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My PhD Degree Workload - My Doctorate Degree Work Explained

what's up guys my name's David Swilley

and today I'm going to talk about my

research and its importance in modern

medicine and watch to the end if you're

interested in accumulating this much

data in your own master's thesis or your

PhD thesis and if you have any comments

or any questions put them down in the

comments below I'll get back to you as

soon as I can so guys with that said

let's get into it let's have a look at

my research so I was actually extremely

lucky in terms of the research title

that I got for my PhD and I was looking

at how factors in your own blood can

affect skeletal muscle growth so

isolating factors from human platelets

and putting them on to skeletal muscle

stem cells and what stem cells are are

their cells that affect the regeneration

of your your skeletal muscle and so what

this graph here shows is that in the

white bars you can see there's an

exponential amount of papers being

published with platelet-rich plasma and

the black parents down the bottom barely

visible they are and papers being

published on plate rich plasma on

skeletal muscle so the topic that I'm

actually interested in it's a hot topic

it's very new in the field and these are

recent papers and reap review articles

that were published on plated based

regeneration on schooling to the muscle

now these review articles are public

face taun clinical trials and what they

show is that there's inconsistent

results so there's also an inconsistent

preparation methods and the timing of

application is very important and that's

not standardized with the clinical

trials which is why as you can see in

this graph here they show roughly 50/50

either positive or negative results so

some people say it's bad some some

people say it is nothing at all and some

people say there's actually a positive

effect on orthopedic orthopedic injuries

so standardization of preparation when

they get the whole blood from a human

they spin it down the centrifuge down at

different speeds so that's not

standardized they and they use different

a platelet concentrations so that's not

standardized either so there's different

dosages being used in the patients our

athletes and they use different plainly

agonist so they stimulate the platelets

to release and the growth factors and at

different ratios so there could be good

and bad things mixed in with the

platelet-rich plasma and timing of

application in sport is important as

well so this was the study done in rats

and if you add platelet-rich plasma to

ask a little muscle injury after one or

four days there's more fibrosis which

means worse regeneration and if you use

it on day seven you actually get better

regeneration so this here is how I made

my plated based applications I got for a

whole blood from humans and from mice

and I spun down the whole blood and at a

gentle speed to collect the

platelet-rich plasma or isolated the

player played rich plasma and spun it

down again centrifuges down again a hard

speed to collect the fellowship

platelets at the bottom of the tube

resuspended in a in a buffer and then

activated with thrombin para one or

collagen or sonication which means just

blow off the platelets and then we

removed the cellular debris so what you

have at the end is played a treeless

eight all the growth factors all the

cytokines the good and the bad stuff I

mean in a isolated form now what people

did in the clinical trials was and they

would apply to all different joints all

different orthopedic injuries and just

the plate rich plasma

however we removed the plasma and I just

wanted to see what the players would do

themselves so before we get into it I

just want to show you the structure of

skeletal muscle no I didn't make this

video and I swiped it from YouTube and

however it really emphasizes the point I

want to make such - what we were want to

look at is the muscle stem cells so the

cells are the muscles in green here

- can regenerate a muscle fiber and

after injury so the green signifies pax7

now pax7 is a most

stamps a marker for quiescence which

means they're doing nothing they're just

waiting for an injury they're floating

around on the muscle and moving up and

down just waiting for an injury um yeah

we won't get into the structure the

muscle that's not what we're interested

at the moment it's just going through

how the most fiber moves and the

different structures and internally

however what we want to take from this

video is that on the muscle there's

muscle stem cells Express impact seven

and in this second video I just want to

show here if it would work for me is

that here we are is that after the most

in fiber gets injured as we see here and

the satellite cells Express my OD so in

our graphs we will have my ID in red so

my ADEA means that they're proliferating

so the most so as you can see they're

the most little satellite cell becomes

activated it divides into 2 which is

proliferating divides into 3 into 4 and

some of these muscle stem cells will

Express PAC 7 and lose the mild

expression so they remain in case

there's a future injury M on the muscle

fiber and some will actually express my

genin which is the third and factor that

we're going to talk about and Maia Jenin

and PAC 7 can't be expressed at the same

time and just as you saw there they fuse

into the muscle fiber and fix any areas

that need to be damaged and that need to

be fixed so Maya genin is an expression

for differentiation so they

differentiate into the most fiber form

myotubes

and fix any area that needs to be

repaired so my research questions with

this in mind is number one how does

played it really safe so the method that

we showed earlier and how does this

affect a proliferation and B

differentiation of myoblasts and number

two how does play the real estate affect

other cell types and number three can we

optimize platelet realization now what

do I mean by optimizing plated

realization so here's a study showing -

they got platelet-rich plasma and here's

a second study they did the same thing

however in the first they remove

