STEM

Overview

In my personal opinion, STEM is by far the most unique subject taught here at MAMS and it is one of the primary reasons I came to this school. During STEM 1, each student works on their own individual research project that they create and complete all on their own. My project is about studying the effects of curcumin on memory in relation to levels of amyloid precursor protein using zebrafish. Keep scrolling to learn more about my work.

Abstract

Memory is a primary aspect of neurology and brain function. An estimated 5.4 million people in the U.S. are diagnosed with Alzheimer’s disease making it the leading cause of dementia. Alzheimer’s disease is a neurodegenerative disorder caused by the accumulation of amyloid-beta peptides (Aβ). Aβ is derived from amyloid precursor protein (APP), which is a prominent protein in the central nervous system. Curcumin, a compound that majorly componentizes the common household spice turmeric, can be protective against the buildup of Aβ due to its ability to adhere to Aβ and inhibit its aggregation. The purpose of this study is to observe how increasing amounts of curcumin directly influence memory through its impact on Aβ, using Danio rerio as a model organism. Using a sample of 24 zebrafish (before vertebrae development), they were put through a habituation test where a stimulus is initially conducted once to all zebrafish then repeated once after curcumin treatment. The second time the stimulus is repeated, their response times are recorded. Four groups were given increasing amounts of curcumin, with one group receiving none (control group). Using the response times, the effects of curcumin on memory were determined based on the correlation between the quantity of curcumin and the time of their immediate reaction. Based on the results of the data, the correlation most closely related to an exponential function. As the quantity of curcumin given to the zebrafish increased, the time of their immediate reaction to the stimulus decreased.

Phrase 1: Researchable Question

Phrase 2: Hypothesis

How does curcumin affect memory in relation to APP levels in zebrafish?

If curcumin induces a decrease in APP then there will be an increase on memory in Danio rerio.

Background

Infographic

One major symptom of Alzheimer's disease is memory loss making it the leading cause for dementia worldwide. Although AD cannot be fully cured, it can very much be prevented or treated to subside the symptoms of dementia.

Amyloid-beta (Aβ) accumulation is a determinant of cognitive decline, in AD patients, caused by post-translational modification (PTM) and trafficking of amyloid precursor protein.

Curcumin is the compound in turmeric and is a generally universal nutraceutical. It prevents AD pathogenesis due to its abilty to adhere to peptides and enzymes reducing Aβ generation.

Danio rerio, formally known as zebrafish, are an accurate model organism in depicting neurological disorders due to their clarity in modeling human brain functions.

Procedure

Infographic

In the initial plan, around 25-30 zebrafish would be used to test with and the mechanism for experimentation would be a maze. They would start on one end with a reward chamber holding fish food on the other end, with one correct pathway they must follow. The fish would each be given a standard five minutes to initially navigate the maze and to embed the structure in their minds. The fish would then be split into five different groups given four increasing amounts of curcumin per group, with one group receiving non to behave as the control group. After being given about an hour to consume the turmeric, each fish would once again be put in the maze, however, they would be timed for how long it takes to get to the reward chamber.

The fish were kept in a bowl at about 78º F and were tested on the fifth day of development, to ensure the experimentation was done prior to vertebrae development. First, using a pipette, each fish was removed from the bowl and placed into a well tray with each well having a depth of 0.6cm and a radius of 1.0cm. The volume of each well calculated to be around 1.05558cm3. Upon placing seven fish in these wells, they seemed to die, perhaps due to suffocation. In addition, each fish was incredibly difficult to capture with a pipette and when being moved, they seemed to be losing general energy, based on visual observation. Because of this, it was determined that the initial procedure would not work. After additional research, the idea of a habituation test was concluded on as an experimentation mechanism.

Habituation simply refers to the reduction of a response in reaction to a repeated stimulus. For this, each fish was placed in larger individual wells where they would remain for the remainder of the testing. First, the pipette was used to circle the inside border of the each well once as an initial stimulus for the fish to naturally react to. After this, each group was given a different amount of turmeric to consume. 24 fish were split into four groups of six with one receiving no turmeric acting as the control group. For the other groups, 10mg of turmeric was mixed into 10mL of water and a suspension was created by constant stirring as the three independent amounts were extracted with a micropipette. One group received 25µg, the second received 50µg, and the third received 100µg. After one hour of consumption, the stimulus of the pipette being circled around each well was repeated, however, this time, the reaction time was recorded. The dependent variable was the amount of time that each fish takes to return to a still position after a known stimulus is conducted. With these results of curcumin quantity and time of immediate response, the correlation between curcumin and memory can be analyzed.

