
just kind of approaching
Dr. Mwananyanda and saying,

1115
00:52:44,850 --> 00:52:46,760
I'm really interested in doing research

1116
00:52:46,760 --> 00:52:50,370
and he wanted to know what I wanted to do.

1117
00:52:50,370 --> 00:52:53,360
And so I think that the
biggest hill to overcome

1118
00:52:53,360 --> 00:52:55,770
is that first question,
finding a faculty member

1119
00:52:55,770 --> 00:52:57,190
who's doing something you're doing.

1120
00:52:57,190 --> 00:52:58,050
And that's what I did.

1121
00:52:58,050 --> 00:53:01,530
And so SSU significantly
repaired me in that.

1122
00:53:01,530 --> 00:53:04,430
I got comfortable asking
those kinds of questions

1123
00:53:04,430 --> 00:53:06,760
of people and asking to make space

1124
00:53:06,760 --> 00:53:08,963
for me in somewhere to grow.

1125
00:53:09,840 --> 00:53:12,020
And then once that happened,

1126
00:53:12,020 --> 00:53:14,390
working with him was a
significant opportunity

1127
00:53:14,390 --> 00:53:17,220
for me to engage in the scientific method

1128
00:53:17,220 --> 00:53:20,170
and start thinking about how
to think about experiments

1129
00:53:20,170 --> 00:53:22,640
and whether or not that
would be interesting for me.

1130
00:53:22,640 --> 00:53:24,720
So SSE really did prepare me,

1131
00:53:24,720 --> 00:53:26,270
both the Biology and Chemistry Departments

1132
00:53:26,270 --> 00:53:27,750
had great opportunities available.

1133
00:53:27,750 --> 00:53:29,520
I know several people

1134
00:53:29,520 --> 00:53:32,320
who worked with other
faculty like Dr. Tracy

1135
00:53:32,320 --> 00:53:35,340
where I had students working
with her and Dr. Jason Brown.

1136
00:53:35,340 --> 00:53:37,490
And so there are plenty of opportunities

1137
00:53:37,490 --> 00:53:39,560
if you're interested
at SSU to get involved.

1138
00:53:39,560 --> 00:53:41,663
And I really encourage you to pursue them.

1139
00:53:44,620 --> 00:53:46,780
- We'll end with one last question.

1140
00:53:46,780 --> 00:53:49,380
Did you notice lethargy
or other physical issues

1141
00:53:49,380 --> 00:53:51,580
with the rats that were
sensitive to sodium?

1142
00:53:52,860 --> 00:53:55,107
- They were a little tired
of the day after surgery,

1143
00:53:55,107 --> 00:53:59,600
but (laughs) most people
are, but no in all honesty

1144
00:53:59,600 --> 00:54:01,900
would leave the rats a
five day recovery period

1145
00:54:01,900 --> 00:54:04,260
following the cannular
implantation into their brain

1146
00:54:04,260 --> 00:54:08,320
to facilitate rest and relaxation

1147
00:54:08,320 --> 00:54:11,800
so that the study can proceed
unobstructed in the rats,

1148
00:54:11,800 --> 00:54:14,050
display all normal behavioral parameters.

1149
00:54:14,050 --> 00:54:15,720
They eat the same amount of food.

1150
00:54:15,720 --> 00:54:17,150
They drink the same amount of water.

1151
00:54:17,150 --> 00:54:20,670
They aren't scoring
higher on any pain scales

1152
00:54:20,670 --> 00:54:22,730
that we can assess them with.

1153
00:54:22,730 --> 00:54:25,250
So they appeared to be
functioning entirely normally

1154
00:54:25,250 --> 00:54:26,900
but you never know,

1155
00:54:26,900 --> 00:54:29,163
there's always the
undiscovered factor, right?

1156
00:54:34,550 --> 00:54:36,643
- So I'll pass you over to Dr. Gordon.

1157
00:54:39,040 --> 00:54:43,560
- And with that, I'd like
to thank Jesse Moreira

1158
00:54:43,560 --> 00:54:46,720
for being our alumnus speaker.

1159
00:54:46,720 --> 00:54:50,040
I think you're a great
model for other students.

1160
00:54:50,040 --> 00:54:52,800
I'm glad we had students
asking questions about

1161
00:54:52,800 --> 00:54:54,910
how they get involved in research

1162
00:54:54,910 --> 00:54:59,900
and what motivated you and how
did you become so successful?

1163
00:54:59,900 --> 00:55:03,840
So I think you've given
them a lot to think about

1164
00:55:03,840 --> 00:55:05,870
and you've given all of
us a lot to think about.

1165
00:55:05,870 --> 00:55:10,670
So we want to thank you for
being our alumna speaker.

1166
00:55:10,670 --> 00:55:13,550
And we also want to wish
you well in your career

1167
00:55:13,550 --> 00:55:17,570
and success in your upcoming
dissertation defense.

1168
00:55:17,570 --> 00:55:20,670
Thank you very much for
participating in this,

1169
00:55:20,670 --> 00:55:23,820
our 42nd Annual Darwin Festival

1170
00:55:23,820 --> 00:55:27,820
and our first online webinars series

1171
00:55:27,820 --> 00:55:30,850
for Darwin, and so historic moment for us.

1172
00:55:30,850 --> 00:55:33,270
And we are very proud of you

1173
00:55:33,270 --> 00:55:35,140
and we wish you well in the future.

1174
00:55:35,140 --> 00:55:36,070
Thank you.

1175
00:55:36,070 --> 00:55:37,363
- Thank you so much.