This summer I had the great opportunity to work as a fellow within the American Physiology Societies Summer Undergraduate Research Fellowship program. I was able to study under Dr. Michelle Gumz who focuses on circadian rhythms in the kidney at the University of Florida. Circadian rhythms help us wake up and brush our teeth in the morning and fall asleep the same time every evening. Four distinct classes of genes called Per, Cry, Bmal, and Clock, regulate these rhythms. Our lab studies the regulation of Per1 and its effects on blood pressure as a circadian rhythm. Mean arterial blood pressure decreases at night when humans sleep, called “dipping” and peeks when we first wake in the morning (Solocinski, et al., 2016). My research project is designed to study the effects of removing the Per1 gene globally from the 129/sv/s1s4 mice background. After the mice are developed we inserted a radio-telemetry device to monitor their hourly blood pressure. With that data we run specific statically analysis, including cosinor analysis. Cosinor analysis is a program to studies the mesor, amplitude, period, and acrophase of the mean arterial pressure (MAP) of each animal. The mesor, midline-estimating statistic of rhythm, is the midpoint on the y-axis and based on the distribution of MAP across the circadian rhythm. The amplitude is the distance between the mesor and the maximum/minimum point of oscillation. The period is the measure of a full circadian wave. The acrophase is the distance along the cycles peaks or crests that fits the measurement of the circadian rhythm. Each variable allows us to observe the MAP in a circadian pattern to determine the variation between WT and KO 129/sv/s1s4 mice. After I complete the analysis of the blood pressure data from the 129/sv/s1s4 mice we will then run western blot and immunohistochemistry analysis to observe the different proteins within the kidney.

Working in a biochemistry research lab everything is measured in micro values and we work with small portions of protein and DNA to test for specific genes. The process of discovering new proteins expressed in wild type or knockout kidneys is an exciting experience. We develop new conditions for western blots, which is a detection process for select proteins within a sample by using antibodies. Each antibody needs individual concentrations and washing methods to discover the correct ratio to produce a viable band of protein. In addition to study the changes within different proteins levels it is also important to observe the physiological response within the animal models. Measuring hourly blood pressure points allows us as researchers to recognize how the animal is adjusting towards the global removal of the Per1 gene. Once we understand the physiological response and how the body adapts to gene changes we can then apply this result to human studies. Blood pressure regulation is a major contributor to heart disease. If we are able to better comprehend the circadian clock gene regulation within the kidney we could improve cardiovascular outcome.

Our procedures for western blots and immunohistochemistry can take two to three days to see results. It can be concerning when we do not produce correct results, but as a lab we make adjustments and start the protocol over again. Through the optimizing process it can take weeks to produce a blot with results from our Per1 knockout mice population. However, once we optimize the antibodies we can test multiple samples to gain data for publication. Working alongside Dr. Gumz and her PhD students, it creates a reliable environment for undergraduate students learning basic scientific procedures. Working with a team allows me to make mistakes and learn how to correct different procedures to perfect western blot, immunohistochemistry, and telemetry protocols. The life of a scientist can be very rewarding when you are able to optimize protocols and receive results that can determine different gene regulations. Within the Nephrology Department at the University of Florida we attend weekly meetings to discuss other research from a variety of department labs. This allows me to learn about different lab protocols and apply their procedures to our current studies to produce ideal results. This summer research fellowship was a wonderful opportunity for me to improve as an undergraduate researcher and make connections with students across the country that are also interested in pursuing science.

References

1. Solocinski, K., Holzworth, M., Wen, X., Cheng, K., Lynch, I. J., Cain, B. D., . . .Gumz, M. L. (2016). Desoxycorticosterone pivalate-salt treatment leads to non-dipping hypertension in Per1 knockout mice. Acta Physiologica,220(1), 72-82. doi:10.1111/apha.12804

Amber Miller is a rising senior majoring in Health Science at the University of Florida in Gainesville, Fl. She is a 2017 student in the Undergraduate Summer Research Fellowship that is funded by the APS. Amber works in Dr. Michelle Gumz’s lab at the University of Florida that focuses on circadian genes and its effects on blood pressure. She is currently applying to medical school this summer with plans to become an academic physician in the field of physiology.