Alec Dupont Junior, biomedical science major Auburn University
My Research Project
My project involved examining the adaptation of skeletal muscle to resistance exercise in mice that had been depleted of muscle stem cells (satellite cells). Generally, muscle growth is accompanied by an increase in protein synthesis and the differentiation of satellite cells into muscle nuclei. During this project, we examined if growth happens without the addition of satellite cells into muscle. As certain clinical populations have reduced satellite cell content and muscle mass, our project aimed to provide insights into how muscles respond to a growth stimulus with the loss of this cell population.
We used Progressive Weighted Wheel running (PoWeR) as a model for resistance exercise. PoWeR involves voluntary running activity of the mice in weighted running wheels. The weight placed on the running wheel is gradually increased over the course of four to eight weeks, overloading the musculature and causing a growth response called muscle hypertrophy. Using a genetic mouse model that allowed for the selective depletion of satellite cells, we compared sedentary- and resistance-exercised mice in groups of satellite cell-replete (vehicle treated) and -depleted (tamoxifen treated) mice. We compared muscle hypertrophy and other physiological adaptations between groups to determine the effects of satellite cell depletion. At the completion of this project, we hoped to gain a further understanding of the role satellite cells play in muscle growth.
Realities of Research
My main focus for the summer was using muscle tissue from the PoWeR mice, and making it possible to obtain data and useful information. I accomplished this through immunohistochemistry, a laboratory technique where we cut cross sections of the muscle and stain them for proteins of interest. This staining allowed us to visualize the sections under the microscope, image them and quantify the images using different forms of software. This technique presented certain challenges because the tissue must be carefully prepared and stored to prevent degradation. Poor quality tissue introduced variability outside of what is normal to the mice models. For example, having to overcome challenges and work to optimize a stain meant visualizing newly formed RNA in muscle nuclei. The stain can appear too dull and the quality would not be high enough to draw conclusions unless the procedure was optimized. Overcoming these challenges provided stunning images and reliable data. We found that although satellite cells were not absolutely required for muscle growth in response to weighted wheel running, there was a decrease in growth in the satellite cell depleted mice.
Life as a Scientist
The day-to-day life of a research scientist presented me with a constantly changing experience that was more engaging than the traditional classroom setting. There was always a new aspect of the project to investigate. It was incredibly satisfying to see your work come together in data that tell a cohesive story. The process of getting there was occasionally tedious though. For example, we’d normalize our data to the number of fibers in the muscle cross section and when the software couldn’t count for us, we were forced to count by hand. When the sections were between 600 and 800 fibers in a study with 48 mice, that part of research tended to drag. But that was only a minor inconvenience to a necessary bump in the road towards a satisfying research project.
Alec Dupont is a junior at Auburn University in Auburn, Alabama, studying
biomedical science. He is a 2019 Undergraduate Summer Research Fellow (UGSRF)
working under Dr. Charlotte Peterson at the Center for Muscle Biology at the
University of Kentucky in Lexington. Alec’s work is funded by the American
Physiological Society’s UGSRF program and a grant from the National Institute
of Health to Dr. Charlotte Peterson and Dr. John McCarthy (AR060701).
Jayachandra Kura Junior, Applied Physiology and Kinesiology University of Florida 2019 UGSRF Fellow
My Research Project
This past summer, I worked in Dr. Joshua F. Yarrow’s research lab at the Malcom Randall Department of Veteran Affairs Medical Center. Dr. Yarrow’s lab explores the effectiveness of pharmacologic and exercise treatments following spinal cord injury (SCI). For the specific SCI we studied, the posterior end of the 9th thoracic vertebrae was surgically removed, exposing the spinal cord underneath. A machine delivered an impact causing hindlimb paralysis. My research used Sprague-Dawley rats that were given either a 1) SCI, 2) surgical control (SHAM), 3) SCI + Bodyweight Supported Treadmill Training, or 4) SCI + Passive Bicycle Training. We scanned the distal femurs at baseline, two weeks and four weeks after SCI using a micro tomography (microCT) scanner.
In order to observe the effect of each treatment on the spongy cancellous bone, a technician would individually draw a region of interest (ROI) in the transverse view of the femur (Fig. 1) to include the internal trabeculae while excluding both the growth plate and solid cortical bone. However, repeating this on more than 100 slices for every sample at every time point is very time intensive. Instead, I worked to adapt a registration procedure for the spinal cord injury model. The registration was created by using two scans at different time points are aligning them in 3D. An ROI was created at baseline and then applied to the two-week and four-week scans, reducing the amount of labor required. I then compared the data from registered images to data from nonregistered images. I also helped to develop a script that allowed the computer to automatically draw the ROI with minimal manual correction, which further improved efficiency.