and tgf-beta no tgf-beta causes fibrosis

so poor muscle and regeneration and

tgf-beta am is you don't want to when a

muscle is regenerating that's pretty

much the gist of it so they remove

tgf-beta from platelet-rich plasma and

they find that it does even better that

platelet-rich plasma alone in terms of

skeletal muscle regeneration now with

that said we're gonna go into the first

part of my first research question which

was how to play the race age affect

skeletal muscle proliferation so the

proliferation of muscle stem cells on

skeletal muscle so to do that we got

some mice and satellite cells we

proliferate them for 24 hours with a

live marker which is the edu here and we

find - and collagen and Parowan

stimulated played a treeless age and so

we stimulate them with these two common

platelet agonists we collectively they

say if we put it on the cells and they

proliferate nicely and here's a dose

response and some more real estate gives

better proliferation and the third graph

here is more plated concentration gives

a more proliferation so if you use a

more realistic and be more platelets you

get more proliferation very clear code

message very obvious there's nothing

more we can do with this here except

looking into the mechanisms behind why

this is happening

so platelet real-estate stimulates

muscle stem cell proliferation very

clear code obvious message um but we're

going to go ahead with the physiological

levels of platelets which is 2.5 times

10 to the 8th and so what we have here

on the left side is platelet realization

composition so we looked at all these

different growth factors that can be key

not all of the growth factors and

platelet real estate and however the key

growth factors that we think are

important such as player derived growth

factor and the vascular vascular

endothelial growth factor so the PDGF

and VEGF

we find that there's a lot of it in

Parowan stimulations played real estate

which is the bar on the right so guys

for the purpose of this presentation I'm

going to assume that you know what a PCR

is so I ran a real-time PCR on our

myoblast RNA after they were exposed to

play the real estate and we found that

various myogenic markers are increased

and especially VEGF and PDGF and we

applied it to our cells and we found

that the protein was also increased was

translated into protein for PDGF and for

GM so what this tells us is that

platelet read as age increases PDGF and

VEGF expression on an mRNA level and on

a protein level in proliferating

myoblasts or proliferating muscle stem

cells M so it's in the planar realistic

competent composition and it increases

and their expression in the myoblasts M

having this in mind we find that

inhibition of VEGF and PDGF in our

skeletal muscle stem cells after the

given played in real estate as a

dose-dependent loss of proliferation so

what this tells is the VEGF and the PDGF

and in played a real estate can be

mitigated through the application of our

inhibitors which means they're important

factors in liberation of my blasts now

guys if you can remember back to the

video we had earlier maya Jenin was the

third factor that caused there was

expressed during differentiation and

here we show that when you differentiate

the muscle stem cells they actually they

don't differentiate in presence of

played a realization and we back this up

a PCR which is all these factors on the

top however this new addition to the

graph here when you add it to a late

stage of differentiation they actually

differentiate better in the presence of

plated realization so what we find is

it's bad for fusion however it's good

for proliferation and late stage

differentiation and when we inhibit VEGF

and PDGF during differentiation we find

that there's a loss of myotube formation

there as well in the presence of planar

realism so guys what we did next was we

got single muscle fibers from mice and

we found that in the presence of planar

realization the expression of pax7 and

my ot was increased in the presence of

plate realization so the totally

satellite cell number was increased and

the total amount of my OD was massively

increased M cyclin d1 and markov

proliferation was increased and Scribd

so america for differentiation was also

increased both on the satellite cells

and the fibers and in our isolated

satellite cells so here we have

satellite cells that were

differentiation of off of the muscle

fibers in cultured dishes we find that

real estate drives muscle stem stem cell

proliferation and differentiation in our

fiber model and on these satellite cells

off of the fibers so guys in conclusion

we find that planar realization is

highly effective at increasing

proliferation through increasing V air

VEGF PDGF Scribd and cyclin d1

expression during proliferation

however it's bad at the fusion stage and

but it's great during late stage

differentiation in terms of increase in

my Gen expression my tube number and

increasing my Genesis and what this

leads us onto is the conclusion such -

we've shown in vitro and ex vivo that

Parowan activators play lead real estate

increases proliferation and the to

essential components PDGF in PDGF and

are shown to affect both proliferation

and differentiation in terms of mRNA and

protein content and we've shown a

temporal effect of plate of real estate

on my chip formation and finally we've

identified for the first time that

plated Real Estate's effect on myoblast

lineage progression and ex vivo is

through scrip the marker scrip M so the

only one went on to do in vivo works in

mice we injured their limb muscles and

we found that embryonic an embryonic

myosin heavy chain or newly regenerated

fibers is increased in terms of size and

we find that there's less IgG

infiltration so less necrosis in the

muscles after five days of injury with

the plated real estate

accelerates skeletal muscle regeneration

of the tibialis anterior muscle in

people this leads us on to our second

question so how does play the real

estate affect other cell types we find

that skeletal muscle is affected and

quite nicely with the play of real

estate and however how does in fact

