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Home , Chronic stress, Stressor, Stress (biology)

Maia Thompson Independent Study Ms. Howe The Biology of

Introduction As a senior in high school, the last two years have been filled with important tests with scores that reflect the opportunities that will be made available to me as college approaches. Being a student instigates stress quite naturally as classwork is daily, homework is never rare and tests are seen weekly. Just in the last month of May, students have Advanced Placement exams, finals and final projects. Students also have to navigate the social aspect of school, dealing with their own teenager insecurities and finding their self-identities. I took two AP tests, worked on this project, took my class finals, and took my Emergency Medical Technician finals and practicals all while keeping up with regular schoolwork and hanging out with friends. This is a typical schedule for many students.This project is important to me, to show the effect of stress in students as they prepare for tests, hopefully to create awareness of the preparation these students go through and the hard they present with. In this independent study I studied the biological reaction of stress in the body along with an experiment to see if I can decrease the stress of students before an AP using a therapy dog.

Context First, what is stress and why is it so important? Stress often seems like a negative and debilitating reaction from our body, but this reaction was necessary for our survival. Our perceived stress originates from a fight-or-flight response in the body that has helped humans to survive. When there is a perceived threat, we experience an acute stress response. Sensory neurons inform the in the brain which in turn signals the to release adrenocorticotropic (ACTH) and the adrenal glands to release epinephrine into the bloodstream. When the ACTH in the blood stream reach the , the production of is activated. As cortisol is released into the bloodstream, it signals a cascade of fight-or-flight responses in the body. This sympathetic response includes increased rate and respirations, the constriction of the pupils, increased and slowed digestion.

This initial response is called an acute stress response. Often this reaction in the body is an during a time of danger and excitement, heightening the . This stress is temporary and subsides as the danger or excitement is no longer present. The acute stress response in the body is the positive stress response that has helped us to survive and what we still need to continue to survive. is what people most often think of when asked about stress. This type of stress is the reaction in the body that can be debilitating in our daily life. Chronic stress is longer lasting and occurs when are present for a long period of time. These stressors can include family dysfunction, a high-demanding job or traumatic experiences (can range from childhood experiences to PTSD in veterans). According to the American Psychological Association, this prolonged exposure to stressors can cause serious risks such as , , muscle , high blood pressure, and a weakened . The transition from an acute stress response to chronic stress can be seen in the General Adaptation Syndrome identified by . During the Alarm reaction, ​ ​ resources are mobilized and our body goes through an acute stress response. These resources are the sympathetic responses in the body described earlier and the initial increase in cortisol. The second phase is resistance, when the body must cope with the stressors. Cortisol levels in the body stay elevated and epinephrine may increase as well to sustain and blood sugar levels. This is the chronic stress response described earlier with symptoms such as insomnia due to the prolonged elevated cortisol levels. The last phase is exhaustion when the body loses its ability to adapt and “burns out”. Both cortisol and epinephrine levels are low and symptoms such as severe and inability to through the night occur.

Task and Object For this project, my subjects were tested the hour before taking a high-stakes Advanced Placement test. These students, all seniors and juniors, have been preparing all year to take either the AP Physics test or the AP Statistics test. Because they have spent so much time and reviewing the material in class and on their own time, the stakes are high. They also have the opportunity to receive college credit if they perform well enough. This potential to earn college credits not only saves them money, but also looks good on college applications. The object of this experiment was to take advantage of this high stress situation and see if I could lower their cortisol levels using a therapy dog. Not only is this an interesting experiment for me with lots of learning opportunities, but I hoped to relax students before their test and lessen their stress! According to Yerkes-Dodson Law, difficult tasks are performed best with ​ ​ low to moderate levels of while easy tasks are performed best with higher levels of arousal (as seen on the graph below). Taking an Advanced Placement test is most definitely a difficult task, so helping these students to relax will hopefully let them perform to the best of their ability.