Realities of Research
My introduction into research has definitely been equal parts trying and gratifying—trying in the sense that every solution I created seemed to raise a host of other questions that needed to be addressed. I remember when I finally figured out how to register two different time point images, but I then needed to decide what size volume of interest (VOI) to use so the computer knew which landmarks to use to align the two different bones. Intuitively, using a large VOI should provide more datapoints for the computer to use. Doing so caused the solid cortical borders to be well-aligned, but the internal structures weren’t. Repeating this with multiple samples yielded the same results, which suggested that, in bone remodeling, an individual bony landmark’s relative position to the cortical border changes with time. However, using a small VOI caused poor alignment of the two images. This seemed counterintuitive, so when I looked over previous scans of SCI samples, I observed a trend of severe bone loss occurring below the injury site. The registration procedures outlined in the literature couldn’t directly translate to a SCI model as those outlined procedures required clear internal bony landmarks. Without these data points, the automatic registration software couldn’t produce an accurate alignment.
In this seemingly never-ending cycle of forming new ideas only to eliminate them later on, I didn’t come any closer to developing a script, but I did develop a lot of patience and perseverance. I found research to be inherently challenging, but the setbacks I encountered only made me grow as a person and researcher, and ultimately, made the end result of creating a “mostly” functioning protocol all that more rewarding. I’ve also come to realize that there is never a true “end” in research as there arealways things that can be improved or new questions that can be asked. This opportunity for continual growth was really exciting and intrinsically motivating.
Life as a Scientist
Compared to my past work experiences, research has by far been the most enjoyable. Never did I have a bout of the “Sunday scaries,” where I was enveloped with the existential dread of going to work the following day. In contrast, my work environment was low-stress and was dictated entirely by my own drive and will to work. The lab was filled with diverse, interesting individuals and I enjoyed the conversations I had and the relationships I formed with my labmates. Although there was always monotonous data entry, most of the work I did within the lab was challenging and fun. I always felt the work I was doing was meaningful.
I recall a conversation I had with my labmate who’d recently graduated: I had jokingly asked what it was like not having class. He laughed and said, “I spent the last four years—every fall, spring and summer semester—taking classes and working here at the hospital. If you think about it, when you’re an undergraduate, you’re basically working 70+ hours a week with all the stuff you do, so you really never have to think about anything except for school. Now that I’ve graduated and work 40 hours a week here in the lab, my work ends when I leave. But I remember going home and sitting on my couch not knowing what to do with myself, thinking, ‘Man, time to find some hobbies.’” Being out of school, if only briefly, allowed me to finally begin to appreciate this. Now sitting on my own couch trying to find things to do, I’ve found this freedom to be exciting and paralyzing. I definitely feel that the physician/scientist career path is like a pipeline and there’s constant pressure to continue moving towards the end. To be honest, I haven’t put much thought into the adult I want to be outside of my career or really explored the things I find fulfilling. I’m just thankful for the opportunity to have had these experiences, both in and out of the lab, and believe that this summer was largely beneficial for my growth not only as a researcher, but also as a person.
L Arsuaga, J & Villaverde, Valentín & Quam, Rolf & Martínez, I & M Carretero, J & Lorenzo, Carlos & Gracia, Ana. (2013). Arsuaga et al. 2007.
“Establishment of a Rat Model of Spinal Cord Injury (SCI).” Neural Regeneration Research, www.nrronline.org/viewimage.asp?img=NeuralRegenRes_2016_11_12_2004_197145_f1.jpg.
Jayachandra Kura is a junior majoring in applied physiology
and kinesiology and minoring in Japanese at the University of Florida in
Gainesville. He is a 2019 American Physiology Society Undergraduate Summer
Research Fellow (USGSRF) working in Dr. Joshua F. Yarrow’s lab at the North
Florida/South Georgia Medical Center in Gainesville, Fla. Jayachandra’s
fellowship is funded by the American Physiological Society and the Department
of Veterans Affairs. After graduation, Jayachandra plans to pursue a career as
a physician scientist.
Caleb Smith Senior, Applied Health Science Messiah College 2019 UGSRF Fellow
My Research Project
When exposed to a high-fat diet (HFD), both human and rat models show inflammation in the brainstem. The specific area of concern is called the dorsal vagal complex (DVC) which is responsible for maintaining homeostasis, or the balance, of energy and gut function. Specific neuronal cells in the brain, called astroglia, along with inflammation, help to control the DVC.
Previous studies have shown models that experienced a short period of energy regulation after a 24-hour period of excessive eating when exposed to a HFD. Therefore, the purpose of this study was to determine how that energy balance is restored during exposure to a HFD through the activation of the astroglial cells. As part of this study, control and HFD chow were fed to a rat model for one, three, five and 14 days. The brainstems were removed and cut into thin slices and the astroglial cells were tagged with proteins that illuminated under specific lighting. This process, known as immunohistochemistry, allowed for the density of astrocytes and physical characteristics—like size and shape—to be analyzed. To process the role of the astrocytes in this energy metabolism regulation, small tubes called cannulae were surgically inserted into the DVC in order to directly administer fluoroacetate, a drug that inhibits the function of astrocytes. Once the rats recovered from surgery, a five-day control and HFD exposure were fed to the rats while food intake and body weight were measured twice daily.
While data was still being collected, preliminary data confirmed the role of astrocytes in metabolic regulation during HFD exposure. That meant astrocyte activation was necessary in controlling metabolic balance when exposed to HFD. Ultimately, painting the picture of how energy balance is controlled will be essential to producing a therapeutic drug that can help treat obesity.