other cell types so here's C 2 C 12 they

are skeletal muscle cells and then we

want to look at the h 9 C 2 s which are

hair cells now if you can remember back

to Erin here we've got the dose response

usually the concentration and we find

that there is a very poor effect of

physiological levels of platelet

realization on ER h 9 c 2 s however when

we add and super physiological levels of

played at real estate we find - and

there's a massive increase encouraging

myocyte and proliferation which is on

our h9c - cardiomyocytes or harsh and

stem cell proliferation

then we differentiate our h 9 c 2 s and

on day 4 and on day 7 we see an increase

in differentiation on these cells and so

we see this an increase in my general

which is also America for cardiomyocyte

differentiation and played it real

estate seems to stimulate cardiomyocyte

differentiation more so on day 7 than on

day 4 and again there's an increase in

cyclin d1 and there's an increase in fee

EGF expression so it seems to work

through the VG and a pathway to

stimulate cyclin d1 on a mRNA and a

protein level and it seems to inhibit

and differentiation by the igf-1 pathway

which is important for skeletal and

carry myocyte and hem differentiation so

this seems to be mesh mediated through

the VEGF and pathway we also looked at

skin cells we looked at h hih cats and

humans or fiberglass or keratinocytes

and fiberglass so on top we see this is

better differentiation in our

fibroblasts and we see on the bottom

that there is an better terminal

differentiation with the Hackett's

metadata here is not shown on bosch we

actually did am human keratinocytes so

the exterior of the skin cells and they

differentiate better with

realization and on to our third question

can we optimize play the realization so

is the room to optimize it and if you

can remember back to earlier in the talk

when they removed this tgf-beta factor

from related and platelet-rich plasma

they found less fibrosis in the skeletal

muscle and less necrosis means better

regeneration so next we got the blood

from all these knockout mice and we

knocked a factor that an aunt of

affected platelet function and we found

that the most promising M blood from the

transponding one knockout mouse and

positively positively affect my oblast

proliferation and we looked at the

skeletal muscle and profile of these

mice and found that they're relatively

normal as compared to wild-type mice and

so we wanted to see how this

transplanted one knockout mouse how its

blood can induce proliferation better

than in the wild-type so um what we did

first was look at the muscle fibers from

these mice we found that and transponder

one knockout satellite cells proliferate

ur proliferate faster than wild-type and

bought when you look at the satellite

cells off the fiber and on the fiber we

find that there's a very normal and

proliferation profile and however

there's more satellite cells at a time

point four th of culture so after 48

hours there is more satellite cells on

the fibers than first is the wild-type

even though they have a very healthy and

expression profile during injury next

what we looked at was the satellite

cells off the fibers so we actually

didn't find any difference in

proliferation and when we looked at the

South blood cells off the fiber on the

left hand graph here but when we look at

the right hand graph we see that the

trauma spawned in one knockout and

myoblasts have actually less capacity to

differentiate but this is rescued when

we add plated real estate and we

repeated this on second muscle so we

look at the EDL on the left and the BP

on the right so the the EDL is the

extensor digitorum longus and we

repeated it for a differentiation as

well on the baby so the biceps muscle

and we also find on the VB that there's

less differentiation in the wild-type

and in the transponder one knockout but

it's rescue coplanar real safe and a

typo in 72 we find the same results

there's a healthy proliferate there's a

healthy differentiated profile on the

fibers however there is a total there's

more numerous total amount of satellite

cells on the fibers after 72 hours of

cellular culture in the presence of

played a realization so guys what we did

next was we added the platelet

realization from the mice themselves we

looked at the Trump responded one

knockout Mouse we got the potato

realization from these mice and we added

it to their own and wild-type and Muslim

fibers and we found that on the knockout

mice with their own knockout blow did we

find the most elaboration on the top

graph at timepoint 48 and at the bottom

graph time point 72 we find it

unanimously has the same amount of

proliferation and so in conclusion we

find the PDGF and VHF are essential for

driving muscle stem cell cyclin d1 and

proliferation so cyclin d1 is the

proliferation marker increase in my DP

GF v GF and script we find the same on

cardiomyocytes it's driven through VEGF

and on the TSB one myoblast we find T

sp1 is important for differentiation and

in the knockout we find that plate of

research increases this differentiation

so in conclusion those miscommunications

between the clinics and the laboratories

we find novel in vitro data the planar

real estate effects

proliferation of skeletal and cardiac

maya blasts as well as human dermal

fibroblasts and our in vivo data shows

the plain area say promotes regeneration

proteomics revealed that there's a high

level of TSP one in Parowan activated

platelets and removing this through the

knockout can promote further

proliferation of higher blasts and so in

conclusion and we find that play real

estate can be used as a factor of

increasing different cellular responses

M after injury it's important to point

out that this is my first and second

year and date of my PhD so the third

year and the

second half my second year war is not

included in this because it's not been

published so guys with that said hope

you enjoyed the video and I hope you

learned something from it and most

importantly if you need any help with

any of the techniques I've covered today

please please please comment down below

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