We all know how tired we get
if we have a bad night of sleep,
and if we have a couple of those in a row
it really gets exhausting.
So, can you imagine if you had
to experience daytime sleepiness
together with nighttime insomnia
for months and years?
That's what people with sleeping sickness experience.
Sleeping sickness is actually an infectious disease
caused by a parasite called Trypanosoma brucei.
This parasite is transmitted
by the bite of a fly called the tsetse fly
that is endemic in 36 African countries.
But this parasite is not picky.
It not only is able to infect humans,
but it's also able to infect wild and domestic animals.
So, it makes it not only a health problem in these countries,
but because it affects their cattle,
it also becomes an economic burden.
But the World Health Organization
has done big efforts towards the elimination
of this infection,
and the number of cases has been dramatically reduced.
But there is still no vaccine
against this parasitic infection.
It's almost always lethal if left untreated.
And in fact, the treatment itself is quite complicated.
It requires intravenous treatment,
and it is extremely toxic.
It's believed that about 5% of the people
that have this treatment
die of complications.
And this is all caused by a tiny parasite
that is changing the sleep-wake cycles of its host.
This parasite is not making these people sleep more,
but instead is shifting the timing
at which their sleep occurs.
And the parasite does that by affecting
the circadian clock of its host.
But what do I mean by the circadian clock?
Well, by living in our very rhythmic environment,
with day and night cycles
due to the Earth's rotation,
organisms like us have evolved ways
to anticipate these rhythmic changes.
These are called our circadian rhythms.
Our sleep-wake cycles
are an example of those.
We have hormones and some signals
that make us feel sleepy
during the night time,
and the next morning a different set of signals
makes us feel active and hungry
and ready to enjoy our day.
These rhythms are repeated every day
and they last about 24 hours.
That's exactly where the word comes from,
from the Latin circa diem,
which literally means "around a day".
So, these rhythms are also very interesting
because they are endogenous.
What this actually means
is that they are not really driven
by the presence of these light and dark cycles,
but they are instead really coming from within.
And this was shown by an experiment with plants.
So, plants have circadian rhythms
on their leaf movement:
the leaves go up during the daytime
and they go down at night.
And a French scientist thought that
the plants were not sensing light and they're...
and thus putting their leaves up,
but instead, that the plants were anticipating
that the light was coming
and placing their leaves.
So, he did a beautiful experiment,
which consisted in putting one plant by the window
that would be exposed to both day and night
environmental changes,
and a second plant was placed in a cabinet underneath it,
so this plant was in complete darkness,
isolated from any environmental cue.
What he saw was that both plants
-- the one by the window
and the one inside the cabinet --
still maintained these leaf movements, up and down.
So, this doesn't mean that light is not important;
it's just not... it's not driving these rhythms.
But it's very important to synchronize them.
A simple way to imagine that
is through jetlag.
So, I am originally from Portugal,
but I live and work here in the US.
But every time I travel to Europe to see my family,
I have to travel about 6 hours difference
in time zone.
So, my sleep-wake cycles
take a couple of days to get synchronized
to these new light and dark cycles.
But light is important,
and eventually I become synchronized with the environment
in Portugal.
So, this means that we have circadian rhythms.
Plants have circadian rhythms.
But actually, circadian rhythms go all the way
through the Tree of Life,
to the simpler organisms, like bacteria.
And since I'm so interested in parasitic
and infectious disease,
I wonder if this was just something
that was common in organisms that are free-living
and thus exposed to light and dark cycles.
So, my question is,
do parasites also have circadian clocks?
If organisms like parasites
that are never really exposed to light and dark cycles...
if they also have developed -- evolved --
these circadian rhythms mechanisms.
It turned out that the parasite I was studying,
the sleeping sickness parasite,
was a really good model to answer this question.
And that's because this parasite
is a single-celled parasite,
but it's also extracellular.
So, as you can see, we can culture it in the lab,
and it's by itself.
So, no host cells involved.
So, whatever rhythms I can identify,
they're really coming from the parasite
and not from the host.
But where to start?
Well, I cannot just go and look at the leaf movement
in this parasite, right?
So, instead, I had to take an unbiased approach
and look at gene expression in these parasites,
this gene expression that can potentially
regulate all the downstream rhythmic behaviors.
