sc1ent1fic Undergraduate Magazine
Volume III, Issue I
Fall 2010, Volume III Issue I
Mission Statement: Founded in Spring 2008, Carolina Scientific serves to educate undergraduates by focusing on the exciting innovations in science and current research that are taking place at UNC-CH. Carolina Scientific strives to provide a way for students to discover and express their knowledge of new scientific advances, to encourage students to explore and report on the latest scientific research at UNC-CH, and hopes to educate and inform readers while promoting interest in science and research.
From the Editors To our Readers: This year, we are thrilled to be a part of the Carolina Scientific’s new leadership, passed onto us from the former editors and founders of the magazine. We have watched it grow from an idea into a full-fledged publication, and we hope you find that we’ve carried on Carolina Scientific’s mission statement to the fullest -- to provide an interesting and informative collection of campuswide research. We have enjoyed watching our staff tackle challenging scientific material, and hope that their passion for research and science shines through in their pieces. Enjoy! Rebecca S., Garrick, Rebecca H., and Rohan (From Left to Right) Rebecca Searles, Editor-in-Cheif and Biology Editor, is a senior Biology and Psychology double major. Garrick Talmage, Chemistry Editor, is a junior Biochemistry major and Math minor. Rebecca Holmes, Physics Editor is a senior Physics major. Rohan Shah, Biology major, is a senior Biology major and Chemistry minor.
Special Thanks to our 2010 Carolina Scientific Production Staff! (From Left to Right) Janelle Vecine Kristen Rosano Hema Chagarlamudi Frank Mu Not Pictured: Kati Moore
Fall 2010, Volume III Issue I
Table of Contents 4
Going Viral: How MicroRNAs Help the Epstein-Barr Virus Survive Anuja Mathur
Arctic Bacterial Gluttony Accelerates Global Warming Ashley Mui
Invasion of the Fish Snatchers: The Lionfish Takeover Maggie Hunter
The Study of a Disease That Would Have Ruined Air Bud’s Career Madison Roche
The Quest for the Cure Kara Stout
Growing Up Straight Hema Chagarlamudi
Segmentation and Alignment of Knee MRI Images Keith Funkhouser
Searching for New Physics with the Large Hadron Collider Lenny Evans
Practice Makes Perfect: The Benefits of Tests Kati Moore
It’s All in the Package: A New Way to See DNA Expression Kristen Rosano
EvoDevo: A New Field Examines Evolution’s Role in Development Kyle Roche
UNC Scientists Launch Breast Cancer and the Environment Research Program Yinmeng Yang
Radio Mitigation: Treatment After the Fact Mary La
Probing In-Vivo: In-Cell NMR Ricky Singh and Alex Krois
Kinetochores: Getting to the “Chore” of Chromosome Segregation at Cell Division Rana Alkhaldi
Hybridization: A Toad’s Love Story Shannon Steel and Sonia Bhandari
Nitric Oxide: A Glucose Monitor’s Best Disguise Hetali Lodaya
How Influential is Your Waistline? A New Study of Obesity and Influenza Immune Response Abby Bouchon
Flying High with Drosophila Frank Mu
Fall 2010, Volume III Issue I
Going VIRAL How MicroRNAs Help the Epstein-Barr Virus Survive
Anuja Mathur, Staff Writer
hen you are sick as a child the only things you notice are the symptoms, such as a fever or cough, and that nothing is worth taking that disgusting “cherry” syrup medicine. As you grow older and take some science classes you begin to learn about your body’s immune system and that there are various cells whose sole purpose is to find foreign invaders, attack them, and properly dispose of them. At some point you probably wondered, how diseases such as cancer exist if cells like these exist. Ideally, the body would be able to recognize the tumorigenic cell and kill it, but that is when it gets complicated. The Epstein-Barr Virus (EBV), part of the herpes virus family, is known for causing infected mononucleosis and some cancers such as Burkitt’s lymphoma, Hodgkin’s lymphoma, nasopharyngeal carcinoma and gastric carcinoma. EBV latently infects about 90% of the world’s population, meaning it is essentially “dormant” in the host cell but can reactivate at any time. However, a small percentage of these infected develop the malignancies previously listed. In order to cause these malignancies, EBV must find a way to protect itself from the immune system. Once a cell is infected the immune system may induce apoptosis, programmed cell death. Apoptosis causes the cell to essentially commit suicide, killing the virus and preventing it from proliferating or just remaining in the cell. The virus has to develop a method to prevent the host cell from undergoing apoptosis, and one of the newest fields of research on how it manages to do this focuses on microRNAs (miRNAs). These small, ~22 nucleotide long non-coding RNAs were found in human virus for the first time in EBV . EBV codes for at least twenty miRNAs and these play a dual role in virus survival. First, miRNAs are not detected by the immune system in the same way as viral proteins, allowing the virus to
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live undetected. Secondly, they are known to repress messenger RNA (mRNA) expression, which code for proteins, making miRNAs important gene regulatory molecules. Thus, EBV can control the host cell environment by escaping immune system detection and by controlling the cell itself via protein regulation. It has previously been discovered that one of
Figure 1. Incidence of EBV-related cancers worldwide.
the miRNAs in EBV targets a pro-apoptotic protein, PUMA, and down-regulates it to ensure virus and host cell survival . Many miRNAs in EBV have been discovered, but their function is still largely unknown. I joined postdoctoral fellow Aron Marquitz in the Raab-Traub lab at UNC to determine what other proteins are being regulated, and by which specific miRNAs in EBV. Although there are a lot of different proteins in cells, and one of or multiple miRNAs could target many of them, a list of potential targets can be narrowed down. One way to determine if a protein is a potential target is if there is base-pair sequence complementarity between the protein and a miRNA, which can be found using bioinformatics. In this case, the shortened list included a number of proteins known to be involved with apoptosis. If one of them is regulated by a microRNA then it could shed light on another specific pathway for virus survival. The next step was to test whether the predicted targets were real and led to protein regulation by a miRNA. This can be done through a variety of methods, such as western blotting and reporter assays. The protein of greatest interest was Bim, known to have
Figure 3. An example of complementarity between several miRNAs and the Bim sequence, allowing miRNA protein regulation.
a role in causing cell death, and the amount of Bim was seen to consistently decrease when the cell line was infected with a combination of miRNAs compared to the control, indicating that there was some effect of miRNAs. Data indicates that both Cluster I (including eight microRNAs) and Cluster II (including about 12 microRNAs) down-regulate Bim, and even more so when both were combined together. Further experiments were done to determine which microRNAs in the cluster were causing the downregulation but it seems that a combination is required and it is not just one miRNA that targets Bim . Bim is known to be part of the BH3 family of proteins, which are involved in the cellular signaling pathway that tells a cell to undergo apoptosis. Thus, a combination of miRNAs in EBV may be down-regulating Bim in order to prevent apoptosis and promote virus survival. More research has to be done to determine if there are particular miRNAs that have more
of an influence than others, and to continue looking at other potential target proteins. Investigating the targets of miRNAs is making clear how viruses such as EBV survive in spite of the body’s typical immune response. Understanding the exact cellular mechanisms can elucidate the pathways that are affected by viral infections. The hope is that knowing which proteins are affected will lead to targeted treatments for cancers associated with EBV, such as nasopharyngeal carcinoma and gastric cancer. An inhibitor for the miRNAs that have a role in down-regulation could allow the host cell to undergo its regular process of apoptosis, thus ridding the body of the infected cell and virus. This could be an effective method of anti-EBV and anti-cancer therapy.
Anuja Mathur is a Senior Biology Major
Figure 2. The role of the protein Bim in apoptosis; a foreign substance initiates the signaling pathway via caspases, turning on Bim. Bim inhibits anti-apoptotic proteins and forms pores in the mitochondria to release Cytochrome c, eventually leading to apoptosis
1. D. Bartel. Cell. 2004, 2, 281-297. 2. E, Choy et al. Journal of Experimental Medicine. 2008, 205, 2551-2560. 3. Marquitz et al. Virology (submitted)
Fall 2010, Volume III Issue I
Invasion of the Fish Snatchers:
The Lionfish Takeover Maggie Hunter, Staff Writer
ith its stark stripes and bizarre, fan-like fins, the red lionfish is a popular saltwater pet and is a familiar sight to most people. But while they may look pretty gliding about in a tank, these little fish have a row of 13 poisonous spines running along their backs that can deliver a painful sting and send a person to the hospital. They also have another dangerous secret: they’re slowly taking over. Working under Dr. John Bruno, UNC graduate student Andrea Anton has set out to solve the mystery of these invasive fish. In 1992, Hurricane Andrew devastated Florida, and in the process it broke an aquarium which released a handful of lionfish into the Atlantic Ocean. This, in conjunction with hobbyists releasing their pet lionfish into the wild, has allowed the fish to multiply and become an invasive species. The lionfish, Pterois volitans, is native to the Indo-Pacific, and in its home range, population numbers are normal. In the Caribbean, though, the fish has become alarmingly successful for reasons that are still mostly unknown. However, Anton has spent the past two summers taking surveys, performing experiments, and gathering data so that the invasion can be better understood . Outside of its native area, the lionfish has been faced with an entirely new set of environmental factors that provide an abundance of resources to allow it to flourish, but have none of the natural controls that could ordinarily keep its numbers in check. It is not known for certain what keeps the lionfish’s numbers down in the Indo-Pacific, but it was originally thought that the presence of predators plays a role. Though there are potential predators in the Caribbean, such as sharks and larger fish like groupers or snappers, Anton’s work has shown that even in areas with high numbers of these predators, there are still high numbers of lionfish. She says that these facts indicate that it is unlikely that anything is preying on the lionfish .
