The Role of Oxidative Stress on Development and Uncoupling Protein Expression in Drosophila melanogaster
Final poster presentation for 2021 Bioenergy Systems REU
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- [00:00:00.720]Hello, my name is Bridget Price.
- [00:00:02.610]Welcome to my presentation on the role
of oxidative stress on development and
- [00:00:06.450]uncoupling protein expression in
Drosophila melanogaster. My research starts
- [00:00:11.320]at the mitochondria. As
implied by its nickname,
- [00:00:13.590]the power has of the cell, mitochondria
play an important role in providing the
- [00:00:17.550]cell with energy via cellular respiration.
- [00:00:20.490]During the final step of cellular respiration,
called oxidative phosphorylation or
- [00:00:24.270]OXPHOS, electrons travel
through the electron transport
chain to pump protons from
- [00:00:29.070]the mitochondrial matrix
to the intermembrane space,
- [00:00:31.920]creating an electrochemical gradient.
ATP synthase can use this free energy
- [00:00:36.570]difference to catalyze
the formation of ATP.
- [00:00:40.440]A by-product of oxidative phosphorylation
are reactive oxygen species or ROS,
- [00:00:45.150]a free radical necessary for normal
cellular function, acting as secondary
- [00:00:49.350]messengers for cellular
- [00:00:52.710]high levels of ROS can be
detrimental to the cell. For example,
- [00:00:56.890]ROS can initiate lipid peroxidation,
which affects membrane phospholipids,
- [00:01:01.290]and can result in the creation
of cytotoxic products.
- [00:01:04.470]Cells have multiple antioxidant defense
systems in place to manage the over
- [00:01:08.790]abundance of ROS,
- [00:01:10.200]such as superoxide dismutases,
glutathione peroxidase and vitamin
- [00:01:14.910]E. A last resort defense mechanism
against high levels of ROS is the
- [00:01:19.500]activation of uncoupling proteins in
the mitochondria. Uncoupling proteins,
- [00:01:23.610]or UCPs are activated by hydroxyl-
- [00:01:25.890]noneal creating an alternative pathway
during OXPHOS for the dissipation of the
- [00:01:30.540]proton gradient without the production
of ATP and ROS as seen here in this
- [00:01:37.500]In mammals UCPs are used to produce
heat during hibernation in brown adipose
- [00:01:42.420]but in Drosophila the
conditions surrounding the
activation of UCPs are less
- [00:01:46.080]certain to better understand the nature
of uncoupling proteins in Drosophila
- [00:01:50.610]and the response to oxygen stress,
larvae will it be exposed to Paraquat, a
- [00:01:55.380]chemical herbicide and a known oxidative
stressor that leads to the
- [00:01:59.430]proliferation of ROS.
- [00:02:01.740]The expression of three of the four identified
uncoupling proteins in Drosophila,
- [00:02:06.180]4B and 4C as well as
pupation and eclosion time and pupation
- [00:02:10.650]height will be evaluated.
- [00:02:14.840]For this experiment, mature adult,
- [00:02:16.940]Drosophila melanogaster from
an outbred population were placed in laying
- [00:02:20.570]condos to lay overnight on grape
agar plates. In the morning,
- [00:02:24.080]50 embryos were transferred to vials,
containing a standard cornmeal-molasses-,
- [00:02:27.650]yeast diet,
- [00:02:28.280]and were allowed to develop at 22 degrees
celsius. During the late second instar,
- [00:02:32.630]or about three days after embryo transfer
200 microliters of even either an
- [00:02:36.950]aqueous solution of Paraquat or tap water
- [00:02:39.650]was applied to the surface of food to
achieve Paraquat food concentrations of
- [00:02:43.370]0, 5, 10, 15,
- [00:02:45.380]and 20 millimolar. Larvae were
allowed to develop otherwise
- [00:02:49.010]normally. Two days after exposure,
- [00:02:51.620]late third instar larvae were collected
from vials and frozen off for RNA
- [00:02:55.820]extraction and
- [00:02:56.840]subsequent qPCR. Pupation
time was noted in vials where
- [00:03:01.570]larvae were not collected.
- [00:03:03.160]Then adults were sexed upon
eclosion with time noted.
- [00:03:06.220]Pupation height was evaluated
after complete pupation using six
- [00:03:11.080]quadrants with the food
being the first quadrant.