Realities of Research
Working in a lab was similar to what I expected while still being very different. In many ways, research in a lab is exactly what you would expect: you make solutions, follow very strict procedures and analyze data for results. The techniques that I learned in high school and college labs were carried over with regards to safety, proper procedure, how to handle materials and how to pipette. I would come in and begin my day the same way by weighing and giving rats shots. In other ways, the lab was not what I expected it to be.
There were long periods of time, whether a few hours or days, where researchers were writing manuscripts to submit to journals, editing their own or other colleagues’ manuscripts, writing grant proposals or reading research happening in someone else’s lab to stay up to date on the current information. Not every minute was spent performing an experiment and analyzing data. The rest of my day involved one or two various procedures, so every day was different. Usually, I would have one or two main goals or techniques for the day. Some days it was immunohistochemistry or analyzing material under a confocal microscope. Other days I performed surgeries on rats or loaded brain tissue onto microscope slides. I was surprised by how similar this lab was to high school or college labs.
In some instances, we had to develop our own techniques. For example, we performed surgery on rats using the procedures and equipment we developed. Other procedures followed strict protocol that had been around for many years, like immunohistochemistry. The lab mentors had a strong understanding of their expectations for the outcome of an experiment, so we were able to successfully perform the experiments and get conclusive results that either supported or refuted the hypothesis. The results were what we had expected. We had a solid background understanding that allowed us to make a very scientifically guided hypothesis. However, that didn’t mean we didn’t have to start over in some cases. There were surgeries that did not go as planned, which resulted in having to start over and try again. Not everything in a lab runs perfectly or goes according to plan. Accidents happen, mistakes are made, and fresh starts were common. Luckily for me, no changes in the overall plan had to be made. Preliminary data suggested that we were going to receive conclusive results.
Life as a Scientist
Over the summer, I was able to dive into the life of a scientist and see what the day-to-day job was like. I was highly surprised by how much time was spent doing activities other than hands-on, standard research much like one would expect from high school or college labs. I couldn’t believe how much time each scientist spent doing work on a computer. In fact, my summer lab seemed to spend about half of the time performing procedures and the other half is spent on the computer doing activities like writing grant proposals, writing articles to be published in a journal, reviewing journal articles, ordering supplies and reading recent research. I just never realized how much time would be spent on these things, but the best part, was performing surgeries on rats. I was able to independently perform hands-on science in a way that, quite frankly, made me feel pretty cool. Who wouldn’t think it sounds impressive saying they’ve given a rat surgery before? I liked being involved in physical work instead of taking care of business on the computer. The surgeries were challenging enough that they required critical thinking, simple enough that I could feel confident in what I was doing and unique enough that every rat’s surgery was a little different. Plus, it was neat to see the success of the surgeries I performed. On the flip side, the worst part of the job was immunohistochemistry. The process was very tedious and with the large number of samples I had to do, it became quite exhaustive and—dare I say—boring. I would spend days at a time washing samples, mixing them in different solutions, transferring them between containers and very carefully plating them on microscope slides.
The fact is that research does not always involve exciting and intriguing work. The other interesting aspect of working as a scientist was working as part of a lab team. Each person had independent projects that they were working on, but every project related back to the overall theme of the lab and contributed to the overall goal of the study. It was neat to be able to hear from other people about their findings and being able to learn as a collective. It was nice to be able to ask anyone in the lab for their input on a matter because person had an understanding of the science behind almost every project, even if it wasn’t their own. It allowed me to feel independent and like I was contributing my own work while having a support system in place in times of uncertainty or confusion. Ultimately, my summer research fellowship was a wonderful experience that allowed me to engage in hands-on research and experience the daily life of a scientist.
Buckman,L.B. et al. Evidence for a novel functional role of astrocytes in the acute homeostatic response to high-fat diet intake in mice. Mol. Metab4, 58-63 (2015).
Camilleri,M. Peripheral mechanisms in appetite regulation. Gastroenterology148, 1219-1233 (2015).
Clyburn,C., Travagli,R.A., & Browning,K.N. Acute High Fat diet Upregulates Glutamatergic Signaling in the Dorsal Motor Nucleus of the Vagus. J. Amer. Physiol. Gastro. Liver Physiol. 314, 623-624 (2018).
Daly,D.M., Park,S.J., Valinsky,W.C., & Beyak,M.J. Impaired intestinal afferent nerve satiety signalling and vagal afferent excitability in diet induced obesity in the mouse. J. Physiol589, 2857-2870 (2011).
de Lartigue,G., de La Serre,C.B., & Raybould,H.E. Vagal afferent neurons in high fat diet-induced obesity; intestinal microflora, gut inflammation and cholecystokinin. Physiol Behav.105, 100-105 (2011).
Kentish,S. et al. Diet-induced adaptation of vagal afferent function. J Physiol590, 209-221 (2012).
Janssen,P. et al. Review article: the role of gastric motility in the control of food intake. Aliment. Pharmacol. Ther.33, 880-894 (2011).
Troy,A.E. & Browning,K.N. High fat diet decreases glucose-dependent modulation of 5-HT responses in gastrointestinal vagal afferent neurons. J Physiol594, 99-114 (2016).