So, in order to do that,
I would have to first synchronize the parasite population
to day and night cycles,
just like the light and day for us.
But for parasites,
I thought that they might not be really responsive to light.
They are actually always inside our bodies,
so they are not really exposed to it.
But instead, they may be exposed to temperature fluctuations.
Our temperature drops at night
when we are sleeping,
and it increases during the day when we are awake and active,
so parasites that are swimming in our blood
could potentially sense these temperature cycles.
So, to mimic those,
I actually cultured parasites
and shifted every 12 hours
in between incubators
so they would be exposed to day and night temperature rhythms.
Afterwards, I either placed them in the...
kept them in these alternating conditions
or placed them always at 37.
So, in the end, I was trying to
replicate the experiments that the French scientist did
with the plants, either by the window,
exposed to day and night cycles,
or at always 37,
which will be the plant inside the cabinets.
Okay. I tried to synchronize my parasite population,
so now I'm gonna go and check,
what is their gene expression?
But I don't know what genes to look for.
So, instead, taking advantage
of a technique used in biology called RNA sequencing,
I'll be able to not only count the number of molecules of RNA
being expressed through time
but also to which proteins they can potentially encode.
So, this is... can be simply explained here,
by this diagram.
As you can see, at zero hours,
this plant has only two molecules of RNA:
one that is red, one that is blue.
But at 12 hours, the blue molecules are now four.
So, if I plot this data,
I can actually identify that
the red RNA encodes for a protein called protein A,
and there... there's the RNA.
It's called RNA A.
And it's constant through time.
Whereas the RNA blue,
it actually has a higher expression at 12 hours,
and then it drops again.
So, these actually have these rhythms
in gene expression.
So, when I take a look at,
what is the gene expression in my parasites?,
I'll be specifically searching for genes
that have the gene expression pattern
like this RNA blue.
So, when I first looked at what happened in the parasite population
that was alternated between these incubators,
I was very happy to see
that we were able to synchronize the parasite population,
and that's because we indeed found genes that were cycling,
like here, this gene A.
So, you can see that the gene was highly expressed first,
and then it got dropped,
and then it went up again.
And this was repeated on these two days that I measured.
And this was not the case of only this gene A,
but also for these 1,500 genes.
And actually, here, in these plots,
the genes are representing one row for each,
and the color code represents their gene expression level.
So, you can see that, in the beginning,
for example, gene A is marked as red,
and then it becomes blue, and then it gets red again,
and blue, and so on,
which will be an example of, here,
one of the few first rows in these heat map plots.
So, this was what happened when the temperature cycles were present,
so I cannot really be sure
if these are circadian rhythms of the parasite
or just genes that are cycling
because they are responding to the temperature fluctuations.
So, instead, to really be sure
that parasites have circadian rhythms,
we have to look at what the gene expression
of the parasites inside our 37 incubator looks like.
So, excitingly enough,
parasites had gene expression cycling
even in the constant conditions --
even in the absence of any environmental signals.
And this was not only for gene X,
but actually for 1,000 genes
that were cycling in these constant conditions.
So, these are indeed circadian rhythms,
the really endogenous rhythms
coming from this Trypanosoma brucei parasite.
So, this was very exciting.
This really proved that parasites
can indeed have circadian clocks.
But the question now is,
what are these clocks for?
Why are the parasites putting so much effort
in making 1,000 genes cycling throughout the day?
What are these rhythms actually encoding for?
So, to answer this question,
we went and specifically looked at,
what are the genes that are cycling throughout the day?
And what we found was that
most of these genes are encoding for metabolic pathways.
They are either involved in the metabolism
of sugars or carbohydrates.
They're involved in the metabolism
of lipids or fats,
or in the amino acids of proteins.
But this was all at the gene expression level,
so I needed to validate if this was really true
for the parasite population --
if the parasites were somehow in a different metabolic status
throughout the day.
So, I could potentially go and just
look for the expression of these proteins,
but instead I looked at ATP levels.
So, ATP can be seen as
the ultimate energy molecule
that will be the result of all these metabolisms.
So, when I measured the ATP's in this parasite,
I found that the ATP levels
are going up and down throughout the day.