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With no natural enemies to worry about, the lionfish is free to play the role as predator rather than prey. The lionfish is a voracious carnivore and will happily devour a broad range of fish in the Caribbean—examinations of their stomach contents have revealed a total of 41 different species . Unfortunately for these Caribbean prey fish, the lionfish is an expert hunter. It is thought that because these prey fish have not evolved alongside this hungry predator, they have not adapted ways to avoid being eaten by the lionfish, and thus they are easy targets. Part of Anton’s research focused on testing the theory that the lionfish’s prey in the Caribbean really is less adapted than its prey in the Indo-Pacific. With snorkel gear and net in hand, she spent the summer of 2010 catching lionfish and different species of fish that they would consider to be prey. Both fish were then placed in a partitioned cage in the ocean, with lionfish on one side and prey fish on the other,
Photo courtesy of Andrea Anton.
Figure 1. Pterois volitans, a red lionfish.
and their behavior was examined. Her goal was to study the responsive behavior of the prey fish to the lionfish and note their levels of fear. This was done
Figure 2. Anton catching a lionfish for her studies.
in both the Caribbean and the Indo-Pacific, using closely related prey species from each area so that experiments were as accurate as possible. The results showed that the prey fish from the Caribbean did not exhibit nearly as much fear as the prey fish from the Indo-Pacific. It seems that these Caribbean fish do not recognize the lionfish as an immediate threat. In fact, they would sometimes get so close to the lionfish that, had there not been a divider between the two, the prey fish would have quickly been eaten. The lack of fear, or naivety, shown by the Caribbean prey fish could be the reason that they are so easily eaten by lionfish . Not only do lionfish eat many different species of native Caribbean fish, but they eat a lot of them. One study has shown that lionfish can drastically reduce the number of juvenile fish on a coral reef, which has potential to severely disrupt the ecology of the Caribbean . Because they eat fish of commercial importance, lionfish have already begun to impact the local economy and food security of Caribbean countries. Locals have attempted to rid the waters of lionfish by holding rallies where thousands of the fish are caught and killed in one day. However, this has only a shortterm effect and the population numbers eventually pick back up . There is no immediate or apparent solution as to how the lionfish population can be controlled. An-
Photo courtesy of Andrea Anton.
ton says that perhaps the situation will work itself out naturally—over a period of 15 to 20 generations, prey species may succumb to the selective pressure of lionfish predation and adapt better methods of avoiding and escaping the hungry fish. Or perhaps a parasite or disease, which may be what keeps population levels down in the Indo-Pacific, will take hold of the Caribbean fish and lower their numbers. Anton stresses that before the problem can be solved, though, more research is needed to prove with certainty what exactly controls lionfish populations in their native range. With this knowledge, scientists can hopefully Maggie L. Hunter is a better understand a way to sophomore Biology and control the numbers of the English double-major. Caribbean population .
1. 1. Interview with Andrea Anton. 9/28/10 2. J.A. Morris Jr, et al. Environ Biol Fish. 2009, 86, 389. 3. M.A. Albins, et al. Mar Ecol Prog Ser. 2008, 367, 233-238.
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The Quest for the Cure Kara Stout, Staff Writer
ave you ever wondered how many people have been radically affected by the invasion of cancer either personally or through a close friend or family member? The American Cancer Society estimates that in a year’s time 11,028,000 people in the United States are plagued with this disease . Kristy L. Richards, a clinical oncologist, has taken the initiative to fight back by focusing her research on the genetic etiology of lymphoma. Richards’ lab uses a variety of genetic screenings on cell lines and specific organisms with the objective of finding new genetic pathways developing into lymphoma. An undergraduate student, Michael Kingberg, has worked for Richards for the past two and a half years specifically focusing on clinical research with cancer therapy drugs.
the surface of B cells. Supplementing this research, Richards also focuses on epigenetic modifying drugs, specifically hypomethylating agents and histone deacetylase inhibiters . They are concentrating on not merely the lymphoma itself, but the specific interactions between the antibody drugs and cancer cells. Michael Kingberg became in involved in Richards’ lab primarily as a biology major seeking research experience. He works with the first project of the lab analyzing the sensitivity and resistance of CD20 to Rituximab . The CD20 antigen is expressed on nearly all B-cell non-Hodgkin’s lymphomas, and on normal cells when they are in a post-b cell phase. It aids initiation and differentiation of the cell cycle, as hypothesized to be a calcium ion channel. CD20 remains on the surface of the cell even after antibody binding, thus it is not found in circulation . Rituximab interacts with CD20 via a complementary determining region specifically on the cell. The significance of CD20 remaining fixated is so that the monoclonal antibody will not be neutralized in the blood stream before it reaches the Kara Stout is a junior proposed target cell . King- Biology and Global berg’s project involves the si- Studies double major lencing of this CD20 antigen and Chemistry minor. to see if it confers resistance to Rituximab . He uses nucleofection, an electroporation method of transferring RNA into cells via an electric shock, to create resistant cell lines.
Figure 1. The CD20 signal cascade.
Richards’ lab maps polymorphisms that regulate sen-
sitivity to rituximab, a drug used to combat hematologic malignancies. They are applying an ex vivo genetics strategy on CEPH pedigree lymphoblastoid cell lines . One example of this work is the polymorphism in the FCGR3A gene, which is particularly sensitive to rituximab . More specifically, rituximab is a monoclonal antibody commonly approved in the United States for the treatment of B-cell non-Hodgkin lymphoma. It targets the non-glycosylated phosphoprotein, CD20, found on
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1. Altekruse, SF. Cancer Prevalence: How Many People Have Cancer? 2007, http://www.cancer.org/cancer/cancerbasics/cancer-prevalence. 2. Richards, KL. Research Interests. 2010, http://cancer.unc. edu/research/faculty/displayMember.asp?ID=555. 3. Interview with Michael Kingberg. 9/29/10. 4. M.D. Pescovitz. American Journal of Transplantation. 2006, 6, 859-866.
Segmentation and Alignment of Knee MRI Images Keith Funkhouser, Staff Writer
steoarthritis (OA) is the most prevalent form of arthritis in the United States. If you know anyone over 65, it is likely that they have some form of OA . Patients with OA of the knee present with pain in and around the knee and limitation of joint motion. Unlike rheumatoid arthritis, inflammation, if present, is generally mild. The causes of OA of the knee are not always known; however, stresses affecting the cartilage are often important in pathogenesis , and cartilage loss is believed to be the dominant factor in OA of the knee. In order to get a better picture of what’s going on inside the
Figure 1. Dr. Niethammer’s methods allow for computers to rapidly interpret MRI images like these. knee, doctors are increasingly using magnetic resonance imaging (MRI). MRI is a radiologic technique that uses electromagnetic fields to visualize internal structures. Since little can be done to alter the course of OA once diagnosed, recent attention has focused on its identification and prevention, particularly using MRI [2, 3]. During the evaluation of OA, it is helpful to have the MRI images aligned to an “atlas”, or a standard model. This way, the physician knows exactly what they are looking at and their orientation with respect to it. For example, it is much easier to evaluate a patient with a skeleton nearby that looks like them and is oriented similarly. In the same way, the evaluation of knee MRI images can be done much more quickly if the images are “registered”, or aligned, to a standard orientation. In order to solve this problem, Dr. Marc Niethammer, Assistant Professor of Computer Science, has developed a methodology that allows for the simplification of knee
MRI images. When looking at an image, our brains can pick out; we see faces as separate from the background and can pick out particular objects that appear in the image. However, images are seen as nothing more than matrices of numbers to computers. Dr. Niethammer’s work uses complex mathematics to teach the computer which areas of the image are likely bones and which are likely cartilage (Figure 1). The process of dividing up an image into portions is called “segmentation”. After the image has been segmented, it must be registered to an atlas. Computationally, this is a difficult task involving the optimization of functions of several variables. Based on the biology of the knee, different segments of the image are aligned according to different restraints. For example, bones such as the femur and tibia are extremely rigid. Thus, they are only allowed to rotate, reflect, and get proportionally larger or smaller. Cartilage, however, is allowed to be distorted in other non-rigid ways. Each segment of the image is thus aligned to an atlas of the knee, bringing the original MRI image into a reference position which is more easily interpreted by the diagnosing physician. By aligning knee MRI images obtained from OA patients, physicians will be able to evaluate cases with fewer variables involved. These registration methods simplify the analysis of the knee MRI images by bringing the knee into a simpler reference position. In doing so, the physician can more easily identify pathological progression. Dr. Nietham- Keith Funkhouser is a mer’s method is fully auto- sophomore Biostatistics mated and will be improved and Biology double in the future in clinical trials. major.