Design Figuring out the most efficient but accurate method to carry out this experiment was one of the most difficult aspects of designing this experiment. Originally, I planned to individually interview students in a study hall the day before they took an AP test. This method was not only time consuming, but there was concerns that students’ stress levels would not be high enough to cause a significant change if the therapy dog did in fact decrease stress levels. It was decided that the best time to test participants would be immediately before their exam, when stress would presumably be the highest. Independent Variable: I decided to use the presence of a therapy dog as the ​ therapeutic intervention because I was interested in the effect an animal could have on the secretion of chemicals in the body. This therapy dog was my own dog that passed the Therapy Dogs International test to become certified with ​ ​ me. We visit as a therapy team in the special education classes at Longfellow. I was curious if his presence actually lowered stress in kids.

Dependent Variable: In this experiment I will be using a Cortisol Competitive ELISA kit. An ​ ELISA kit is an Enzyme Linked-Immunosorbent Assay that can detect and quantify substances. I will be using this kit to detect the amount of cortisol levels in the saliva of the participants before and after being with a therapy dog and then comparing any change that is found. The Setting: The experiment used Advanced Placement testing as the because there ​ would be many students taking these exams that could participate. Applying this experiment about stress to the students in my own school I thought would have a greater effect and mean more to me than an impersonal subject. Sample Group: Participants that signed up were assigned to either the control or experimental ​ group randomly through each class. Six people signed up from Mrs. Riese’s sixth hour, so I randomly assigned three to the experimental group and three to the control group using a random generator website. I did this for each class, creating a stratified random assignment. ● Control Group: Students without a therapy dog present (no therapeutic interventions) ​ ​ ● Experimental Group: Students with a therapy dog present (therapeutic intervention) ​ ​ Constants: ● Same two conference rooms ● Same questionnaire ● Same therapy dog ● Same time of day-2 afternoon tests Uncontrolled variable: ● Grade level ● # of tests student has already taken/will take ● Pre-existing anxiety from other stressors in student’s life ● /nervousness around dogs ● Different helpers running the experiment ○ Physics: Hanah Geboy, Tyler Thompson and Anica Chestnut ○ Statistics: Hanah Geboy, Tyler Thompson and Mr. Peterson ● # of students in each room (physics had a smaller sample than stats) ● Amount of time interacting with the dog versus time interacting with other participants ● Time of day-students experienced very different mornings ○ If students stayed home for the morning or went to their morning classes

Method Day 1: May 13th, AP Physics C One control group of 3 students in a conference room with two helpers ● One saliva sample taken as they enter the classroom ● Spend 10-15 minutes filling out stress questionnaire ● 25 minutes after the first sample was taken, the second saliva sample taken as they leave One experimental group of 3 students in a conference room with me and one other helper ● One saliva sample taken as they enter the classroom ● Spend 10-15 minutes filling out stress questionnaire ● Time spent with therapy dog ● Second saliva sample taken as they leave 25 minutes after the first sample was taken

Day 2: May 16th, AP Statistics One control group of 9 students in a conference room with Ms. Howe and a helper ● One saliva sample taken as they enter the classroom ● Spend 10-15 minutes filling out stress questionnaire ● Second saliva sample taken as they leave One experimental group of 7 students in a conference room with me and a helper ● One saliva sample taken as they enter the classroom ● Spend 10-15 minutes filling out stress questionnaire ● Time spent with therapy dog ● Second saliva sample taken as they leave

Data Collection Procedure: Students first spit into a plastic cup labeled with their participant number immediately when they came in to the room and then we wrote down their participant number on a white board with the the time they would have to give us their final sample (25 minutes later). After giving us their first sample, the participant received a survey to fill out, and the experimental group interacted with ​ the therapy dog as well. Meanwhile, my helpers and I pipetted the first samples into a labeled 2 ​ ml centrifuge to be stored in a freezer until the samples could be analyzed. After the 25 minutes were up, the participants gave us their last sample that was also pipetted into a labeled 2 ml centrifuge. The participants were then free to leave or stay to hang out with friends and the therapy dog.

Data Reading procedure: All of the samples were frozen until both Ms. Howe and I had enough time to run the ELISA test. The night before the test, I moved the samples into the fridge to thaw and then took them out of the fridge an hour before the test so they could come down to room temperature. Once they were room temperature, I centrifuged the samples for 5 minutes to separate the mucins so the supernatant (clear colorless portion of the saliva) would be easier to pipette. After all the samples has been centrifuged, I followed the chart I made prior to the test to put 100 μL of each standard control and samples into the wells.

I then pipetted 200 μL of the enzyme conjugate into each well, mixing it for ten seconds after.

The samples then had to incubate at room temperature for 60 minutes After incubating, I rinsed the wells with 100 μL of dilutes wash solution 5 times, striking the wells on paper towels between each rinse. After rinsing the wells, I added 200 uL of substrate solution to each well.

The samples then had to incubate for 30 minutes at room temperature. I then added the stop solution (100 uL to each well). Because I didn’t have access to a microtiter plate reader, I wasn’t able to accurately quantify my data. I was however able to compare each sample to the standard gradients (the first row). Standard 0 was the least amount of cortisol (least stressed) while standard 6 was the most amount of cortisol (most stressed). Overall, I saw that the control group cortisol levels increased while the experimental group did not change much.

Survey Interpretation; This survey was given to both groups, asking the students about their ​ current stress and how they handle stress throughout the year. Because my project is focused on stress primarily in students in a school setting, I wanted to learn about how the participants in the study are affected by stress and how they handle it throughout the school year and during AP testing. The answers to the survey are subjective based on each student and cannot be compared between students. Students rating their stress out of 10 will have different ideas of what high stress and low stress is between individuals. This survey was meant to show more what the students felt about their preparedness and their own personal stress rather than the overall stress of the group. I entered the participants’ responses into google forms and used the bar graphs made from those responses to interpret the data given. The following bar graphs include all the participants from both the physics and statistics test, both the control and experimental. Stress Survey Link

Things I learned: I learned a lot about the thought and planning that goes into designing and ​ executing an experiment. While there was a lot of restrictions with time and resources due to it being only a high school experiment, trying to make every aspect of this experiment as accurate as possible was difficult. Not only did I talk to Ms. Howe throughout the process of designing this experiment, but I also talked to other teachers in the school with different backgrounds and expertise. Mr. Peterson was very knowledgeable about the use of human subjects in experiments and the study of stress from both a psychological and biological perspective. Mr. Olson helped me decide how to put my data from my surveys into a visual that can be interpreted. Mr. Phelps helped Ms. Howe and I learn how to use the ELISA kit because he has done similar in his classes and he also let me use his equipment and his room to run the samples. Recruiting participants took a lot more planning than I had planned. The framing of what the experiment consisted of was a huge factor, along with what I would be doing and my involvement. Data interpretation was another aspect of the experiment that I underestimated, I had to learn about basic statistics to accurately measure and interpret my

data. I had two different types of data to quantify, the surveys about stress and the saliva samples.

Understanding chronic stress." American Psychological Association. American Psychological Association. 28 May 2019 . ​ ​

Nature News, Nature Publishing Group, www.nature.com/scitable/ebooks/english-communication-for-scientists-14053993/118519636#b ookContentViewAreaDivID. ​

Yerkes- Dodson Law of Arousal and Performance Applied to Students.” Cdillinghamblog, 12 Nov. 2013, cdillinghamblog.wordpress.com/theory-and-concepts-related-to-teaching-and-learning/theory-ab out-learning/yerkes-dodson-law-of-arousal-and-performance-applied-to-students/.

Sanescohealth.com, sanescohealth.com/stages-of-general-adaptation-syndrome/.