(By Dan Moorhead and Alex Lupolt – Brandauer lab)
Circadian Rhythm, or someone’s “sleep/wake cycle”, is a complex cycle that coordinates our resting and active states throughout a particular day with environmental cues such as light or lack thereof. Metabolism, muscle recovery, body temperature, and many other biological functions show characteristic changes throughout the course of the day. The word “circadian” combines two Latin words, circa, “around/about” and diem, “day.” So how long does one sleep/wake cycle take? About a day.
Throughout evolutionary history, organisms have been continuously exposed to a daily cycling of light and darkness, day and night. It is of no surprise then that many biological processes exhibit daily oscillations that are synchronized with a roughly 24-hour light and dark cycle. In fact, most organisms possess a common molecular machine that governs cellular and tissue circadian rhythmicity through a transcription-translation feedback loop—a likely evolutionary consequence of countless sunrises and sunsets. Humans are photosensitive organisms that are governed by endogenous cellular fluctuations which are regulated by both a central nervous system clock and a molecular clock believed to be present in all cells of the body. Since our light and dark cycles play an important role in regulating proper biological function, disruptions in circadian rhythms (jet-lag, night-shift work, and the movement of daily life indoors) can have deleterious consequences to human health (seasonal affective disorder, psychological disorders, metabolic disorders, cancer, etc.).
In our lab, we work to the rhythms of alternative rock, country music, and no matter what the task, it seems that it takes about a day to accomplish anything. Dissections and snap freezing, aliquotting and pulverizing, Bradford protein assays, homogenizing, and Western Blots. Take any two of these, and if done correctly, the answer to the question “How long will this take?”—Circa Diem. We work rhythmically and timely, as a unit, everything is precise, just like one’s circadian rhythm. But it is ironic, because as we as ponder the effects of bright light on physiology we sit in a somewhat dimly lit, windowless lab, experimenting on the circadian clocks of mice, all while probably messing up our own. In fact, in an attempt to investigate tissue-specific oscillations of molecular clock machinery in order to determine which tissues are more sensitive to circadian disruptions, we will be disrupting our own circadian cycles with a 24 hour experiment, collecting data at 4-hour intervals. This scientific all-nighter is sure to test the power of our internal clocks (and how many Red Bulls we can drink).
We are the Gettysburg College Lipid Lab! We conduct research as part of the X-SIG program, sponsored by HHMI, under the guidance of Dr. Shelli Frey. Though we all have our own individual projects, our work has a universal theme: Lipids. We have battled through DI water droughts, peptide panics, and even the occasional flood of biblical proportions to bring you this blog, in which we hope to share a little about us, our projects, and the scenic lifestyles of a few summer researchers on the Gettysburg College campus. Though it is still somewhat early in the summer, our group already has valuable insights to share, such as 100 ways NOT to make GUVs, why salts + troughs = endless cleaning, and that biological samples do not make friends–they make enemies. Anyway, we hope you find this blog post interesting!
The Research Team
Warren (Alex) Campbell My name is Warren (Alex) Campbell, and I am a rising senior here at Gettysburg College and I intend to graduate in 2015 with a BS in Biochemistry and Molecular Biology with a minor in Neuroscience and a pre-health concentration. After graduation, I plan on pursuing an M.D. Ph.D. in Neurobiology, Stem Cell Research & Regenerative Medicine, or Cancer Biology. I chose to work on the Huntington’s project under Dr. Frey of the Gettysburg College Chemistry Department because the research incorporated both cellular biology and biochemistry with a medically relevant goal in focus. In the upcoming months, I hope to conclude my research and write up my data for publication in a scientific journal.
David Van Doren My name is David Van Doren and I am a Biochemistry and Molecular Biology major at Gettysburg College. I am a rising Junior in the class of 2016 and am interested in graduate school after my four years here in Gettysburg. I began working with Dr. Frey in the fall semester of my sophomore year (in a Chem290 course), which was one of my first introductions to academic research. This summer I am conducting research on the Nanoparticle Project, which I hope will give me some experience to make decisions regarding my future career interests.
Mike Counihan My name is Michael Counihan, and I am a rising junior Chemistry and Music double major at Gettysburg College. I am interested in graduate school and doing research in the field of analytical chemistry. This summer is my first research experience, and I am splitting my time between the Frey and Thompson labs investigating lipid monolayers and nanoparticle surface-enhanced Raman spectroscopy, respectively.