1. M.C. Hochburg. Arthritis and Rheumatism. 1995, 11, 15411546. 2. M. Niethammer. SPIE Medical Imaging, 2010. 3. D.T. Felson. Epidemiologic Reviews. 1988, 1, 1-28.
Fall 2010, Volume III Issue I
Practice Makes Perfect THE BENEFITS OF TESTS
Kati Moore, Staff Writer
f you could ask your professors to change one thing about their teaching methods, you would mostly likely not ask them to give more tests. Yet taking tests has actually been shown to be the most beneficial way to retain information. The benefits of tests are due to what is called the testing effect, which shows that in a group of subjects who study a certain set of material, subjects who are tested on the material will recall it better after a delay than subjects who reread the material. The longer the delay, the greater the difference between the two groups in ability to remember the information . (See Figure 1.) So for the vocabulary test that begins in five minutes, you would probably be better off to reread that list of definitions. But for the test tomorrow or next week, you might Figure 1. Difference in retention by subjects who studied by rereading versus those who were tested. As the time between input and recall want to try testing yourself on the increased, those who were tested on the material recalled more of the words instead. material than those who reread the material. Why is this? That is the question fifth-year graduate student Daniel and that he can modify the experiment slightly and Peterson, who is studying cognitive psychology, see the outcome change as a result of that modifiwould like to answer. To do so, he will attempt to cation. In this way he can begin to determine what determine what variables are responsible for creat- factor makes test-taking so effective. If it turns out ing the testing effect by manipulating those variables that the testing effect actually works differently than to create a very artificial environment in which the other phenomena, and he does not find a negative reverse of the testing effect happens, and students testing effect, he will explore other reasons the testactually do worse on memory tests after being tested ing effect works. “The best experiment,” he said, “is on the material. Such an outcome is called a nega- one in which it’s interesting no matter the outcome.” tive testing effect. Knowing the conditions in which In other words, even if he does not see expected rea negative testing effect occurs will allow him to un- sults, he still hopes to learn something from the data. derstand the reasons the testing effect occurs. One problem with studying by rereading, Peterson If he finds a negative testing effect, the next step, said, is that it is not very productive if the material he says, will be to make sure the results are stable, was read and understood the first time through. Re-
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Carolina Scientific reading also gives students a false sense of security, because a student reads and remembers the material, but the text, Peterson said, “is refreshing [the information] for you. You don’t ask someone who’s learning multiplication tables, ‘Is four times four sixteen?’” . Another reason taking tests might be better than reading is that practice makes perfect. If your goal is to perform well in a sport, you should practice that sport. If your goal is to do well on a test, you should practice taking tests. Given the benefits, why is this not implemented in the classroom? The first hurdle is the impracticality of giving many tests: professors don’t have the time to grade them. Even Peterson says that although he would like to give more tests per semester, he just does not have the time. “If I were a full time lecturer,” Peterson said, “I would absolutely provide more tests for the students . . . give them that feedback, make sure that they were keeping up with the material, and for the memory benefits.” Peterson currently gives four tests per semester. Then there is the fact that students just do not like taking tests. Students as well as teachers see exams just as assessments, instead of as learning tools, when in fact, a 2007 study by McDaniel, et. al. supported the idea that “in the classroom testing can be used to promote learning, not just evaluate learning” . Until students’ and teachers’ perspective on tests changes, tests will not likely be popular study tools.
Figure 2. Recalling information reinforces knowledge of that information more effectively than reviewing, a phenomenon known as the testing effect.
Even if professors do begin giving more tests, multiple choice and true false tests, which are much quicker to grade, are not as effective at enforcing information as short answer and essay questions. The more that the student has to recall on their own, the better . But this also means many hours of grading for busy professors. The solution? If the benefits of testing cannot be implemented on a large scale basis in the classroom, students can still test themselves on their own when studying. “If you’re going to spend extra time studying, why don’t we channel it into something a little more productive,” Peterson said. Instead of simply rereading your notes the night before the exam, try something different. Test Kati Moore is a freshman Biology major. yourself.
References 1. H. Roediger, III, et. al. Psychological Science. 2006. 3, 255. 2. Interview with Daniel Peterson. 9/24/10 3. M. McDaniel, et. al. European Journal of Cognitive Psychology. 2007. 19, 494-513.
Figure 3. Rereading material gives students a false sense of security, says Peterson.
Fall 2010, Volume III Issue I
A New Field Examines Evolution’s Role in Development
Kyle Roche, Staff Writer
ou’ve been told that what you look like is the result of your Mom and Dad’s genomes, but what if that’s only some of the story? Would you look the same if you were brought up under different environmental factors? Phenotypic plasticity has recently been brought into the scope of evolution, particularly its applications within the context of speciation, and adaptive radiation. These effects have been extensively examined by the mem-
bers of Dr. David Pfennig’s evolutionary biology lab. Before exploring the implications that phenotypic plasticity has within evolutionary processes, it is important to first understand the concept of phenotypic plasticity. A phenotype is what an organism looks like as a result of its underlying genome, as well as the genomes interaction with the environment. It follows then, that phenotypic plasticity is the ability of one genome to produce multiple phenotypes
Figure 1. These are all members of the same species; the differences between them are the result of phenotypic plasticity. As shown in the figure below, these differences are significant, and would be expected to influence many characteristics of the individual’s life, including reproduction.
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Carolina Scientific in response to environmental stimuli. Put simply, if at birth you cloned an organism that had the capacity for phenotypic plasticity, placed both of these organisms in different environments, then you might find that the two organisms would look very different after development. If, however, you placed the clones into the same environment, then they would look identical after development. The central dogma for evolution has historically been held as a strictly genetic process involving the change of allele frequency over time due primarily to natural selection. Phenotypic plasticity had always been viewed as a developmental process because these changes did not alter the actual genetic code of an organism, but rather the expression patterns of its genetic code. As such would seemingly not affect the evolutionary processes of genetic divergence and resulting speciation, or adaptive radiation. However, recently phenotypic plasticity has been found to be intricately woven into initiating and expediting evolutionary processes, and in some cases plays a role in initiating and expediting them Phenotypic plasticity gives organisms the ability to quickly and efficiently respond to environmental changes, especially when compared with the mutation model for phenotypic change, which requires a beneficial mutation to evolutionarily respond. By expressing “hidden genes” that are already present within genomes in response to certain environmental stimulus, it is possible for organisms to adapt much more quickly. Additionally, if an environmental stimulus is continually received, then it is possible for a plastic gene to undergo genetic assimilation, a process of in which an environmental stimulus threshold for a gene is lowered to the point that it is always stimulated, and is therefore is no longer plastic. With the introduction of genetic assimilation, it now becomes possible to imagine scenarios in which speciation may occur as a result of phenotypic plasticity. One mechanism in which speciation occurs is through the isolation and breeding of different populations within the same species, also known as reproductive isolation. With that said, it will be helpful to frame phenotypic plasticity and its role in speciation using an example. Let’s say that within a species of big horn sheep, like Rameses, there are two possible environments within a particular mountain range. The first environment is at the bottom of the
mountains, and is warm. The other environment located higher in the mountains, and is colder. When the sheep are born, they either develop thick fur or thin fur, depending on whether they experience a cold or warm environment during development, respectively. In this scenario, speciation can occur via reproductive isolation, because if a sheep is born in the cold, then it will develop a thick fur. Then, as a result, it will most likely interact and breed with other sheep with thick fur. If, over generations, sheep are continuously exposed to the cold, then it stands to reason that through the process of genetic assimilation two populations would result - one with thick fur and one with thin fur. In his work, David Pfennig has outlined numerous possible scenarios in which phenotypic plasticity can play a role within speciation, and evolution as a whole. As a result of his findings as well as the findings of others, the field of evolution is trending away from a purely genetic approach, and beginning to incorporate epigenetic, or above genetic, factors.
Kyle Roche is a senior Biology major and Chemistry minor.