Caleb Smith is a senior majoring in applied health science with
a pre-professional concentration at Messiah College in Mechanicsburg, Pennsylvania.
He is a 2019 Undergraduate Summer Research Fellow (UGSRF) in the lab of Dr.
Kirsteen Browning at the Penn State Hershey Medical Center’s College of
Medicine in Hershey, Pennsylvania. Caleb’s fellowship is funded by the American
Physiological Society. Upon graduating, Caleb hopes to continue into the
medical field by becoming a physician assistant.
For the past summer, I have been working in laboratory of Dr. Sheng UW at the Johns Hopkins School of Medicine. In the Wu lab, we are investigating the etiology of polycystic ovarian syndrome or disease (PCOS/PCOD). One of the major clinical presentations of PCOS is high levels of androgens, a condition known as hyperandrogenism, and the Wu lab focuses on the androgen receptor (AR) which binds to androgen outside of cells and acts inside the cell to express certain genes. The mouse model that we use mimics hyperandrogenism by exposure to dihydrotestosterone (DHT), which is present in low amounts in women without PCOS. To investigate mechanisms of hyperandrogenism and the AR, knockout mice without the receptor are compared to control mice, enabling us to investigate the effects of differential levels of DHT and the role of the AR on fertility, gene expression, protein and RNA levels, adipose tissue, and ovarian morphology (Wu et al., 2014). Despite the prevalence of PCOS in women of reproductive age, and its association with metabolic dysfunction, infertility and hirsutism, the exact cause is not known and effective treatment options are not available. By elucidating the pathophysiology of PCOS, treatment can be designed to target the cause as opposed to only clinically managing the symptoms temporarily.
Realities of Research
Although the prospect of doing research in a lab might sound like it involves the use of expensive equipment and the newest technology (and in many cases this is very true), a large portion of research involves spending hours maintaining and genotyping new litters, waiting for assays and reactions to finish, and pipetting hundreds of samples. Experiments sometimes work, and at other times they fail and must be repeated. Small errors in pipetting or mindlessly forgetting to include a certain solution can cause time-consuming experiments to fail. But, the most critical learning experiences I have had thus far were assessing what went wrong and fixing it by. Results are also not immediate; it takes several weeks before the effects of DHT can be assessed. Confirming the genotypes of our mice is critical to make sure that we are investigating what we claim to be, and ensuring DHT surgeries are done at the correct time requires attention and organization. Preliminarily, the effects of DHT we have observed have been physiologically plausible, although much of the data collection of the study is still ongoing.
Life of a Scientist
Simultaneously, the best and worst parts about doing research in a lab are that the hours are flexible and I can plan my week. But, this also means spending evenings working due to long wait times for experiments that extend past the usual 9 to 5 working day, or because a protocol calls for a certain experiment to be done at a certain time. The independence is exciting, but can also be intimidating as I must be aware of what needs to be done and when. However, research is collaborative. As the youngest and most inexperienced person in my lab, I am always learning how to do new experiments and how to interpret and analyze data. Others are always offering me tips and tricks, supporting me when I fail, and helping me deal with my constant fear of getting bitten by mice. The most rewarding moments of the summer so far have been presenting data to my PI and co-PI, and teaching members of the lab how to complete a computerized tissue analysis that had not been done in our lab before. The most disappointing? Waking up early to complete a western blot protein analysis only for it to fail – not once, but multiple times!
Wu, S., Chen, Y., Fajobi, T., DiVall, S., Chang, C., Yeh, S. and Wolfe, A. (2014). Conditional Knockout of the Androgen Receptor in Gonadotropes Reveals Crucial Roles for Androgen in Gonadotropin Synthesis and Surge in Female Mice. Molecular Endocrinology, 28(10), pp.1670-1681.
Gopika Punchhi is a rising senior at Johns Hopkins University in Baltimore, MD, pursuing degrees in Molecular and Cellular Biology and Public Health Studies. Through the Undergraduate Summer Research Fellowship (UGSRF) program, she is spending the summer working under Sheng Wu, PhD, an associate professor in the department of Physiology at the Johns Hopkins School of Medicine. UGSRF is funded by the APS. Gopika plans on attending medical school to become and gynecologist or endocrinologist, while also continuing involvement in molecular and population-level research in these fields.
Throughout our lives we accumulate damage naturally in our day-to-day activities. Thankfully, our bodies have an incredible potential for repair. Damaged tissues might follow a sunny day at the beach without sunscreen or the micro-tears in muscles resulting from a hard workout, are repaired by our bodies via the orchestration of numerous cellular processes. In response to tissue damage, various proteins will signal to specific cells in the damaged tissue that have the potential to differentiate and repopulate damaged tissue, inducing a regenerative response. These specific cells are known as stem cells .