Projects Huntington’s Disease (Warren Campbell)
Project Overview Huntington’s disease is a progressive debilitating neurodegenerative disorder that affects muscular function (uncontrollable jerking, tremors, rigidity) and cognition (depression, difficulty planning, inability to focus, and reduced awareness). The disease is genetically linked to a single gene, Huntingtin, located on chromosome 4. The gene is dominant, meaning that it there is a 50% chance of inheriting the disease if one of your parents has been diagnosed. This gene codes for a mutant protein, huntingtin, which is understood to be the toxic agent causing neuronal cell death. Approximately 1 in 10,000 people are affected with Huntington’s disease in the US, with no available cure. Current therapeutics, such as antipsychotics, neuroleptics, and dopamine modulators, target the symptoms rather than the disease. New innovative drug therapies in the preclinical stages of development are based on an understanding of the mechanism of the disease. Studying the pathology of Huntington’s via fundamental research has led to a better understanding of the disease on a biochemical and biophysical level. The purpose of my research is to contribute to the analysis and characterization of Huntington’s disease by studying how the mutant huntingtin protein interacts with lipids and cell membranes.
Techniques It is well established (and supported by published work from our lab!) that mutant huntingtin associates with cell membranes. My project is interested in what lipids or membrane components change the affinity of the protein to the membrane and its ability to insert. I focus on two laboratory techniques to study this phenomenon: Langmuir monolayers, and vesicle popping assays.
A Langmuir trough is used to study lipid surfaces that are only one molecule thick, known as a monolayer. Because lipids contain fatty acids, they naturally repel water and spread evenly on a water surface. On both sides of the monolayer is a movable barrier, which can compress the top layer of lipids. During compression, we can measure how tightly the molecules pack, which is unique for each lipid. I study these properties because if a particular lipid changes the affinity of huntingtin to a membrane, it is important to know what changes in physical properties are attributed to the result; then, this can be extrapolated to a physiological cell membrane. The monolayer can also be used to measure how much peptide inserts when injected into the water below the lipid surface.
I am particularly interested in how the mutant protein interacts with a cell membrane, which is composed of a bilayer of lipids. To do this, small nanometer sized lipid spheres, known as small unilamellar vesicles, are synthesized as a simple model for the cell membrane. Inside these lipid vesicles is a dye that can is released with then the peptide inserts. Thus by measuring how much dye escapes when the peptide is added, we can determine how much peptide inserted into the membrane.
Nanoparticle Project (David)
Project Overview Nanoparticles have a large, and growing, array of applications in industrial and medical settings. Their increasing use and vast potential make the characterization of nanoparticle interactions an important task for not only developing technologies, but also for understanding their potential toxicity to biological systems. One way that nanoparticles interact with cells is by either adhering or inserting into the lipid bilayer of the plasma membrane. This interaction can have various disruptive effects, such as changing the fluidity of the membrane, affecting one or more phases of the lipid domain system, or even causing pore formation in the membrane. The Nanoparticle Project aims to examine these nanoparticle to lipid relationships by measuring various nanoparticle interactions on model membrane systems such as lipid monolayers or giant unilamellar vesicles (GUVs), with the intent of relating these simplistic, representative systems to cells that would be affected by similar encounters with nanoparticles.
In order to solely examine the nanoparticle to lipid interaction within a cell, model membrane systems must be created that contain lipids without membrane proteins and carbohydrates, which are commonly found in cell membranes. While monolayers provide useful quantitative data about a two dimensional, single layer of lipids interacting with nanoparticles, my portion of the Nanoparticle Project involves observing three dimensional GUVs in the presence of functionalized polystyrene nanoparticles. I will introduce the polystyrene to these large, spherical bilayers either during the preparation process, or after the GUVs have already been formed. To measure the interaction, I will monitor the membrane in real time using a fluorescence microscope. In this way, I hope to tease out information about how positively and negatively charged polystyrene interacts with this system.
Salts, Nanoparticles and Lipids (Mike)
Project Overview Lipid monlayers are useful for modeling the outer leaflet of a cell membrane bilayer. In Lipid Lab, I am currently investigating the effects of different cations (namely Na+, Mg2+, and Ca2+) on the phase transitions of lipid monolayers. Using chloride salt solutions of these metals in different concentrations as the subphase, I run monolayer isotherms with the Langmuir trough. The presence of the ions changes how the lipids pack as they are pushed together by the trough, making these isotherms quite different from those run on a pure water subphase. Why are we interested in this? It’s important to understand the effect of salts on membranes and membrane/protein interactions as many labs using different buffers (with different salts at different concentrations) for their work – so we are doing this to tease out these fundamental interactions.