Results

Complete data of four variable groups with six fish in each group tested.

Data

Average times of each group with standard deviation and percent difference of SD to average time.

Data

Analysis

The results of the data show for there to be a correlation between the quantity of curcumin that is consumed in comparison with the time of immediate reaction following the repeated stimulus. The time in the table represents the amount of time that the zebrafish moved before coming to a stop after a pipette was circled around their well once. Prior to the action of the stimulus, each zebrafish was at a complete standstill, therefore allowing the time to stay true to their movement. The average standard deviation was about 14.63% which is pretty high, meaning that the data collection was overall not incredibly precise. This likely has to do with the fact that the time was being recorded based on human observation during the period of individual experimentation. This accounts for multiple factors of human error as well as movement error of the zebrafish, as that was also just considered through visual observation, which is neither completely precise nor accurate.

The values of the average data points were graphed on a scatter plot and then two potential trendlines were drawn based on the general shape of the regression. The first standard trendline was that of a linear function for which the data fit somewhat accurately into. The R2 value was 0.8892 meaning that this was not the most precise model for the data, however, it is still within roughly 11% of ultimate precision. The second graph represents the same as the first except with an exponential function going through the average data points instead. This type of regression was determined to be drawn by the visual appeal it had for the data points. For this trendline, the R2 value, 0.9849, was much higher and demonstrated greater accuracy and precision with its calculation. Based on the results it can be said that the exponential function has the strongest correlation with the average data experimented, although the data itself may not be closely in sync.

In addition, a statistical ANOVA test was conducted for which the p-value was 3.11E-12. If the p-value of a data set is less than 0.05, that indicates a significant difference in data meaning that a correlation does exist. Since 3.11E-12 is greatly below 0.05, it can be said the data is valid and does display an accurate representation of what is being tested.

Conclusion

The data overall showed that as the quantity of curcumin given to zebrafish is increased, the memory will also be improved in terms of habituation learning. As a stimulus is repeated, the reaction time of zebrafish reduces due to their cognitive capabilities and adaptive learning. This learning and memory can be further improved with the consumption of the curcumin compound found in turmeric. This outcome fits that of the hypothesis, however, for a different reason than what was discussed. The hypothesis predicts this outcome because of the correlation between Aβ and memory, and curcumin and Aβ. The organisms that were tested, however, were not Alzheimer’s models of zebrafish, therefore not possessing the APP gene that eventually leads to Aβ production. Based on the data, it is clear that there is a direct correlation between curcumin and memory, however, the underlying cause of this is still one that needs to be further researched as it is likely not due to the reason that has already been heavily researched prior to conducting this experiment. In addition, the results have shown to display a strong exponential trend in the relation between curcumin and memory. As curcumin consumption is increased, memory improves along with it.

Extensions

The largest portions of the project that could not be fulfilled as intended were the type of zebrafish that needed to be used. The background research suggests that the zebrafish had to be those of Alzheimer’s model to test the appropriate variables and factors. Alzheimer’s model zebrafish essentially means that they display Alzheimer’s symptoms, such as memory loss, due to the use of an amyloid gene promoter, which induces the APP gene inside of them. This is a crucial aspect of the project considering that the hypothesis discusses the effects of turmeric on memory, specifically due to the connected relation with amyloid precursor protein. For this reason of lack of intended fish, future work would certainly include being able to test with Alzheimer’s model zebrafish and observing whether that creates a difference in the results.

Since the current experiment has not been conducted with the appropriate fish. The next step to this exact study would be to determine if there is any other third-party connection between turmeric and memory, as it is not solely amyloid precursor protein, as shown by the results using the normal zebrafish. To answer this question, additional research can be conducted to determine whether there is another external or internal correlation between the two.

The entire test was run using many fish, however, the entire process can still, in a way, be counted as one massive trial. Because of this, the most obvious future work would be to hopefully conduct this entire test once more to see if the results remain close to what they were the first time or not. In addition, hopefully the use of a lab could be acquired to cut down logistical hassle immensely.

References

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February Fair Poster File

Here is my poster file from the February STEM fair that I printed out to paste on to my trifold.