My research this summer focused on a protein complex that has reported roles during aging and in mediating repair in particular tissues: mTORC1. In muscle, mTORC1 has been shown to be crucial to the activation of muscle stem cells, which are normally dormant, so that they may repopulate the damaged muscle by replacing injured cells with new, healthy cells. Although the role of mTORC1 in tissue regeneration is well understood in muscle, its role during repair is unclear within many other organs, particularly the intestine. The inner lining of the intestine, known as the intestinal epithelium, experiences tremendous damage on a daily basis, in part, as a result of the mechanical stress to the intestinal cells from the passage of food. As such, the intestinal epithelium is one of the most proliferative tissues in the human body, having the capacity to turnover every 5-7 days. My research this summer aimed to understand the role mTORC1 in repairing the intestinal epithelium after injury, and also to understand how this role may differ from its activity in an uninjured context, during homeostasis. We hope that the results from this work will help us better understand the intestinal regenerative process in order to someday have the capability to pharmacologically enhance regeneration in injured individuals, or prevent it, in conditions such as cancer where tissue grows excessively quickly.
Realities of Research
One of the aims of my research project was to focus on the role of mTORC1 during homeostasis. I hypothesized that if the activity of mTORC1 was inhibited, stem cell activity in the intestinal epithelium would be impaired. After genetically manipulating mice so that mTORC1 activity was only depleted in the intestine, we saw that mice lost a dramatic amount of weight and became sickly. We also expected a change on the cellular level. For instance, we anticipated changes in the number of cells actively dividing in the intestinal epithelium as well as changes in the architecture of intestinal epithelial cells. In order to better visualize the number of cells actively proliferating within the intestinal epithelium, I used a cell staining technique called Edu staining which allowed me to visualize each cell undergoing division. Once imaged, I could then quantify the exact number of cells undergoing division within a set amount of time and compare the number of proliferating cells in mice in which mTORC1 was depleted, compared to control mice with normal mTORC1 activity . The Edu staining showed a decrease in the number of cells proliferating in mice depleted of mTORC1 activity, further supporting my hypothesis. In order to further characterize the changes that occur after obstruction of mTORC1 activity, I plan to determine whether intestinal stem cell number is affected following genetic mTORC1 depletion. I also plan to analyze the general structure of the epithelial tissue using a staining technique called H&E staining and quantify the number of cells undergoing programed cell death using cleaved caspase 3 staining.
Life of a Scientist
This past summer, I’ve learned a lot about what it means to do research. Primarily, I’ve discovered the necessity of having keen attention to detail. Prior to working in Dr. Samuelson’s lab, I hadn’t realized how many steps need to go correctly in order to obtain data from an experiment. For instance, to complete a Western Blot, which is an experimental technique used to separate and identify a specific protein from a sample containing hundreds or even thousands of proteins, each step in the process must be executed with acute accuracy and precision. First, the mice had to be treated with the proper experimental treatment. Following the harvesting of the tissue, the protein within had to be extracted and quantified extremely accurately. Finally, the Western Blot could be run, which also has potential for error. If even a single step within the entire time intensive process was completed inaccurately or improperly, the Western Blot likely wouldn’t work and the entire process would need to be redone.
As I near the end of my summer in Dr. Samuelson’s lab, one of my biggest takeaways from working in the lab is that a scientist is never done learning. Sitting in on lab meetings with a team of experienced scientists and PhD students has shown me that even experts in certain fields don’t have the answers to every question. During my ten weeks in the lab, each week was entirely different. While there were several core skills that I learned and expanded upon through practice throughout the whole summer, each week also came with new scientific techniques and new ways of thinking and approaching problems. This constant exposure to new information and lifelong learning is what excites me as a scientist.
Yasmine Abushukur is a senior at the University of Michigan studying both French and Bimolecular Science. This summer, she worked in Dr. Linda Samuelson’s lab thanks to funding from the APS Undergraduate Summer Research Fellowship. This upcoming school year, she plans to continue working under Dr. Samuelson’s guidance, studying gastrointestinal physiology. After graduation, she plans pursue a career as a physician-researcher.
When we go from a lying down position to standing up, our blood tends to pool in our legs and has to fight against the force of gravity to travel our brain. Overcoming the force of gravity is the greatest challenge the cardiovascular system faces. Luckily for us, our body is able to detect when such changes are to occur and can effectively change blood pressure to compensate for the change in posture. The body has many different sensory inputs that contribute to homeostatic regulation of blood pressure during these postural changes. The vestibular system, baroreceptors, chemoreceptors, and higher order brain centers (hypothalamus and cerebral cortex) are all examples of the body’s many sensory inputs (Yates et al. 2014). If these homeostatic responses perform inadequately, then conditions such as orthostatic hypotension can be the result (Yates et al. 2017). Orthostatic hypotension results from a drop in blood pressure due to a change in posture, where insufficient perfusion of blood with the brain occurs. My research project this summer focused on the cardiovascular responses that are necessary to maintain normal biological homeostasis during anticipated imposed postural changes. Homeostasis is defined as the tendency of biological systems to maintain relatively constant conditions in the internal environment while continuously interacting with and adjusting to change originating within or outside of the system (The Free Dictionary, n.d.).
In this study, my lab investigated the activity of neurons in the RVLM (rostral ventrolateral medulla), located in the brainstem, in awake adult female cats. There has been significant research to support the claim that neurons in the RVLM play in important role in blood pressure regulation in both animals and humans (Yates et al. 2017). RVLM neurons are necessary for cardiovascular responses to maintain homeostasis during postural movements. The neurons in the RVLM were identified by their changes in firing rate correlated with the cardiac cycle and their location. The experiments in the lab involved recording the activity of neurons in the RVLM during imposed 40º head-up tilts that were preceded by a light cue. The light cue occurred 10 seconds before the 40º tilt, and the heart rate and firing rate of neurons were determined for 5 second time periods before and during the tilts.
Our hypothesis is that in neurons that exemplify cardiac related activity there is an increase in neuronal activity prior to the onset of the 40º imposed tilt, in the time interval after the light stimulus as compared to before the light. This would indicate that when the animal is able to anticipate when the change in posture is about to occur, the animal’s cardiovascular response system will increase heart rate and blood pressure to prepare for the postural change. This would be an example of a feedforward mechanism rather than a feedback. In a feedback mechanism, the animal would have already undergone the change in posture and body positon in space before the cardiovascular response system could compensate heart rate and blood pressure. This research project will ultimately further our knowledge of cardiovascular homeostasis and may lead to new treatment options for orthostatic hypotension and also a treatment for cognitive mechanisms that can cause changes in blood pressure such as stress and anxiety.
Figure 1: An example of the data that was collected and analyzed during one of the tilt table experiments. The x-axis shows the change in time as the table went from a nose down position, to a 40º head-up tilt. The neuronal firing was isolated along with the heart rate through the use of an EKG. The sun cartoon indicates when the light stimulus was shown with the five second time intervals appropriately labeled before and during the head-up tilt. Credit: Dr. Bill Yates’ Lab, UPMC Department of Otolaryngology.
Realities of Research
I was extremely grateful to take part in this research opportunity this summer as it really allowed me to grow as an undergraduate researcher. The reality of research is that you get out of it what you put in. If you’re motivated and passionate about what you are doing, then I believe that anyone will be satisfied with the outcome of their experience. I became enamored with learning about physiology after completing Dr. Yates Honors Human Physiology class. After completing his course, I was eager to investigate what research was like on the physiological spectrum. Over a course of 10 weeks, I completed dozens of experiments and analyzed large amounts of data. We performed single-unit in vivo recordings using electrophysiological techniques and procedures. When experiments ran to completion and a respectable amount of usable data was collected, the day was considered successful. Another reality of research is that not every experiment is going to go as planned. There were experiments where either the animal wasn’t cooperating on the table, an electrode bent, or we just simply couldn’t isolate any good neurons to record from. When problems like these would occur, we would simply end the experiment and try again the next day. Because there are a great deal of experiments left to perform and more data analysis to complete, there are no final results or conclusions yet to be made. However, we do predict that the firing rate of the cardiac-related neurons will increase after the light stimulus is given, prior to the onset of the tilt.
Figure 2: The tilt table that is used in all of the single-unit in vivo recordings. During an actual experiment, the curtains would be drawn and the lights would be dimmed to allow for the light stimulus to appropriately work. The machines in the back are used to find and isolate a neuron in the RVLM and to change the position of the table. Credit: Dr. Bill Yates’ Lab, UPMC Department of Otolaryngology.
Life of a Scientist
I sincerely enjoyed my daily routine as a scientist in the Yates lab. I was able to set my own schedule and I developed meaningful friendships with everyone I worked with. It was a very-low stress working environment and most days I would follow the same routine. A huge part of my project this year was analyzing the data collected from the experiments. When I wasn’t running any experiments I would be on the computer analyzing large amounts of data. Analyzing data is something that a lot of undergraduate students have trouble with, mostly because it takes a lot of time to learn and pick up. I was very surprised by how quickly I was able to pick up the skill and it allowed the lab to make huge amounts of progress throughout the summer. My fondest memory of my time in the lab didn’t actually happen in the lab. On a Friday afternoon after work, everyone in the lab went out to a Pirates game at PNC Park in Pittsburgh. It was my first one and it was really fun to see everyone outside of the lab doing something together. Overall, I had a great summer and I’m very thankful that APS gave me the opportunity to see what full-time research was actually like. This summer experience was very rewarding and has motivated me to pursue my continued interest in biomedical research.
Yates, B. J., P. S. Bolton, and V. G. Macefield. 2014. Vestibulo‐sympathetic responses. Compr. Physiol. 4:851–887.
John Bielanin is a rising senior majoring in Neuroscience and minoring in Chemistry, with a certificate in Conceptual Foundations of Medicine at the University of Pittsburgh in Pittsburgh, PA. He is a 2018 Undergraduate Summer Research Fellow working in Dr. Bill Yates’ lab in UPMC’s Department of Otolaryngology at the University of Pittsburgh. John’s Undergraduate Summer Research Fellowship (UGSRF) is funded by the American Physiological Society. Outside of school and work, John enjoys reading, writing music, and spending time outdoors. After graduation, John plans on applying to medical school while continuing to pursue research opportunities in his gap year.
Over the summer I have been working at Emory University with Dr. Tansey and other lab members looking for potential causes of Parkinson’s disease (PD). Parkinson’s disease is an illness that damages important parts of the brain and nervous system. This can cause a loss in efficient body movement. We believe that a specific protein, something that the body naturally makes, called LRRK2 may play a role in this disease because there is an increased amount found in PD patients compared to healthy controls. Therefore, we have studied this protein by trying to pin point its location in the body and learning how it causes other PD symptoms such as inflammation. This research will not only provide answers for PD, but can be used to learn more about other neurodegenerative diseases. The goal is to one day cure such debilitating illnesses for everyone who is unfortunate to develop them.
Realities of Research
One of the biggest things I have learned about working in a lab is that your plan of action can suddenly change, while the goal stays the same. However, there are many times where you must go back to the “drawing board” and erase or insert something new. To me, going back and finding out that you have to try something new is a good thing. You are improving on your research and hopefully it will bring you closer to significant results. I also had other learning opportunities such as improving on specific lab techniques. During my BCA assay (Bicinchoninic Acid assay), which is a protocol used to standardize amounts of proteins in a sample, I had to perfect my pipetting skills for the assay to work. I practiced for hours to get the exact amount of solution every time I withdrew from a tube before I actually ran the real assay. This summer, my project started off bumpy because of an experiment not going as planned. I was taking tissue sections of kidney to stain and investigate for the colocalization of LRRK2 with the primary regulator NFAT (Nuclear Factor of Activated T-cells) and pNFAT (NFAT promoter) status in the nucleus. We were looking at these specific cells because they are involved with the immune system which LRRK2 seems to help regulate. However, after my staining protocol, the kidney cells would essentially combust. After many days of trial and error, we decided that there was not enough knowledge of kidney staining in our lab, and instead of spending time and resources to figure it out we moved on and started looking at a new location. There were plenty of times where we had to re-evaluate our plan, and as I said before I only see them as learning opportunities. Sometimes research is not always going to be straight forward where you make a hypothesis, go through an experiment, and at the end collect an answer. It can take a lot of time and creative thinking to get where you need to go.
Damaged kidney cells.
Life of a Scientist
Being a part of a lab is great for many reasons. One reason is that I prefer the type of work style that it offers. Those aspects include being able to have flexibility within your schedule and not having to be constantly micromanaged. Yet, I am still offered enough guidance to steer me on the right track. I love that every day is another day pushing towards a goal that you most likely had a hand in setting. I am also surrounded by people who all want to see each other succeed. Although everyone in the lab may be working on their own personal agendas, people are constantly helping others with their projects whether it’s by lending their skills for a certain assay or giving an extra hand to make timely experiments go by faster. I would go on to say that the entire science community within your field begins to feel more familiar as your work progresses, as well. I value this idea of a connected community within my workspace. Overall, my experiences in my lab this summer were positive and resourceful. Of course, I have witnessed complications occurring in the lab such as assays not going as planned or having to re-due them because of minor mistakes. Human error constantly occurs and I have learned that you can only work with it and make sure to try as hard as possible to get accurate, significant results. Knowing that I could make a career into doing what I love, conducting science and answering important questions that benefit humanity, gives me the motivation of becoming a neurologist in the future.
Chayla Vazquez is a rising junior at Emory University in Atlanta, GA where she is majoring in Neuroscience and Behavioral Biology, with a minor in Ethics. She is working at Emory over the summer with Dr. Malu Tansey as an Undergraduate Summer Research Fellow (UGSRF) funded by the American Physiological Society. Chayla strives to become a biomedical scientist and utilize her skills in research to help people who struggle with cognitive defects.
Throughout the summer of 2017, I worked in the invertebrate physiology lab under Markus Frederich, investigating how the enzyme AMPK affects the salinity stress tolerance of European green crabs. Using crabs collected from the Maine intertidal areas, which often experience variances in salinity and temperature, I focused on injecting and activating this enzyme. By doing so in settings of different salinities or temperatures I was able to see how the enzyme plays a role in ion regulation and stress tolerance, giving insight into what makes this species such successful invaders.
Participating in this research experience has been extremely valuable. I have been able to acquire lab skills which are imperative to my future as a scientist, while also receiving a unique opportunity to develop a very strong understanding of a very specific topic that is very exciting. Coming into my summer research experience, I felt nervous and overwhelmed thinking that I had so much to learn in so little time, but very quickly I got into a comfortable position of diligent work, where I learn something new every day. Scientific research involves patience, and while everything might go right the first time, I have learned that it may not be because of a mistake, but that a different approach needs to be taken.
Being a scientist is a surreal experience because it is such a meticulous practice. The smallest things can cause large alterations in data, and knowing that makes me appreciate the practices of scientific research that much more. Every day involves thinking outside the box, and a true dedication of time. The best part is learning something new every day, and while it may be exhausting to sometimes go multiple days with nothing making sense or things not working, there comes a moment where something new is discovered, which is extremely rewarding. I am truly appreciative of the lab that I work in because everyone is willing to help each other, working together to answer questions, and learn from one another. My advisor specifically has been very influential in making this summer experience one of the best things I have ever done, and has been a strong influence for my love for research.
Pierce Lancor attends the University of New England in Biddeford, ME. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) and worked in Dr. Markus Frederich’s lab over the summer at the University of New England. After graduating, she plans to continue her education and research toward a masters and PhD in zoology.