- [00:03:15.640]It was expected that Paraquat exposure
would delay development because oxidative
- [00:03:19.540]stress leads to a less
efficient mitochondria where
development would take a
- [00:03:23.500]back seat to more critical
- [00:03:26.530]It was also hypothesized that Paraquat
exposure would lead to higher expression
- [00:03:30.220]of uncoupling proteins because elevated
levels of ROS would activate uncoupling
- [00:03:33.940]proteins for cell defense. In figure one,
- [00:03:37.870]you can see the effects of Paraquat on
eclosion time of both male and female
- [00:03:41.710]An increase in Paraquat dosage
correlated with an increase in eclosion
- [00:03:45.610]time for both sexes,
- [00:03:47.110]with the eclosion time
between the control and 20
millimolar being delayed 11
- [00:03:51.970]hours for females and seven
hours for males. In figure two,
- [00:03:55.630]you can see the effects of Paraquat on
the expression of uncoupling proteins
- [00:03:59.260]with respect to the control gene
Actin. There's no significant trend,
- [00:04:03.280]but there is suggestion that Paraquat
dosage influences the expression of UCPs.
- [00:04:08.530]there was evidence of sample contamination
during qPCR with negative controls
- [00:04:12.400]fluorescing like positive controls,
so further testing is necessary.
- [00:04:16.900]In figure three, you can see the
effects of Paraquat on pupation time,
- [00:04:20.560]which follows a similar trend t
the eclosion delays. In figure four,
- [00:04:24.430]you can see an observation of puption.
- [00:04:26.440]supporting the quantitative results
in figure three. The number of pupae,
- [00:04:30.400]the darker brown marks, decreases
with increasing dosage.
- [00:04:34.990]In figure five you can see the effects
of Paraquat dosage on pupation height.
- [00:04:39.220]There's a loose correlation between
pupation height and treatment, with
- [00:04:42.540]pupation high decreasing with dosage.
- [00:04:45.850]The increasing concentration of paraquat
on pupation and eclosion time suggests that
- [00:04:50.060]oxygen stress influences the productivity
of Drosophila metabolism in such a way
- [00:04:54.400]that development is delayed. A decrease in
pupation height with increasing Paraquat
- [00:04:58.420]dosage supports the idea that oxidative
stress impacts the metabolism as it is
- [00:05:03.700]likely they have less
energy available energy to travel
- [00:05:07.780]above the food for pupation. In addition,
- [00:05:10.720]no statistically significant trend
was identified in expression of any
- [00:05:13.810]uncoupling proteins but preliminary
data suggests that ROS can influence
- [00:05:17.600]expression of UCPs. Uncoupling
- [00:05:20.830]proteins can serve as important oxidative
stress regulars in the mitochondria.
- [00:05:25.570]In Drosophila melanogaster
exposure to Paraquat
- [00:05:28.570]to elevate natural levels
of reactive oxygen
- [00:05:30.640]species causes delayed development as
seen from lengthened time to pupation and
- [00:05:34.990]eclosion with increasing
Paraquat dosage concentration.
- [00:05:38.410]The expression of UCPs are less clearly
correlated to Paraquat exposure,
- [00:05:42.310]contradicting expected models of
UCP activation and the developmental
- [00:05:47.170]results, indicating that further
experimentation is required.
- [00:05:52.410]This experiment has implications for
future research. In the immediated future,
- [00:05:56.460]further replication of this is experiment as required
as evident in inconsistencies in
- [00:06:00.920]qPCR UCP expression
data. In the short term,
- [00:06:04.610]lifespan evaluations would be a
natural extension of this experiment.
- [00:06:07.430]As studies have indicated that low,
- [00:06:08.970]exposure to Paraquat can increase
mitochondrial activity and therefore
- [00:06:12.530]lifespan. In addition,
- [00:06:14.330]given that the impacts of Paraquat on
physiological processes like pupation and
- [00:06:21.290]it would be of interest to evaluate
the fecundity. In the longer term is
- [00:06:25.280]experiment can be used to gauge response
to Paraquat dosage in order to execute
- [00:06:29.420]experiments were elevated ROS production
and UCP expression is of interest.
- [00:06:33.950]From a long-term perspective,
- [00:06:35.300]uncoupling proteins in Drosophila can be
used as a model for understanding the
- [00:06:38.960]nature of human uncoupling proteins,
- [00:06:40.880]which have implications in
degenerative diseases and aging.
- [00:06:45.910]Thank you to NSF grant DBI 2 0 5 0 5 7 4.
- [00:06:50.890]Also thank you to the Montooth and Meiklejohn
labs at UNL for being welcoming
- [00:06:55.270]and providing all the support and
guidance necessary to make this project
- [00:06:58.300]possible. Thank you and have a good day.
- [00:07:01.660][Entire Poster View]
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