And in fact, this happened in the conditions
that were in alternating incubators,
but also in the condition that's always at 37.
So, you can also see that
the timing of these higher ATP concentrations
also matches the higher...
that you have more genes being expressed
of these metabolic pathways.
So, there seems to be a big importance
of circadian rhythms in regulating
the metabolism of these parasites.
This was very interesting
because of course I'm very passionate
in understanding how the biology of these parasites work,
but also, in the end,
I'm interested in trying to find ways to fight them.
And because we know that most of the antiparasite drugs,
they actually act on these metabolic pathways,
it could be that just by
simply administering the drug at specific times of the day,
we might find the parasites more susceptible
to these treatments.
So, I set out exactly to test this question,
to try to answer it.
And I first started by... go and...
synchronizing this parasite population,
again, with our temperature cycles.
Then, to each of these cultures,
I gave an increasingly higher drug concentration
and hoped for the increasing higher drug
to kill more parasites,
and that's exactly what I... what I saw.
So, if I give very little or no drug,
100% of the parasites remain alive,
but as I increase the drug concentration,
these parasites start dying.
And this was a drug called suramin,
which in fact is known to act upon the glycolytic pathway,
and it's used to treat sleeping sickness parasites in the field.
So, this was very exciting -- the assay was working.
So, I could then ask whether these...
exact death of parasites through this suramin drug
was similar throughout the day.
So, when I looked at what happened at 4 hours,
I was very disappointed,
because it was exactly the same.
You can see that these curves look the same
whether I treat parasites at 0 hours
or whether I treat them at 4 hours.
So, this could have just meant
that my hypothesis was wrong altogether,
that there are no benefits in treating parasites
at different times of the day,
and that their metabolic pathways changing
is not really correlated with the treatment.
But instead, I didn't take that as a no,
because these are just two time points
in the 24-hour day.
So, instead, I went and look at what happened at 8 hours.
And as you can see,
I was extremely excited,
because there is now a shift.
So, parasites are requiring
a higher drug concentration
in order to be killed.
So, for killing the same amount of parasites,
now the drug needs to be at a higher dose.
This was very exciting.
And so, when I finished...
for all the time points throughout the day,
I can then transform this plot
into the dose that I require to treat half of the parasite population.
So, you can see here that if I treat at 0 or 4 hours,
I need half of the amounts of drug
to kill the same number of parasites.
So, if I treat them at 8 hours instead,
I would need 2.5 times more drug.
And this really means that there is
a potential for improving the treatment
by simply administering at different times of the day.
So, altogether,
I was able to show that Trypanosoma brucei parasites
have circadian rhythms
and that these rhythms regulate the parasite metabolism,
making it more susceptible to drug treatments
at different times of the day.
But as you can imagine,
parasites probably didn't evolve these circadian rhythms
in order for us to use it against them.
I can think of that parasites
have evolved these rhythms
to anticipate when the vector is coming to bite at the end of the day,
to be prepared and thus enhanced transmission.
But I can also think that maybe parasites
have evolved circadian rhythms
in order to predict when the strong immune response
is gonna happen,
because our immune system
is also cycling throughout the day.
I can think that parasites may have evolved circadian rhythms
in order to anticipate when the nutrient influx
is gonna happen --
we only eat during the day, right?
So, there are many different reasons
for why circadian rhythms may be so important
for parasites.
But what we know now is that they have them --
Trypanosoma brucei has circadian rhythms --
and there... and there is a potential to use the metabolic rhythms
that it has to treat them
at specific times of the day.
So, I think it's worth it to confirm
that our results from the cultures
really translate into differences in mouse models.
And what we found for suramin
may be also true for other, different drugs available to treat this infection.
Also, it's worth it for us to go and look at,
what are the genes that actually
regulate these circadian rhythms?
What are the few genes that regulate
the expression of these cycling 1,000 genes?
And if we identify them,
can we actually use these genes as potential drug targets
to disrupt the circadian rhythms of these parasites?
Finally, what about other parasites?
So, do parasites have clocks?
It's a very general question.
We should look at what happens
in circadian rhythms
in malaria parasites, in Leishmania, in Toxoplasma,
all these other infections
that are very bad for both humans and animals.
Do these parasites also have circadian clocks,
and can we use that against them?