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.
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.
About 30% of the world’s population has hypertension due to a recent surge of salt in our diets (1). According to the American Heart Association, the average American eats more than 3,400 milligrams of sodium daily, while the ideal limit is 1,500 mg per day for most adults (2). So, how does your body know when you have ingested salt? Your brain has specialized cells that monitor the amount of sodium (and other ions or substances). Some of these cells are located in the Organum Ventral Lamina Terminus (OVLT). OVLT is one of a few special section of the brain that lacks a complete blood brain barrier that allows substances in the blood to pass more freely from the bloodstream to the brain. So, this section of the brain essentially has the equivalent of “x-ray vision”; it can “see” the particles in the blood, such as salt, and send messages throughout the body to initiate responses. One of these responses may be to increase blood pressure, which is known as salt-sensitive hypertension. Salt-sensitive hypertension refers to an increase in arterial blood pressure to an increase of salt intake.
Figure 1. Rat diagram with optogenetic set-up.
My project utilized a new innovative tool known as optogenetics to inhibit or “turn off” OVLT neurons during dehydration and acute NaCl loads. Optogenetics works by shining a light at different wavelengths at a region of the brain where a light activated or inhibited ion channel was injected via a virally-mediated delivery system. In my experiments, water intake, urine output, and blood pressure (via telemetry) were measured in rats dehydrated overnight or infused with a concentrated NaCl solution with or without the laser on to inhibit OVLT neurons. I found that when these neurons were inhibited, they would decrease the volume of water intake of the rats. Upon further investigation, if OVLT continues to prove to be a major contributor to salt-sensitive hypertension, we hope more methods will be developed and implemented in humans suffering from this disease.
Figure 2. Leica Vibratome used to take serial sections of brain to determine injection location. Credit: Dr. Sean D. Stocker’s Lab, University of Pittsburgh Medical Center
Research is a very rewarding occupation. Although experiments can be long and time sensitive, the outcome is worthwhile. As long as you are careful and perform experiments honestly, any result can be used to learn. Further, I was amazed by the techniques I used to complete my experiments. The science and technology behind them is very interesting and innovative. The use of optogenitics is relatively new and entails complex surgeries and sciences. Unfortunately, with complexity comes consequences, and I have hit some bumps in the road. The technology used on the rats was very fragile and could not be fixed if broken. In addition, since it is not possible to alter salt concentrations without altering other aspects of the rat, we had to test the secondary stimuli to see what affect they had on the rats’ behavior. I am not finished collected data yet for this summer, but the results appear promising. In addition, I still need to test more animals and determine if the injected virus hit OVLT in order to define my conclusions.
Upon arriving, I was most surprised by the dynamic of the lab. All lab members are working on separate projects, but with related goals. Work is mostly independent, yet the goal is interdependent. Working with a lab team was a fulfilling experience and helped me gain a better understanding of my project. I was able to learn new things and make better sense of my project through communicating with my team members. The best part of my research is the technology and innovation associated with it. One of my favorites is the way we analyzed water intake by applying basic principles of circuits. During experiments, the rats are in cages with metal floor, plastic walls, and water bottles with metal spouts. A wire connected the floor to the spout of the water bottle, creating an incomplete circuit. When the rat takes a drink, it completes the circuit because it is touching both the metal spout and floor. This causes a voltage to occur, which is recorded on a computer application. This data is used to determine when the rats were drinking, and it can approximate water intake because duration of time at the water bottle spout correlates with the amount of water the rats ingested. In contrary, the worst part of my research was the delicacy of the animals. It is very disheartening when the head caps fall off the rats and no longer can be used for experiments. Another incommodious aspect was the time sensitivity of experiments. I have to give the rats time between experiments, so some days were much slower than others. All in all, the positive aspects of research have outweighed the negative, thus far. It has further peaked my interest in a future of biomedical research, and I would recommend anyone interested in the sciences with a love of problem solving to engage in research!
Choi HY, Park HC, Ha SK. Salt Sensitivity and Hypertension: A Paradigm Shift from Kidney Malfunction to Vascular Endothelial Dysfunction. Electrolytes & Blood Pressure 13:7-16, 2015 [14 July 2017].
How Much Sodium Should I Eat Per Day? [Online]. American Heart Association. https://sodiumbreakup.heart.org/how_much_sodium_should_i_eat?utm_source=SRI&utm_medium=HeartOrg&utm_term=Website&utm_content=SodiumAndSalt&utm_campaign=SodiumBreakup [14 July 2017].
Rachel Maile is a junior majoring in Biological Systems Engineering and minoring in Biomedical Engineering at Virginia Polytechnic Institute and State University (Virginia Tech) in Blacksburg, VA. She is a 2017 Undergraduate Summer Research Fellow (UGSRF) working in Dr. Sean D. Stocker’s lab at the University of Pittsburgh Medical Center in Pittsburgh, PA. Rachel’s fellowship is funded by the APS and has enabled her to further her appreciation for research. After graduate, Rachel plans to perform biomedical research, using her engineering background and physiological expertise.