ABSTRACT

The Illinois Junior Academy of Science

This form/paper may not be taken without IJAS authorization.

CATEGORY Biochemistry STATE REGION # 6

SCHOOL Niles North High School IJAS SCHOOL # 6038
CITY/ZIP Skokie, IL SPONSOR CELL PHONE # 8475022672
SPONSOR Christi Camel SPONSOR E-MAIL chrcam@d219.org

MARK ONE: EXPERIMENTAL INVESTIGATION ☐ DESIGN INVESTIGATION ☐

NAME OF SCIENTIST* Ellen Chibirko GRADE 10th

NAME OF SCIENTIST GRADE
NAME OF SCIENTIST GRADE
NAME OF SCIENTIST GRADE

* If this project is awarded a monetary prize, the check will be written in this scientist's name, and it will be his/her responsibility to distribute the
prize money equally among all participating scientists.

PROJECT TITLE Disruptions of our Circadian Rhythm

Purpose: Cognitive illness is an unfortunate aspect of aging which is thought to be influenced by many factors in a human's
younger years. One such factor that has yet to be explored is our circadian rhythm. We are not exploring lack of sleep, but
more so obeying our light cues by sleeping at nightfall and being active during the day. If the proposition that disrupting our
circadian rhythm leads to earlier onset neurodegenerative disease is found to be true, such information can be displayed and
advertised in order to encourage a superior general understanding of cognitive decline and maintaining longevity.

Procedure: Drosophila Malngastoer will be separated by sex. All trials conducted will have a control and treatment group. The
control groups will be placed near a window where there will be little to no change within their natural environment. The
treatment group will be wrapped in tin foil and placed in a quiet area as the sun rises and later, unwrapped and placed in an
orbital shaker lit with halogen light bulbs as the sun falls. After 5 consecutive days of this treatment, both groups will be tested
for their negative geotaxis. With a minimum of 3 trials with each sex being conducted, average times in which the fruit flies
navigate their way up the tube and patterns in which they travel will be recorded and observed for future comparison.

Conclusion: The majority of both male and female fruit flies within the control group had not made it to the top of the tube
at all. Only control group 1 (female) had an average of 16.14 seconds with an 8 percent rate of accuracy (this means the
mean only accounts for 8 percent of the fruit flies and the other 92 percent had not traveled at all.) All treatment groups,
both male and female have shown a similar pattern in which they have an average of 10-15 sec/15 ml. The rates of
accuracy were different among the treatment male and female populations. They were much higher among males
(Average 76%) and much lower among females (Average 13.2%). From observation and final measurements, female
populations were much higher than those of male populations in both the control and treatment groups. The control group
of females had an exponentially higher population than that of the control group of female fruit flies. From these
calculations and qualitative data, the fruit flies receiving treatment were much more active and higher in population than
the control group. The behavior of the fruit flies does not prove our hypothesis but presents interesting patterns.
 SAFETY SHEET
The Illinois Junior Academy of Science

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and/or advisor must sign in the indicated space. By signing this sheet, the sponsor assumes all responsibilities related to th
project.

Safety and the Student: Experimentation or design may involve an element of risk or injury to the student, test subjects and
others. Recognition of such hazards and provision for adequate control measures are joint responsibilities of the student an
the sponsor. Some of the more common risks encountered in research are those of electrical shock, infection from pathogen
organisms, uncontrolled reactions of incompatible chemicals, eye injury from materials or procedures, and fire in apparatus
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the chart below, list the principal hazards associated with your project, if any, and what specific precautions you have used
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entitled "Safety Guidelines for Experimentation'' before completing this form.