Techniques My project mainly utilizes the Langmuir trough as described above. The current lipid under investigation is DPPC (dipalmitoylphosphatidycholine). To illustrate our results, we overlay the isotherms from the different salt concentrations over that of DPPC on pure water and compare the liftoff points (when the packing lipid molecules start to cause a change in surface pressure), plateaus (equilibrium between the liquid-expanded and liquid-condensed phases), and collapse points (when the lipids cannot pack any tighter and “collapse” into a third dimension, the subphase).Changes to the isotherm properties of DPPC when NaCl is added to the water subphase.
A week in the lab
Each week follows a general format, to provide some structure to the chaos of science. Each day we do our duties in the lab: cleaning, preparation, and running experiments. Then more often than not something goes wrong, and you eat, sleep, and repeat. If only science was search instead of REsearch, the whole process would be much more efficient.
To add some pizzaz to the week, we have brown-bag lunches. Here, all the X-SIG research labs gather together and discuss their projects to other labs who are unfamiliar with your work. This helps you get a sense of the great work that other students and faculty are working on every day, not to mention help you learn some new science. After all, the more you know, the less you don’t know.
On Wednesday–the almighty hump day–we are privileged to have bountiful access to phenomenal pastries courtesy of the almighty supreme chef Frey. I can’t stress enough how painfully good these pastries are. I know what you’re thinking, “Oh whatever, I have had good pastries before.” No, you’re wrong. These are better. Tears will run down your face as the first bite touches your unworthy mouth. The ancient Mayans predicted the coming of these delicacies from star alignment. These delicately crafted goods are so perfect, they cure diabetes as you eat them. These pastries brought my dog back to life–don’t use the chocolate ones. Also, did you know these pastries are the secret to world peace? I guess what I am trying to say is, Professor Frey makes some decent snacks for a Wednesday.
At the end of every week, our lab group gathers together and discusses their work for the week, including what experiments were run, what was found, and any problems that occurred. It is great to be updated on the work your fellow Lipid Lab crew, and can be helpful for brainstorming ways to troubleshoot their issues. During the week we are responsible for reading a relevant scientific article to our projects or lipids in general, where we analyze and discuss the paper. Finally, when comes the weekend, we can enjoy the recreational pleasures of Gettysburg.
Summer life on Campus
Gettysburg summers are surprisingly full of opportunities and activities outside of the lab. Recently, there was Gettysburg Fest, a week long festival that found its way into campus by the weekend. Three stages in the center of campus provided ample music from local rock bands, brass bands, and a Beatles cover band. The weather is wonderful – bright, warm summer days that make the campus picturesque. Even when it gets too hot, you can cool off with a sweet treat from Mr. G’s Ice Cream shop (less than a mile from the Science Center!). Of course, Gettysburg wouldn’t be Gettysburg without its annual flood of tourists in the first days of July to relive the historic battle over 150 years ago. And when you want to relax, there’s nothing better than lounging in an Adirondack chair and reading a book in the warm sunshine. Well that’s all we have for you guys, have a nice day!
I am Abby Bull and I am a rising junior in the physics department. This past semester, I took Biophysics, a cross-disciplinary course in the Chemistry and Physics departments, and became very interested in the research that is currently taking place in the field of biophysics. Because of this, I chose to do research with Professor Andresen this summer on DNA in solution with different concentrations of ions.
DNA has many odd and interesting quirks about it. One of which is that it aggregates (clumps together) when +3 ions are present in solution. This is especially interesting because DNA aggregates in this case with very small concentrations of +3 ions but this phenomenon will not occur with any amount of +1 or +2 ions alone. We have DNA samples, made by Professor Andresen’s collaborators at George Washington University, with different concentrations of +1 (sodium) ions in it along with cobalthexammine as the +3 ion in solution. I am conducting my experiments using the Induced-Coupled Plasma Optical Emission Spectrometer (ICP for short). Using the different wavelengths of light of various elements as a base, the ICP outputs the concentrations of different elements in solution. We hope to discover more about the physics behind DNA aggregation caused by the +3 ions and determine the relative concentrations of ions around DNA when it is aggregated.
In addition to my project with DNA, Steve Kenyon is working in the lab on the engineering side of experiments. He is constructing magnetic tweezers from the ground up. Magnetic tweezers are a biophysical technique that measures the forces keeping biological macromolecules together by pulling them apart using magnets.
If you want to know more about either of our projects for the summer, our lab has a group blog of our own at: