Working in front of our lab’s fume hood

Author: Melih Karabacak | Major: Biology | Semester: Fall 2023

My name is Melih Karabacak and I am in the Fulbright College of Arts and Sciences. I am a Biology major with a Chemistry minor and I am conducting research under my mentor Dr. Jeannine Durdik in the Biology department. My future plan is to continue this research until my graduation in Spring 2025, then attend medical school. This blog is written for my Fall 2023 semester of funding.

During my Sophomore year, I was motivated to begin my research endeavors and emailed the head of the Biology department for any current faculty staff that were looking for help in their lab. Through some discussions, I eventually chose to take part in Dr. Jeannine Durdik’s research lab. Specifically, I was interested in their topic of choice: understanding the complex relationships between a macrophage’s immune response and its age, temperature, and species.

Through my experiences in Dr. Durdik’s lab, I have learned that once macrophages are stimulated, they undergo a specific, standard pattern in metabolic change; this change is measured in PER (glycolysis) and OCR (metabolic respiration). Furthermore, this standard pattern can be denoted by four specific points: Basal, Activation, Nitric Oxide production, and Glycolysis peak, together known as BANG points. I have also found out that through Dr. Durdik’s past experiments, macrophages that were put under fever temperatures had BANG points far earlier and at elevated values compared to those at normal temperatures. This correlates with the tendency for macrophages to have improved function at fever temperatures, however, the underlying changes in transcription levels for this process was still unknown. Thus, I wanted the aim of my experiment to be able to gather RNA from macrophages at each of the BANG points (at both normal and fever temperatures), and conduct analysis for the functional molecules and metabolic pathways that were being utilized to improve immune function.

Moreover, in recent years, it has become more difficult for people to reach ideal fever temperatures, primarily due to decreasing average body temperatures in the past 100 years, and this temperature naturally decreasing with increasing age. Nevertheless, artificial fever temperatures (eg. a warm blanket) have shown to still improve immune responses. Therefore, in order to improve the efficiency and stimulation of the relationship between fever temperatures and improved macrophage function, I will need a thorough understanding of its regulatory pathways.

This past semester, I have spent most of my time primarily getting ready to conduct my experiments. I was going to perform a total of three independent runs in which I collected RNA from each BANG point, however, I needed to first harness the skills and knowledge to efficiently pull this off. Initially, I learned culturing methods and how to count and subculture my cells in the lab. This consequently allowed me to figure out the doubling time of my cells and calculate how long I would need to grow them to reach ideal experiment numbers. Next, I spent a considerable amount of time expanding and refining the procedures for my experiments, specifically in terms of calculating the amount of materials I needed; these included the number of cells, volume of stimulants, and amount of Trizol solvent to harvest RNA. Furthermore, to conduct my experiment, I would be using a machine called a Seahorse. This instrument would allow us to load up cells, add stimulants, and observe changes in PER and OCR under different temperatures. Unfortunately, before joining this lab, I did not have any experience in operating this machine. However, Dr. Durdik and another graduate student, Kenneth Taylor, were planning on using this instrument for one of their other projects; hence, by shadowing them and following their guide, I was able to learn how to prepare cells in advance, load them into the Seahorse, begin an experiment, and collect the cells once completed.

At the end of the semester, Dr. Durdik, Kenneth, and I decided to finally perform a run of our BANG experiment. However, we were unfortunately unable to harvest RNA from the last two points due to the metabolic changes never appearing in the Seahorse. Nevertheless, I have learned many valuable skills this past semester, and hope to perform my experiments in the Spring. In the meantime, I will be working with Kenneth in order to troubleshoot what went wrong during our first run so we can avoid it in the future.

I ran into many challenges this past semester, both in terms of skill and knowledge. However, I was fortunate enough to have my mentor Dr. Durdik and her graduate student Kenneth to guide me in solving them. However, one significant problem that I learned to fix myself was communication and time management. Although this past semester may have been busy for me, I hope to visit the lab more often in the Spring through better scheduling, thus allowing me to become a better researcher by efficiently communicating with the rest of my team. Furthermore, I hope to finish my BANG experiments and RNA analysis next semester, hence preparing me to  publish my research and present at conferences.