Possible hazards Precautions taken to deal with each hazard

Goggles will be worn when
Flying organisms handling fruit flies
● Will be anesthetized through freezer
● Caution will be taken with cold surfaces
through gloves

Specific safety practices related to materials requiring endorsement sheets should be detailed on the specific
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Please check off any other possible endorsements needed. Include these documents in your paper and on your board.
_____Humans as Test Subjects –for any projects involving humans including survey administration;
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SIGNED Ellen Chibirko

Student Exhibitor(s)
SIGNED

Sponsor *
*As a sponsor, I assume all responsibilities related to this project.
Disruption of Circadian Rhythm
(Experiment by Ellen Chibirko, Niles North High School)
TABLE OF CONTENTS:

Abstract: Page 1

Safety Sheet: Page 2

Title: Page 3

Table of Contents: Page 4

Acknowledgments: Page 5

Purpose and Hypothesis: Page 6

Review of Literature: Page 7-11

Materials and Methods: 12-14

Variables: Page 15

Results: Page 16-17

Conclusion: Page 18

References: Page 19-21

 Acknowledgements
Through a Scientific Inquiry class offered by Niles North High School, I have been able to obtain lab settings
and equipment. My mentor has been our professor, Christine Camel, who has introduced me to scientific
methods that include separating fruit flies and applying orbital shaking machines.
 Purpose and Hypothesis
Completely inverting the flies' already developed circadian rhythm will cause them to perform

significantly worse on the climbing activity than the flies who maintained their initial circadian

rhythm. When monitored under confocal microscopy, the population of dopaminergic neurons will be

significantly lower in the group of flies that have disrupted their circadian rhythm than in the flies

who maintained their initial circadian rhythm. Within C. Elegans, if their circadian rhythm is

inverted, they will pose more C-shaped bends according to our chart than in C. Elegans with normal

circadian rhymes. In both the C. Elegans and fruit flies, sex will not play a role in nuerdegeration

when the organisms circadian rythme has been changed. Both results for C. Elegans and fruit flies

will prove that changes within a developed circadian rhythm leads to formation of neurodegenerative

disease. A twenty four hour circadian rhythm which is facilitated by biological cues such as light

(assuming the individuals initial circadian rhythm has been facilitated this way through infancy) is

essential to maintain for longevity.

 Review of Literature

Have you ever found it excruciatingly difficult to close your eyes in attempts to fall asleep a

few hours earlier than usual and wake up before the sun has even risen? This is not a coincidence,

and what accounts for this is our body’s biological cycle called the circadian rhythm lasting roughly

24 hours which you have already developed complementary to that of the rising and falling sun

(Gnocchi & Bruscalupi, 2017). Another interesting topic explores the unfortunate aspect of

neurology. Neurodegenerative disease is known to be much more evident within people of older age

and this concept has and is being researched in a number of ways, whether through case studies, large

surveys, etc. What defines neurodegeneration can be broken down into the loss of structure or

function within nerve cells. There are many different classifications of neurodegenerative disease

such as Alzheimer's, Parkisons, and many more but they are all very similar as many of the same

proteins, cells, and parts of the brain are involved. Cures to such diseases are yet to be found, so until

then, ways to prevent neurodegeneration from occuring in the first place are being actively

researched. Dialing back to the circadian rhythm, a number of studies have shown that disruption of

this cycle as an infant may lead to psychiatric diseases into adulthood (Yates, 2018). But is this all

there is to know about circadian rhythm? I suspect not. Can disrupting our recommended sleeping

habits according to our body's biological systems cause neurodegeneration? Will we maintain

longevity no matter how we choose to sleep, or can our sleeping habits be the cause of our cognitive

decline?
To study circadian rhythm, model organisms have proven to be excellent candidates for such

research. When human experimentation becomes marked as unethical, model organisms are studied

with the expectation that they will allow for discoveries to be made that are applicable to the human

population. Although model organisms are quite different from humans, the common descent of all

living organisms makes these applicable discoveries possible.

When studying neurodegenerative disease such as Alzheimers, invertebrate models such as C.

Elegans, Drosophila Melanogaster, Ciona Intestinalis, etc, have proven to be excellent test subjects

for correspondent research applications. Looking further into C Elegans, major research applications

include: localization of amyloid deposit, effect of aging on the size of amyloid plaques, Aβ toxicity

and behavior correlation, and effect of dietary deprivation in AD. Similar applications are used on

different model organisms but one advantage of the C. Elegan is its short life span. Short lifespan

allows for results to be harnessed much more quickly than that of an organism with a long lifespan.

Another advantage is the worm's transparency. This makes studying developmental processes such as

cellular differentiation much easier than in another organism (Marta Di Carlo, 2012). Lastly, C.

Elegans express roughly a 24 hour circadian rhyme facilitated by light and uniquely, temperature.

When targeting neural impairment within these organisms, many techniques are used to measure

symptoms of such impairment.

Looking further into Drosophila Melanogaster (fruit flies), Drosophila Melanogaster are better

used for assessment of modulators of BACE1 or APP metabolism, localization of Aβ peptide, and the

determining the role of tau protein in AD. Like C. Elegans, fruit flies exhibit short lifespans as well

as a 24 hour circadian rhythm facilitated by light. Fruit flies have been especially useful for
discoveries within the field of neurodegenerative disease as the fly has a similar neuronal

composition to that of the vertebrate central nervous system while carrying an estimate of a whopping

100,000 neurons to monitor (Marta Di Carlo, 2012). An easy way brain activity is measured after

treatment or detection of a specified gene is by something called the climbing activity which

questions whether the flies' negative geotaxis has been impacted by the treatment or gene detected. A

decline of negative geotaxis is commonly associated with impaired brain development. Through

more advanced microscopy, the number of dopaminergic neurons are counted and compared.

Complementary to its name, dopaminergic neurons synthesize neurotransmitter dopamine.

Conveniently, dopamine happens to be one of the primary hormones associated with circadian

rhythm providing an excellent case as to why these invertebrates are quality model organisms for

such procedures. This data should be sufficient enough for a data table that reflects significant

changes within the flies locomotor behavior as well as the size of their dopaminergic neuron

population (Maria Cecilia Barone et al., 2013).

Measuring a decline of neurodegenerative disease is very doable in both fruit flies and C.

Elegans. Fruit flies are tested on the sharpness of their negative geotaxis whereas c elegans

interestingly enough, can be measured by the C - Shaped bends they present after treatment.

Although this is ideal within circumstances that require fast and budgeted results, this method (called

thrashing) is very error prone, especially for those new to such methods (Buckingham & Sattelle,

2009). When concentration of these C. Elegans are measured in comparison to the dish it is held

under, applying these same parameters for every step of measurement should stay within our

calculated confidence interval. This method is not exclusive to c. elegans, but can be used on any

nematode that swims in liquid by applying the organism's specific morphometry.

 To get a deeper appreciation for our 24-hour biological clock, we must know the history of

what makes this inherent trait so great. Evolution ties us to one common ancestry amongst all living

organisms making circadian rhythms a trait similar within almost all organisms. For example; flies

have shown a circadian rhyme cycle of around 22-28 hours: the leaf cycle of Phaseolus coccineus

showed roughly a 24.4 hour circadian rhythm: and humans have shown a circadian rhythm lasting

roughly 24 hours (all of which are counseled by light cues). Generally, our circadian rhythm provides

optimal times in which our bodily processes take place, almost like a monitor for our homeostasis.

Many hormones associated with these processes are melatonin, insulin, leptin etc. (Gnocchi &

Bruscalupi, 2017). No matter how one sleeps, our body’s biological clocks will be activated by light

cues believed to be primarily recepted and detected through ganglion cells in place of rods and cones

inside the retina. This indicates that although a person may be persistent within the hours in which

they sleep which obeys one rule of circadian rhythm, not waking and sleeping to the sun's rise and

fall may be considered a disturbance to this aspect of health.

Additionally, more studies have shown that the circadian rhythm is not just an arbitrary

assortment of times in which we have learned to sleep, but a necessary cycle that should be

maintained for proper development as a newborn into infancy and overall longevity. Newborns spend

roughly 60-70 percent of the 24 hour day sleeping and as they age, progress into a diurnal pattern

with less, but higher quality sleep. Through the development of the light bulb, maintaining circadian

rhythm is much more difficult than it was 140 years ago and therefore, sleeping habits in newborns

must be monitored very closely. This is proven by studies conducted on young mice in which they

recieve abnormal light exposure. The results showed that genes in the peripheral clocks of the lungs,
spleen, and heart had been negatively affected into adulthood. Other studies have also shown that

similar disruptions within young mices’ circadian rhythm instigate anxiety-like behaviors as well as

bipolar disorder as they age (Jaqueline Yates, 2018).

In closing, circadian rhythm is surprisingly a lesser known topic yet unquestionably

important. Bodily processes are essentially built upon these times. With neurodegenerative disease

causing untimely suffering and death, the findings of this procedure may mark the beginning of a

preventative method. Testing the relevance of circadian rhythm will work on model organisms such

as fruit flies almost just as well as a human subject. With a significant quantity of neurons with

similar structures to that of a human and a negative geotaxis that we may use for all sorts of

measurements, fruit flies can present very applicable data. What we will find will answer the question

as to whether being an early bird or night owl affects your health negatively. A significant decline in

the average person's cognitive health frequently occurs from ages thirty to forty. Extended research

has also asked whether sex plays a role within cognitibe decline anchoring more towards females

recieving the short end of the stick. Discovering such factors of neurodegenerative disease will bring

hope to newer generations as they can expect an overall better quality of life and longer lifespan than

those of older generations.

 Procedures and Materials:

First experiment: (run a minimum of 3 trials of this experiment)

1. Take two tubes of female fruit flies and label one “control” and the other “treatment”
2. Take the tube labeled “control” and leave it near the window with clear sunlight. Don’t touch
this until 5 days have passed.
3. At sunset, place the treatment tube within an orbital shaker at a speed of 20.
4. At sunrise, take these fruit flies and wrap them completely in aluminum foil.
5. Repeat this cycle for 5 days consecutive days.
6. After treatment has occurred, place a high quality camera to capture the flies within both test
tubes.
7. Begin to record and gently tap both test tubes for all the flies to descend to the bottom of the
tube
8. After fruit flies have stopped traveling to the top of the tube, conclude your recording
9. Calculate the rate at which the fruit flies were able to navigate their way to the top in both the
treatment and control group (distance/min)

Second experiment: (run a minimum of 3 trials of this experiment)

1. Absolutely the same as our first experiment, but only using male fruit flies.

Third experiment (run a minimum of 5 trials of this experiment) (can only be a possibility in April of
2022)
1. Take a population of c elegans and record on camera the c elegans for a 24 hour period near a
clear window. Observe and record their sleeping patterns (movements) through a speed up
version of the video.
2. Repeat step 1 twice.
3. Split the population into two petri dishes.
4. Label one petri dish control and the other treatment.
 5. According to your observations from the speed up video, infer what an inverse circadian
rhythm would be for the c. elegans.
6. At the time of when the c. elegans have been observed to sleep, leave the petri dish labeled
control near a clear window for the rest of this experiment.
7. At the same time, in a room with light, place the treatment tube within a machine that shakes
the petri dish rapidly for one second every time a camera detects movement (if this isn’t
possible, have it automatically shake for 20 seconds). This will continue until sunrise (look up
what time sunrise and sunset is for maximum accuracy).
8. Next take aluminum foil and wrap the petri dish of c elegans who are under treatment.
9. Repeat this cycle for 5 days consecutive days.
10. After treatment has occurred, place both the control group and treatment group of c elegans in
pure water quickly in a petri dish. Start recording at an angle to which all the c elegans
movements are visible.
11. Stop recording at 60 seconds.
12. Calculate the number of c shaped bends in each group per 30 seconds. This may take a long
time so count every c elegan who bends second by second.

Materials;

Fruit flies trials

High resolution camera

Timer
Orbital Shaker
Male and female fruit flies with no altered genes
(6) 12’ inch tubes
Petri dishes (for separation of the different sexes)
Microscope (for separation of the different sexes)
Test tube shaker
Aluminum foil

C Elegans trials

Male and female C. Elegans with no altered genes

High resolution camera
Timer
Petri dishes
Petri dish shaker
Aluminum foil
1.5 inch tall glass cup
 Variables

The independent variables were times in which they will experience darkness and go to sleep. This was

manipulated by covering the flies with tin foil. What this independent variable targeted was

something we would measure after 5 nights and 4 days of treatment which was the time in which it

takes the flies to travel up 15 ml when transferred to a clear tube. This measures their negative

geotaxis which has a positive correlation to brain impairment. Essentially the only factor that

remained constant was the food they received. Our control group was left alone near a window to

experience what a “natural” pattern of light cues would look like.

 Results
C - Control
T - Treatment
1 - Trial 1
2 - Trial 2
3 - Trial 3

Sets of data points labeled as ( - ) indicate no flies had traveled upwards of 15mL

C1F Time traveled up tube: 5. 67, 11.6, 16.0

Average - 11.09 sec/15ml with 8 percent accuracy

Pop: 25

C2F Time traveled up tube: -

Average - 0 sec/15 ml with 100 percent accuracy

Pop - 43

C3F Time traveled up tube: -

Average - 0 sec/15 ml with 100 percent accuracy

Pop: 26

C1M Time traveled up tube: -

Average - 0 sec/15 ml with 100 percent accuracy

Population 3 (where many died from indents)

C2M Time traveled up tube: -

Average - 0 sec/15 ml with 100 percent accuracy

Population 4

C3M Time traveled up tube: -

 Average - 0 sec/15 ml with 100 percent accuracy

Population 2

T1M Time traveled up tube: -

Average - 0 sec/15 ml with 100 percent accuracy

Population 2

T2M Time traveled up tube: 4.2, 7.48, 7.56, 8.04, 8.11, 10.04,11.68, 11.97, 24.77

Average - 9.13 sec/15 ml with 64.2 percent accuracy

Population 14

T3M Time traveled up tube: 8.15, 12.03, 24.41, 25.38,

Average - 17.49 sec/15 ml with 66.6 percent accuracy

Population 6

T1F Time traveled up tube: 5.13, 7.04, 8.17, 10.59, 10.59, 11.74, 12.41, 12.68, 17.15,
17.5, 17.5, 19.97 (flies who had already made it up had went down and started
flying gaina so data may be distorted)

Average - 12.53 sec/15 ml with 12.4 percent accuracy

Pop 97

T2F Time traveled up tube: 3.53, 5.40, 6.86, 9.10, 10.72, 12.73, 23.2, (flies who had
already made it up had went down and started flying gaina so data may be
distorted)

Average - 10.22 sec/15 ml with 13.2 percent accuracy

Pop 53

T3F Time traveled up tube: 5.73, 6.47, 7.08, 7.78, 8.34, 8.84, 9.76, 10.81, 11.25,
24.31, 26.2, (flies who had already made it up had went down and started flying
gaina so data may be distorted)

Average - 11.5 sec/15 ml with 14.2 percent accuracy

Pop 77

Confounding Variables that may have had an effect on our results -

● T1M had many of their initial population dead due to the food not being pressed properly. This is a trial that does not indicate statistical significance to
our findings. This could have been prevented if their food had been compressed more thoroughly

● Flies, especially in the TF groups, were traveling 15 ml and some did so continuously. Some data points may or may not be a result of the same fly
traveling. This may or may not have been prevented by having a shorter time limit for our recordings and only measuring the times of the first 3- flies
times.
● Their environment should have stayed the same but switched at different times of the day. The control group should have been placed within the orbital
shaker and tin foil aswell.
 Conclusion
Conclusion - The majority of both male and female fruit flies within the control group had not made it to the top of
the tube at all. Only control group 1 (female) had an average of 16.14 seconds with an 8 percent rate of accuracy
(this means the mean only accounts for 8 percent of the fruit flies and the other 92 percent had not traveled at all.)
All treatment groups, both male and female have shown a similar pattern in which they have an average of 10-15
sec/15 ml. The rates of accuracy were different among the treatment male and female populations. They were much
higher among males (Average 76%) and much lower among females (Average 13.2%). From observation and final
measurements, female populations were much higher than those of male populations in both the control and
treatment groups. The control group of females had an exponentially higher population than that of the control group
of female fruit flies. From these calculations and qualitative data, the fruit flies receiving treatment were much more
active and higher in population than the control group. The behavior of the fruit flies does not prove our hypothesis
but presents interesting patterns. What may account for our results that are essentially insignificant is the difference
within their environments. Our hypothesis simply changed the times in which they were exposed to light and dark,
therefore, the control should have been placed within the orbital shaker during the day and wrapped in tin foil
overnight.
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