The Role of Oxidative Stress on Development and Uncoupling Protein Expression in Drosophila melanogaster

Bridget Price Author

08/02/2021 Added

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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 homeostasis. However,
[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
[00:01:34.530]diagram.
[00:01:37.500]In mammals UCPs are used to produce heat during hibernation in brown adipose
[00:01:41.640]tissue.
[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,
[00:02:05.880]UCP4A,
[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 physiological functions.
[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
[00:03:41.350]Drosophila.
[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.
[00:04:07.750]Experimentally,
[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
[00:06:18.770]eclosion,
[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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The Role of Oxidative Stress on Development and Uncoupling Protein Expression in Drosophila melanogaster

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