References 1. Pfennig, D. W., Wund, M. A., Snell-Rood, E. C., Cruickshank†, T., Schlichting, C. D., & Moczek, A. P. 2010. Phenotypic plasticity’s impacts on diversification and speciation. Trends in Ecology and Evolution 25: 459–467. PDF
Fall 2010, Volume III Issue I
Radiomitigation: Treatment After the Fact
Mary La, Staff Writer
ausea and vomiting. Acute burns. Anemia, study and research funding. A research group headed bleeding and a severely weakened immune by Dr. Norman Sharpless, Associate Director for Transsystem. These are a few of the trademarks of lational Research at the UNC Lineberger Comprehenexcessive radiation exposure. Improving the treatment sive Cancer Center, has described the first successful options for the acute radiation syndrome is a major area radiomitigant in a mouse model system. They were of research, with impacts not only in disaster response, able to identify this radiomitigant based on the fact that but also in helping cancer patients cope with the side amitotic (non-dividing) cells are more resistant to DNA effects of radiation therapies. Currently, treatments are damage of the type caused by radiation exposure  chosen depending on several factors, such as the ra- . diation dosage, and whether the exposure was external The agents that the Sharpless group has studied inor internal. Treatments immediately after exposure are designed to bind internally circulating radioactive agents to inactivate them, and therapies against infection are required a few days to a month after exposure . There are even radioprotectants, a class of compounds that can help decrease the effects of radiation exposure if taken before the exposure occurs . However, radioprotectants are effective only if radiation exposure is expected beforehand (e.g. before radiation therapy), unlike in accidental irradiation. Should a radiation exposure victim survive, consequences of the incident can Courtesy of www.unchealthcare.org. still be seen years after, ranging from continued low red blood cell Norman Sharpless, MD, Associate Director for Translational Research at and white blood cell counts (anemia UNC Lineberger Comprehensive Cancer Center. and neutropenia, respectively) to the passage of genetic defects from one generation to the next. hibit the activity of enzymes called CDK4 and CDK6, No currently available treatment addresses one of which control the cell cycle (Figure 1.). CDK4/6 sigthe most critical consequences of radiation exposure nals influence the division of, among other cell types, – bone marrow damage. Bone marrow is principally bone marrow hematopoietic stem cells that produce responsible for generating blood cells; damage to bone blood cells. The identified CDK4/6 inhibitors arrest the marrow results in anemia, thrombocytopenia (low cell in the G1 phase of the cell cycle, before the cell has platelets) and neutropenia, the latter causing immu- had a chance to synthesize copies of its genetic material nosuppression. The search for radiomitigants, medi- and prepare for division. The authors term the use of cations that can be taken after radiation exposure to CDK4/6 inhibitors to cause a transient cellular arrest in reduce bone marrow damage, is the subject of much the G1 phase pharmacological quiescence (PQ).
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Figure 1. The cell cycle. Notably, CDK4/6 acts to control the cell’s progression from the G1 phase (normal cell functioning and metabolism) to the S phase (duplication of genetic material in preparation for cell division).
A series of studies were conducted to evaluate the limits of this radiomitigant’s efficacy. After confirming in vitro that the cell cycle could be halted in the G1 phase to confer protection against DNA damage, various dosage schedules of the radiomitigant were used in mice before or after total body irradiation (TBI). The outcomes of this experimental group were compared to control mice that were given TBI, but were not administered CDK4/6 inhibitors. These compounds were shown to increase chances of survival when used 4 hours before TBI, concomitant with TBI, and even up to 20 hours after TBI. Other approaches to radiomitigation have provided short term protection in mice, but by keeping damaged cells from dying, these efforts have led to the later development of bone marrow failure and leukemia . However, the mice that survived TBI due to the PQ approach did not demonstrate excess leukemia or bone marrow failure when observed for several months post-TBI. Another concern was whether or not the drug would allow malignant cells to live after treatment, such as when radiation is applied as cancer therapy. To address this concern, mouse models with induced melanoma were given the radiomitigant at the time of radiation treatments. The CDK4/6 inhibitors did not stop tumor growth, suggesting that the tumors studied were not dependent on the activity of CDK4/6 for proliferation. Since these agents did not induce PQ in the tumors, the tumors were as sensitive to radiation therapy as tumors in mice that did not receive the radiomitigants. Tumorbearing mice that received CDK4/6 inhibitors at the time of radiation, however, showed a marked reduction
in bone marrow toxicity. In this way, these radiomitigants can ameliorate the most important side effects of radiation-induced DNA damage, while still leaving the tumor vulnerable to potentially curative radiotherapy . The authors were careful to warn, however, that it is likely that the proliferation of some types of cancer may be decreased by CDK4/6 inhibitors, and in these types of cancer, such agents would decrease the effectiveness of radiation therapy. CDK4/6 inhibitors are can be given as pills, and are relatively nontoxic and chemically stable, which makes possible their stock-piling to handle emergency radiation exposures. Treatment of radiation exposure with CDK4/6 inhibitors, or other compounds that induce PQ, could be more successful and cost-effective than current radioprotectant therapies, especially in the hours post-exposure . Radiomitigant-mediated PQ also sees potential application in minimizing the marrow toxicity resulting from chemotherapy and radiation therapy against cancer. Much more work remains in determining radiomitigants’ behavior in primates and humans, whether radiomitigants hinder the longterm inheritance of genetic defects resulting from DNA damage, and identifying the most effective forms of CDK4/6 inhibitors. However, this vital field of research should facilitate the development of more effective responses to radiologic crises and radiation-mediated cancer therapies, well into the future. Mary La is a senior Many thanks to Dr. Nor- Chemistry and Comman Sharpless and Dr. So- puter Science doubleren Johnson for their crucial major, and a Hispanic Studies minor. guidance in this article. References 1. NJ Chao. Experimental Hematology. 2007, 35, 24-27. 2. TK Gosselin TK, et al. Clinical Journal of Oncology Nursing. 2002, 6, 175-180. 3. SM Johnson, et al. Journal of Clinical Investigation. 2010, 120, 2528-2536. 4. Drug Mitigates Toxic Effects of Radiation in Mice. News Room - UNC Health Care. 2010. <http://news.unchealthcare.org/news/2010/June/drug-mitigates-toxic-effects-of-radiation-in-mice?searchterm=radiomitigation>. 5. F Herodin, et al. Blood. 2003, 101, 2609–2616.
Fall 2010, Volume III Issue I
Kinetochores: Getting to the “Chore” of Chromosome Segregation at Cell Division Rana Alkhaldi, Staff Writer
s cells progress through the cell cycle and approach cell division, genetic information encoded in the form of DNA begins to condense together to form chromosomes. Once condensed, the chromosomes must be segregated equally so that the two daughter cells receive the appropriate amount of genetic information necessary for proper growth and
Figure 1. A picture of a chromosome (condensed DNA). The chromosome is in blue, and the red spots are the kinetochores on each sister chromatid.
survival. This process occurs mainly with the help of two essential structures. The first of these is the centrosome, the organelle that sends out protein filaments (called “microtubules”) to attach to the chromosomes and pull them to opposite poles of the cell. The second is the kinetochore, the region of the chromosome that receives these microtubules (Figure 1). These components of mitosis, or cell division, have long been known to be key players in the alignment of chromosomes in preparation for the splitting of the cell. However, scientists have struggled to determine the exact mechanism that drives microtubules to append to the kinetochore; these biologists have also striven to unravel the mystery of how microtubules consistently pull exactly half of the chromosomes to each cell pole every time the cell divides. These are the questions Dr. Edward D. Salmon, Ph.D., a professor in the Biology Department at UNC, hopes to address with his research . Over the last four decades of thorough research in
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this topic, Dr. Salmon’s lab has contributed extensively to progress in learning how microtubules attach to the kinetochore and how this attachment allows chromosomes to separate during mitosis. One of the discoveries of his research, done in collaboration with Dr. Andrew Murray of Harvard, was the detection of a protein, called Mad2, which operates as a “checkpoint” in mitosis; this protein binds at high concentration to kinetochores that are not yet fully attached to the microtubules. This effectively protects the cell from moving into anaphase – the stage of cell division in which the cell and its chromosomes split (Figure 2) – before all the chromosomes are connected to a pole of the cell . In a subsequent discovery, his lab showed that a motor protein called dynein removes the binding site of this Mad2 protein from kinetochores, thus allowing the cell to proceed into anaphase . These observations were major strides in the path to understanding the intricate mechanisms of cell division. In more recent research, Dr. Salmon’s lab provided evidence that the core microtubule binding sites within kinetochores depend critically on a complex of proteins called Ndc80. This protein complex is required for microtubule attachment, correction of errors in those attachments, control of Mad2 concentrations at kinetochores, and generation of force for the movement of chromosomes during anaphase. Over the past few years, the Salmon lab, in collaboration with Dr. Kerry Figure 2. A picture of Bloom’s lab at UNC, a cell during anaphase. has developed a map of Microtubules are colored the proteins at the mi- green, and chromosomes are crotubule-kinetochore shown in red.
Figure 3. A depiction of the kinetochore-microtubule attachment site. The microtubule in its depolarized (shrinking) state is shown on the top, and in its polarized (growing) state on the bottom. The tension is high during depolymerization, and low during polymerization.
attachment site in order to learn more about this complex. Together, the two labs engineered a method of two-color fluorescence light microscopy to measure on a nanometer scale the separation of proteins. In human cells and budding yeast, they used antibodies that fluoresced at two different colors to label proteins, and thus measured the average distance between proteins at the microtubule-kinetochore juncture. This represented the first time that the protein structure of kinetochores of humans and of budding yeast was successfully mapped [4, 5]. Perhaps even more noteworthy from this study was the discovery of the change in the structure of the human kinetochore when taxol, a common cancer drug, is added to the cell. â€ƒ Taxol disrupts the natural dynamic assembly of microtubules and, in doing so, effectively prevents the cell from dividing. Microtubules typically grow and shrink many times per minute, and this instability generates tension once they attach to the kinetochores (Figure 3). This tension is theorized to be one of the forces the cell uses to pull chromosomes apart. Taxol inhibits this feature of the microtubules, decreasing the tension at the kinetochore-microtubule attachment site and impeding the cell from progressing into anaphase. While this is significantly useful for fighting an out-of-control tumor, its effects on healthy cells is detrimental; as taxol stops the cell from proceeding to the end of mitosis its presence ultimately leads to the cellâ€™s destruction. Dr.
Salmon and his colleagues also found that taxol alters the structure of the kinetochore; specifically, the Ndc80 complex buckles towards the inner portion of the kinetochore, possibly due to the activation of the waitanaphase checkpoint. This checkpoint indicates to the cell that it is not ready to enter anaphase, an action that impedes the progression through mitosis and, in the case of taxol, kills the cell. Comparing the structure of the kinetochore in a normal cell versus a cell exposed to taxol was crucial to learning in greater depth the functions of the various proteins at the kinetochoremicrotubule interface, and particularly in elucidating an additional function of the Ndc80 complex. Although the exact process of how the Ndc80 complex inactivates the Mad2 checkpoint is not fully understood, the importance of this complex of proteins in regulating this switch has at least become clear . â€ƒ Simply listening to Dr. Salmon describe his research demonstrates his deep curiosity in this subject and his drive to pursue further research in the topic. Although the exact process of the checkpoint regulation in kinetochores has yet to be entirely understood, Dr. Salmon and his colleagues have made remarkable advances in bringing us closer to that answer. The unequal separation of chromosomes during cell division as a result of failed checkpoints or failures to correct errors in microtubule attachment can lead to a variety of genetic disorders, birth defects, or even certain types of cancer; thus, understanding this process in detail could lead to monumental advancements in an array of fields of biology. With his ongoing research, Dr. Salmon will certainly aid us in progressing forward towards a deeper comprehension of this complex, yet undeniably Rana Alkhaldi is a senior intriguing, mechanism of Biology major and Arabic minor. mitosis.
References 1. Interview with Edward D. Salmon, Ph.D. 9/30/10. 2. J. C. Waters, et al. J. Cell Biol. 1998, 141, 1181-91. 3. B. F. Howell, et al. J. Cell Biol. 2001, 155, 1159-72. 4. X. Wan, et al. Cell. 2009, 137, 672-84.
Fall 2010, Volume III Issue I
Nitric Oxide: A Glucose Monitor's Best Disguise
Hetali Lodaya, Staff Writer
he human body is equipped with various defenses to ward off almost any foreign substances. This has posed a problem to scientists for years – potentially efficacious treatments such as transplant organs and internal biosensors, used to measure parameters such as blood glucose or vitamin levels, are often not as efficient because the body hampers their function . Decreased efficiency can be a result of electrical failure, sensor membrane degradation, infection, and the envelopment of the device in fibrous tissue as a result of the foreign body reaction (FBR), as depicted in Figure 1 . The FBR occurs during wound healing after a sensor is implanted – the body develops tissue that is thick and fibrous, forming a “capsule” around the sensor. Sensors rely on diffusion from nearby blood vessels for measurements, and the FBR capsules cut off sensors from input sources . A sensor modified to decrease capsule thickness or formation would therefore likely have increased efficiency. In par-
ticular, the development of electrochemical sensors for glucose monitoring, used by many diabetics, has been hampered: because of the FBR, they generally have to be replaced every 3-4 days . Researchers in Professor Mark Schoenfisch’s laboratory at UNCChapel Hill are working to improve the biocompatibility of glucose sensors, specifically by addressing the FBR. According to Professor Schoenfisch, “decreased capsule thickness is… very likely to improve performance of biosensors” by increasing the capillary density around sensors, and thereby increasing the ease with which sensors measure the materials diffused around them . The Schoenfisch group concentrates on the use of nitric oxide (NO) as a coating for sensors to improve their function. NO is a very simple molecule that plays many roles in the body. In particular, its involvement in improving wound healing and battling microbial infection is of interest when considering it as a candidate for reducing the FBR . Its short half life also ensures that it will not diffuse in the body too far away from the site where it is needed. NO has many qualities that suit it to protect an in vivo biosensor from the FBR and from bacteria – the challenge is to deliver it in an efficacious way . The first question the Schoenfisch lab asked involved understanding the theoFigure 1. Several examples of the foreign body reaction with different degrees of severity. retical use of NO to Arrows indicate the different thicknesses of capsules, with A being the largest capsule and prevent increased C the smallest. capsule size caused
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Carolina Scientific membranes, providing a delivery method that also potentially increases sensor permeability and lifetime – the hope is that nanoparticle-enhanced sensors would only have to be replaced once a week . Professor Schoenfisch states that dummy sensors will be used first to see whether “continuous NO release is important – we want to look at the FBR as a function of NO flux” . Once the best delivery schedule for NO has been determined, nanoparticles will be doped into real sensors in an effort to understand whether, with the help of NO, they truly result in sensors with longer, sustained performance and greater biocompatibility. Biosensors are an essential tool in the medical arsenal, particularly for measuring blood glucose levels. Although countless diabetic patients use these sensors every day, scientists have struggled to improved their efficiency and ease of use in the face of the FBR. As demonstrated by the Schoenfisch group, nitric oxide, delivered with the help of nanoparticles, could be a candidate to decrease the FBR capsule thickness, increasing blood flow around sensors and thus improving their function. Nanoparticles and NO are potentially the perfect cloak for sensors to hide behind as they continue to help the body.
Figure 2. A diagram showing the various biologically relevant properties of nitric oxide.
by FBR. A simple NO releasing probe was used to measure the FBR and diffusion across the probe. It was found that NO both decreased the thickness of the FBR capsule and increased diffusion of the desired indicator, H2O2, across the probe . With this evidence that NO could improve glucose sensor biocompatibility, the Schoenfisch group next needed to find a way to deliver NO in the context of an actual sensor. The Schoenfisch group has been exploring a novel delivery method for NO from a sensor: nanoparticles loaded with NO and integrated into a sensor membrane. The group has already shown that these particles release NO efficiently, and are now starting experiments to see what effects nanoparticles carrying NO will have on the FBR and capsule formation [5,6]. These nanoparticles will be doped into sensor
Hetali Lodaya is a freshman undecided major. References
1. Biocompatibility. 2007, http://www.biology-online. org/articles/closer_nature_biomaterials_tissue/biocompatibility.html. 2. Paul and Schoenfisch, in In Vivo Glucose Sensing (John Wiley and Sons, 2010). 3. Interview with Mark H. Schoenfisch, Ph.D. 10/11/2010. 4. Hetrick, Evan M. et al. Chemical Society Reviews. 2006, 35, 780-789. 5. Hetrick, Evan M. et al. ACS Nano. 2008, 2, 235–246. 6. Johnson, T.A. et al. Nitric Oxide. 2010, 22, 30-36.
Fall 2010, Volume III Issue I
Arctic Bacterial Gluttony Accelerates Global Warming Ashley Mui, Staff Writer
s any doctor will tell you, never underestimate the power of bacteria. Though microscopic, short lived, and seemingly unimposing, microbes have important roles in abiotic and biotic cycles. Bacteria play a key role in the global carbon cycle. As bacteria breathe they convert carbon to CO2, accelerating the rate of global warming. The cause of global warming is the release of greenhouse gases that cause radiative forcing, which occurs when the amount of solar energy that enters the atmosphere is less than the amount radiated back into space. The most frequently discussed greenhouse gas is carbon dioxide, which is emitted by fossil fuel consumption, cement production, aerobic respiration, and decomposition. Since bacteria are a natural source of CO2, they create a positive feedback cycle in global warming. Bacteria respiration rates in-
crease due to global warming, and greater CO2 release rates intensify the global warming process. New research conducted by Dr. Rose Cory of the Department of Environmental Sciences and Engineering explores why Arctic bacteria may be adding a new CO2 contribution in response to Arctic warming. The hallmarks of the Arctic tundra biome are cold climate, simple vegetation structure, a short season of growth, and a detritusbased sink for energy and nutrients . The Arctic tundra fluctuates annually as a source and sink for carbon, depending on the balance between photosynthesis and soil decomposition. During the brief summer when photosynthesis dominates, Arctic tundra plants (sedges, heaths, grasses, mosses) convert CO2 into other forms of carbon that are incorporated into their bodies. With the arrival of subzero Arctic temperatures in the winter, these plants die, but
Figure 1. Arctic permafrost has showed signs of accelerated thaw.
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their bodies are not decomposed as rapidly as in warmer climates. Rather, most plant biomass freezes and is buried, making their carbon in the form of dead organic matter (DOM) inaccessible to be returned as CO2 the atmosphere . Tundras contain about 160 gigatons of carbon as decomposing organic matter, equivalent to about 27% of the carbon content of Earth’s atmosphere . It has been noticed that Arctic global temperatures have increased at a rate faster than the rest of the Earth . The increased solar radiation has caused thawing of the Arctic permafrost, making the DOM accessible to Arctic bacteria. When aerobic bacteria decompose the permafrost DOM, they convert the detrital carbon into CO2 through respiration. This forms a positive feedback cycle - increased temperatures promote permafrost thawing and increased soil decomposition, which causes further CO2 emissions. To
Figure 2. Dr. Cory will be returning to the Toolik field station for the next two summers.
Figure 3. The photochemical reaction chamber that was used to incubate soil cores.
make matters worse, weather patterns in the Arctic have shifted as a result of global warming. According to Dr. Cory, in 2007 this caused the first known lightning storm and tundra fire in thousands of years, creating patches of burnt soil. Dr. Cory’s research studies the effects of sunlight and fire exposure on rates of Arctic soil decomposition by bacteria. Her research group suspects that once the dead organic matter is released from the freezer-like conditions in the permafrost, exposure of this material to sunlight or fire can “cook” the organic matter, making it more palatable to bacteria. If the bacteria prefer the organic matter “cooked” by sunlight or fire, they may respire more of it, thereby converting more of the carbon to CO2. Soil cores and DOM-rich water samples were collected from the Arctic Long Term Ecological Research Station (Arctic LTER) in Alaska. Some of the soil cores consisted of burnt soil from fire afflicted areas while others contained soil from unaffected areas. All cores were sterilized of bacteria. Half of the cores and water samples were then ex-
posed to a 24-hour period of ultraviolet (UV) radiation. All samples were re-cultured with the original bacteria and incubated in darkness for one week. Three measurements were taken to determine the growth of bacterial activity and consequential CO2 output. First, respiration rates were determined using a membrane inlet mass spectrometer that measures O2 concentrations. These results were converted to CO2 concentrations using known chemical relationships. Second, the quality of the organic matter was analyzed using a fluorescence spectrometer to determine the initial and final types of organic content of the samples. Third, the bacterial growth rates were measured through cell counts, implementing both flow cytometry (electronic detection) and microscope counts. Results confirmed that the soils exposed to UV radiation had greater amounts of soil decomposition. Bacterial growth rates and CO2 output rates were both 2-3 times greater in the exposed samples when compared with unexposed samples. Additionally, preliminary data seems to show greater decomposition in the burnt cores, though this data is still being processed. These findings are indicators of the mechanisms behind the Arctic tundra’s transitioning state, which can have serious implications for climate change science. The thawing of Arctic permafrost may outweigh compensatory factors combating increased CO2 release, such as higher photosynthetic production due to warmer Arctic temperatures . Dr. Cory has National Science Foundation
Figure 4. Soil core from the Toolik field station.
funding for the next three years to complete this project, and plans to return to the Arctic LTER for the next field season.
Ashley Mui is a junior Environmental Science major and Computer Science and Math minor.
1. The Tundra Biome. March 2007. <http://www.ucmp.berkeley.edu/exhibits/ biomes/index.php>. 2. Oechel et al. Nature. 1993, 361, 520522. 3. Monfray, Claire, et al. Geophysical Research Letters. 1997, 24, 229. 4. Interview with Rose Cory, Ph.D. 09/29/10.
Fall 2010, Volume III Issue I
The Study of a Disease That Would Have Ruined Air Bud’s Career
Madison Roche, Staff Writer
hen thinking of golden retrievers, a famous dog comes into mind for most people: Air Bud. This extremely athletic, basketball -playing dog can sprint, score baskets, and even play defense. However, to Dr. Joe Kornegay and his research team, what comes to mind is a disease found in golden retrievers called golden retriever muscular dystrophy (GRMD). This disease is one that could have destroyed Air Bud’s basketball career. Joe Kornegay, DVM, PhD, came to the UNC- Chapel Hill School of Medicine in 2006 to continue his studies of the spontaneous canine disease, GRMD . GRMD is analogous to a disease in humans called Duchenne Muscular Dystrophy (DMD). These diseases affect the striated muscles of both humans and dogs. More specifically, they cause skeletal and cardiac muscle deterioration. GRMD and DMD are caused by genetic mutations in the dystrophin gene . According to Dr. Kornegay, the goal of his research is not only to advance veterinary medicine, but also to better under-
Figure 1. GRMD dog at 15 months of age .
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stand DMD in humans . Dr. Kornegay explained, “If you go up to 35,000 feet, so to speak, and look at the role that animal models can play in human disease and the evolution of molecular biology over the last 20 plus years, [the development of the human genome] has allowed for the molecular and the genetic basis for many human diseases and their counterparts in dogs to be defined .” GRMD represents one of these animal models that allow scientists to research the disease. This leads us into the discussion of how exactly these genes are delineated and how defining them allows for advances in medicine. In 1981, while Dr. Kornegay was in his residency at the University of Georgia College of Veterinary Medicine, he was asked to consult on two male golden retrievers that had signs of muscle disease. In reviewing the literature, he saw that five other golden retrievers, all of which were males, had been reported to have similar clinical signs in other papers and case reports [1, 2]. During the laboratory studies of these dogs, researchers found similar pathologic changes in the muscles. Therefore, it was assumed that the dogs had the same genetic disease. Furthermore, the fact that all five dogs were male suggested that this disease was an X-linked trait. A pattern of X- linked inheritance means that the causative gene is on the X chromosome. Because males only have one X-chromosome, the inheritance of a single diseased allele is sufficient to cause the male to have the disease. While the inheritance of one X chromosome in a female will make her a carrier, she will not have the disease . When Dr. Kornegay began his studies in the early 1980’s, the dystrophin gene had not yet been discovered. Ultimately, he and his
Carolina Scientific collaborators were able to show that affected dogs have a mutation in this gene, in keeping with DMD in humans. The question now is, in what direction is this research going? There are two main divisions in research of GRMD: pathogenesis (studying the mechanism of the disease itself) and treatment develJoe Kornegay, opment. DVM, PhD “Our research has several – Principal approaches, broadly speaking,” Investigator and Dr. Kornegay describes. “One is Director, National we want to better understand the Center for Canine disease such as why do particular Models of DMD features [of the disease] occur? (NCDMD) As an example, some muscles are affected more so than others. Why is that? Obviously, if you can determine those mechanisms you might be able to manipulate the muscles that are more severely affected to behave more like the ones that are more moderately affected. So we look at the pathogenesis of problems in dogs to understand the disease and extrapolate our findings to DMD .” In other words, Dr. Kornegay and his colleagues first find out as many details as possible about the disease, and then use this information to help develop potential treatments. While there is no cure yet for GRMD or DMD, Dr. Kornegay says things are on their way. Currently, there are a few treatments, but none of these are able to stop or reverse the progression of GRMD or DMD . In general, there are three types of treatments: molecular, cellular, and pharmacologic . The two most common molecular treatments being studied worldwide are gene therapy and gene correction. Gene therapy involves inserting the dystrophin gene into the muscle through use of plasmids or viral vectors . On the other hand, gene correction involves introducing oligonucleotides which help by either inducing repair mechanisms or by skipping exons to correct the nucleotide sequence. An example of cell- based therapy is injecting normal stem cells into an affected muscle. Lastly, pharmacologic therapies consist of drugs that target the dystrophic effects on the muscle. One of the main drugs being used in DMD is prednisone, which has been shown to have unexplained benefits. “Prednisone doesn’t cure anybody and there are side effects,” Dr. Kornegay said. “It helps—it can delay the progression of the disease, and it should be used, but everyone will say we want to do
Figure 1. The above pictures are muscle biopsies taken form 6-month-old dogs with GRMD showing changes in response to chronic 2 mg/kg daily oral prednisone treatment. A,C, and E are untreated GRMD controls.
better than that .” In working towards finding a better treatment option, Dr. Kornegay and his team currently house a colony of dogs in an animal care facility in Hillsborough . There is no doubt that progress is being made every day in the hope that we can alleviate human suffering, and enjoy many more healthy Air Buds to come.
Madison Roche is a sophomore Biology major and Chemistry minor.
References 1. Kornegay, Joe. “Biosketch of Dr. Kornegay.”2010, <http://www.med.unc.edu/pathology/faculty/biosketchof-dr-kornegay>. 2. Interview with Dr. Joe Kornegay, DVM, Ph.D. 9/28/10 3. MedlinePlus. “Sex-linked Recessive.” 2008, http:// www.nlm.nih.gov/medlineplus/ency/article/002051.htm. 4. National Center for Canine Models of Duchenne Muscular Dystrophy. “Background Information” , 2010, <http://www.ncdmd.org/background.cfm>.
Fall 2010, Volume III Issue I
Growing Up Straight GROWING UP STRAIGHT Hema Chagarlamudi, Staff Writer
hat do embryonic development and cancerous tumors have in common? It may not be the first thing that comes to mind, but one simple answer is blood. Blood supply provides oxygen and other essential nutrients to an organism, and it is essential for most life processes including early embryonic development. However, blood supply is also essential for the formation and abnormal growth of cancerous tumors. At the vascular development lab at UNC, Dr. Victoria Bautch is interested in the vehicle of blood – the blood vessel. Dr. Bautch initially became interested in blood vessels while she was working with mouse development as a post-doctoral fellow in New York. Through a “series of accidents”, Dr. Bautch created a transgenic mouse that exhibited tumors in the blood vessels . Since then, Dr. Bautch has been studying various as-
Courtesy of http://www.bio.unc.edu/Faculty/Bautch/Lab/
Figure 1. An image of half-developed mouse embryo with all the blood vessels visible in dark blue.
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pects of vascular development in mice. More recently, Dr. Bautch’s lab, composed of postdoctoral fellows, graduate and undergraduate students,
Figure 2. A simplified schematic of blood vessels in a mouse retina.
has been working on the relationship between several extrinsic and intrinsic factors that regulate blood vessel growth. The importance of extrinsic factors, mainly a signal molecule known as VEGF, has already been established by various studies. VEGF is made and released by the cells or tissues that want the blood vessel. The blood vessel then grows toward that signal. Since VEGF is not secreted by the blood vessel cells, it functions as an “extrinsic” factor. Dr. Bautch and her team have been working on how a blood vessel knows to grow in a straight line toward an extrinsic signal from the point of its origin. This study has shown that a key factor regulating this process is Flt-1. This molecule is a protein which is produced by the blood vessel cells and hence, is called an “intrinsic” factor. In normal vessel growth, a few cells grow toward the VEGF signal while the cells around these growing cells secrete a lot of Flt-1 protein and remain stationary. Flt-1 is a signal that inhibits the growth of these cells, allowing for a linear growing motion of the entire vessel. In order to understand this process better, a blood vessel can be imagined as a blob of cells when it first forms. If all the cells in this blob extended and grew toward VEGF, an abnormal vessel with contorted angles would form instead of becoming
Figure 3. Images of emerging vessel sprouts (marked with asterisks). The vessel sprout in image D does not grow straight because it lacks Flt-1. The sprout shown in vessel H is “rescued” by the presence of soluble Flt-1 around it.
a straight vessel . In this way, Flt-1 creates a “corridor” in which the growing vessel cells, known as a blood vessel sprout, can extend in the proper direction . In experiments where mutant cells cannot make Flt-1, these vessel sprouts start to form, but as they grow their direction becomes increasingly random. As a result, these vessels are unable to reach their target tissues properly . When asked if there have been any challenges or setbacks in this study, Dr. Bautch laughs and exclaims, “Oh, Yeah!” Since the publication of the Flt-1 study, Dr. Bautch and her lab have been trying to understand how the various cells of a vessel communicate with each other. For instance, when a vessel senses VEGF, why do some cells take on the role of “moving” cells and other choose to remain stationary? In other words, they are studying how inter-cellular communication determines which cells do not secrete Flt-1 and which ones do. This is a specific area that requires more research in order to find a satisfactory explanation . In collaboration with other studies, the Flt-1 study illustrates the potential for new therapies that can combat cancer by inhibiting tumor growth. For instance, a recent study published by a different lab showed that tumor blood vessels express very low levels of the Flt-1 protein. This study suggests that the absence of normal levels of Flt-1 is likely a factor in the abnormal growth of tumors. This means that normal blood vessel growth can be promoted through the regulation of Flt-1. While
it is important to keep in mind that this is still a work in progress, Flt-1 shows great promise for the development of novel therapeutic drugs for various diseases . On a more personal note, Dr. Bautch is passionate about research and enjoys traveling whenever possible. She advises all undergraduate students to get involved in research at UNC by getting in touch with professors and speaking up. She also encourages students to find a career path that they are passionate about. “You are going to be good at what you love to do,” says Dr. Bautch .
Hema Chagarlamudi is a junior Biology major.
1. Interview with Victoria Bautch, Ph.D. 09/28/2010. 2. J.C. Chappell, et al. Developmental Cell. 2009, 17(3), 377-386.
Fall 2010, Volume III Issue I
Searching for New Physics with the
Large Hadron Collider
Lenny Evans, Staff Writer
lose to 200 meters below the border of France and Switzerland lies the Large Hadron Collider (LHC), a huge tunnel 27 kilometers in circumference, in which protons are accelerated closer to the speed of light than any previous manmade accelerator has achieved. The LHC also collides these protons at an intensity higher than that of previous accelerators. Protons are collided at various points along the ring, where the high-tech detectors lie. There are two general purpose detectors, called ATLAS and CMS, and two specialized detectors, ALICE and LHCb. All of these detectors are looking for signs of new physics. One of the main goals of the LHC experiments is to look for the Higgs boson, a particle that is theorized to give mass to all the other particles. The LHC is also searching for supersymmetric particles,
which are particles that are similar to the known particles but have spin differing by one half. These particles are theorized to exist as they solve the “hierarchy problem,” in which calculating the Higgs mass using the laws of particle physics requires two numbers that are barely different to subtract from each other. Other questions such as why the fundamental forces act differently from each other and the nature of dark matter (unseen matter that cause galaxies to spin faster than expected) are also questions that the LHC hopes to answer. The ATLAS and CMS detectors are designed to be as sensitive as possible across a variety of scenarios so that should any unexpected phenomena happen, the detectors will be able to gather as much information as possible about these events . The detectors are often made up many compo-
Figure 1. The relative size and parts of a CMS detector.
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Carolina Scientific simple. Sometimes electrons are not stopped completely by the ECAL, and will deposit some of its energy into the HCAL. Events like this need to be studied so that electrons are not accidentally identified as hadrons, which would implicate impossible decays. Because the particles are colliding at such high energies and at extremely high intensities the way this radiation affects the detector must be considered as well . â€ƒ While I was at the LHC, I was involved in examining the effect of this radiation on some of the lead tungstate crystals used to detect electrons and light that enter the CMS detector. For the first time, I was able to see efficiency changes in the detector that were consistent with effects seen in preliminary research with the crystals. From this, we determined that efficiency corrections would need to be included Figure 2. A Higgs Event. as a calibration measure . â€ƒ After the detectors are characterized and undernents. The CMS detector, for example, has a region called the inner tracker, which uses a magnetic field stood well, the physics can be studied. Events that to determine whether a particle is charged or not, and are not consistent with known physics will be charto determine where the particle came from. Right acterized and analyzed with particular care, and outside of the inner tracker is the electromagnetic could lead to the discovery of a new particle. This calorimeter (ECAL), which uses lead tungstate crys- can confirm or refute certain theories that predict tals to detect and measure the energies of all light particles with certain interaction. This will lead to a and electrons produced in collisions. Outside of this deeper understanding of the fundamental laws of the region is the hadron calorimeter (HCAL), which de- universe and will hopefully help explain some unextect particles made up of quarks, the particles that plained phenomena that have been observed. make up protons and neutrons. The outermost part of the detector are the muon chambers, which detect muons, an unstable particle similar to the electron. Because the muon is much more massive than the electron, and is often produced with high energies, it is not stopped by any of the inner detectors . â€ƒ But before any discovery can be made, the detectors must be understood perfectly. Currently the LHC is operating at half its design collision energy and is working its way up to its design intensity, to make sure that all parts of the accelerator and detector are working harmoniously. In past particle physics exLenny Evans is a periments, particles like the pi_0, W and Z bosons senior Physics and have been created and have had their decay energies Math double major. and half-lives measured well. The LHC should see these particles like the other experiments, and if the LHC detectors see the particles with a different de- References 1. CERN public.web.cern.ch. cay energy, this is indicative that an energy shift cor- 2. CMS Collaboration. Phys Rev. rection needs to be included into the calculation of 3. Tabarelli. J. Phys. Conf. Ser. 160 (2009) decay energies. However, the calibration is not this 4. L.T. Evans. CMS Internal Note
Fall 2010, Volume III Issue I
It's All In The Package
A New Way to See DNA Expression
Kristen Rosano, Staff Writer
tudents learn early about the four bases of DNA – adenine, guanine, thymine, and cytosine – that make up who we are. But it is becoming increasingly apparent that gene expression is determined by so much more than the combination of these four bases. One important regulator of genetic expression is how genes are packaged within the cell – that is, how DNA is folded on itself to fit into the nucleus of a cell. This packaging is an issue central to the research being conducted in Dr. Jason Lieb’s lab. As Dr. Lieb explains, “We’re trying to answer a fundamental question about how DNA is read out by cells, and a big part of that involves how the DNA is packaged” . Imagine trying to reduce two meters of DNA into each microscopic cell; this is where DNA packaging comes in. Packaging affects expression through gene accessibility. Those genes that are more hidden within the packaging mechanism have a lower expression and those more accessible have a higher expression. When DNA packaging goes wrong, diseases such as cancer – which can arise when packaging comes undone – may arise. One study found the binding specificities – DNA sequences that transcription factors prefer to bind to – of 112 DNA-binding proteins . This information was de-
termined primarily through the use of DNA microarrays, which contain about 20,000 spots of unique, short DNA sequences. For each of the 112 proteins, the protein is applied to the microarray to see which sequences it binds to the most readily. The study also analyzed the effect mutations in six transcription factors had on nucleosome occupancy and transcription. In each of the transcription factor mutants analyzed, there was generally an increase in nucleosome occupancy in the NFR of the targeted gene, which corresponded to a decrease in transcript levels. The most basic packaging mechanism is the nucleosome. A nucleosome consists of a core of eight histone molecules – proteins whose primary function is to bind to DNA to package it. Around each histone core is a strand of DNA containing 147 base pairs that is wrapped nearly two times, like DNA thread on a protein spool. These nucleosomes are separated by linkers, stretches of DNA that are not wrapped around a histone core. This level of packaging is often called “beads on a string,” in reference to the appearance in electron micrographs. To access the genetic code, proteins separate segments of DNA from the nucleosomes, so that they can be copied – or transcribed – into messenger RNA (mRNA). DNA closer to a linker – and near the end of the 147 base pairs wound around a nucleosome – is more easily accessed. These nucleosomes’ location and frequency in the genome help to determine transcript levels. Areas with very few nucleosomes, called Nucleosome-Free Regions (or NFRs), generally have higher expression of mRNA because transcription factors – proteins that bind to DNA to begin transcription into mRNA – can bind more easily to DNA that is not tightly packaged in nucleosomes. Dr. Lieb’s lab has conducted a number of studies on nucleosomes which showed that: a) an increase in nucleosome occupancy in NFRs leads to a decrease in transcript levels, b) the DNA sequence itself plays a large role in nucleosome positioning and occupancy, and c) evoFigure 1. Three nucleosomes, containing 8 histone proteins each, and lutionary differences in gene expression DNA wound around them. among species is partly due to changes in
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Carolina Scientific nucleosome occupancy. The study also revealed something very interesting about a particular transcription factor: Rsc3. This protein is part of the protein complex RSC that creates NFRs in the promoters of genes, making them more Figure 2. An example of a accessible. Rsc3 transcription factor binding to binding sequences DNA. were 16 times more likely to be found in NFRs than within genes. Furthermore, a mutation in Rsc3 caused an increase in nucleosome occupancy in promoters containing Rsc3 binding sites. Therefore, it appears that Rsc3 is involved in maintaining NFRs, which promote transcription. These data indicate that Rsc3 uses its binding sites to bring the rest of the RSC complex to the promoters to create NFRs. So what affects nucleosome organization? There are multiple known factors, one of which is the sequence of the DNA itself. One of Dr. Lieb’s studies examined the importance of this factor . This study compared genome-wide nucleosome occupancy in both in vitro and in vivo experiments. Because the in vitro experiments used purified octamers and DNA, the nucleosome positions could only be due to DNA sequences, while the in vivo experiments included all other factors, such as transcription factors, that go into nucleosome occupancy in a living cell. The results showed that the in vitro and in vivo experiments were highly correlated, indicating that the DNA sequence plays a large role in determining nucleosome occupancy. The correlation between average nucleosome occupancy per base pair of the two experiments was 0.74. This study also revealed that nucleosome depletion was observed near transcription factor sites, which confirmed the conclusions from the previous study, that nucleosome depletion is related to increased transcript levels. A third study compared the transcript levels and nucleosome occupancy of genes in two yeast species: anaerobic S. cerevisiae and aerobic C. albicans . The goal was to see how nucleosome occupancy and transcript levels of genes involved in respiration compared between these two species because one uses respiration and the other does not. First, the transcript levels of a wide range of genes were measured and the genes were split into three
categories. Category 1 genes are growth genes involved in normal cellular processes, so had high transcript levels in both species. Category 2 genes are those involved in specific states or in stressful conditions, so had low transcript levels in both species. Lastly, category 3 genes are those that had high transcript levels in C. albicans and low transcript levels in S. cerevisiae, indicating that they are involved in respiration. With these categories in mind, nucleosome occupancies were measured for each category of genes both experimentally – with purified chicken histone proteins and DNA from both species – and with a computational model. Category 1 genes had low nucleosome occupancy, accounting for the high transcript levels. Category 2 genes had high nucleosome occupancy, and category 3 genes had low nucleosome occupancy in C. albicans and high occupancy in S. cerevisiae. Ten other species were examined in the same way and the same pattern was found. This pattern leads to a noteworthy conclusion. The relationship between low nucleosome occupancy and high transcript levels is evolutionarily conserved. Because in vitro methods with purified DNA were used, the DNA itself must have encoded nucleosome occupancy, regardless of continuing gene transcription. This allows us to assume that evolutionary changes in DNA led to changes in nucleosome occupancy to provide the most ideal transcript levels for the species. These findings come, interestingly, in the 100th anniversary year of the Nobel prize-winning discovery of the histone protein . Though the research does not relate to a specific disease, as is typically thought of medical research, it is leading to a better general understanding of the cell’s functions, which has far-reaching implications to further research in a wide range of medical fields because of its fundamental nature. “Much of what we know about the mechanisms of human disease is in how the cell works,” Dr. Lieb clarifies. DNA packaging, and more specifically nucleosome organizaKristen Rosano is tion, is another layer of informaa freshman Biology tion on top of the DNA code, that major and Chemistry is critically important for life. minor.
1. Interview with Jason D. Lieb, Ph.D., 9/30/10. 2. G. Badis, et al. Molecular Cell. 2008, 32, 878-887. 3. N. Kaplan, et al. Nature. 2009, 458, 362-366. 4. Y. Field, et al. Nature Genetics. 2009, 41, 438-445.
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UNC Scientists Launch Breast Cancer and the Environment Research Program Yinmeng Yang, Staff Writer
esearch scientists and community members will partner to study pregnancy, obesity and breast cancer disparities. Basal-like breast cancer is an aggressive subtype of breast cancer that is more prevalent among young African American women with no targeted treatment . Basallike breast cancer has unique risk factors. For example, previous research studies have demonstrated that full term pregnancy reduces the lifetime risk of breast cancer, but the opposite is true for basal-like breast cancer. Pregnancy is associated with an increased risk of this subtype of disease . Newly funded research at UNC strives to better understand the unique factors driving development and progression of basal-like breast cancer. Researchers, in collaboration with the National Institute of Environment Health Sciences (NIEHS) and the National Cancer Institute of Breast Cancer (NCI) and the Environment Research Program, will study how relationships between age, race, parity, and obesity impact the risk and progression of basal-like breast cancer . NIEHS and NCI cofounded the Breast Cancer and the Environment Research Program (BCERP) in 2003, a $35 million, seven-year study investigating the environmental exposures that render women susceptible to breast cancer . In a new wave of research grants under this program, the program will support Dr. Melissa Troester (PhD)Assistant Professor of Epidemiology in the UNC Gillings School of Public Health, and Dr. Liza Makowski (PhD), Assistant Professor of Nutrition. The investigators have been awarded a five-year, $2.2 million grant by BCERP to study how pregnancy, inflammation and obesity may promote the development and progression of basal-like breast cancer. Obesity is characterized by a body Melissa Troester, mass index (BMI) of 30 or above. PhD, It is becoming a global epidemic, Assistant with more than 1 billion overweight Professor of adults, posing high risks for chronic Epidemiology illnesses like diabetes and certain forms of cancer . In non-Hispanic whites, almost 25% of the population
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between the ages 20 and 39 are obese . For non-Hispanic black Americans, the prevalence is twice as high. Obesity has complex relationships with cancer risk that also depend on when obesity sets in. Obesity may also increase the risk of only some types of breast cancer. Some studies have suggested that an increase in adipose tissue and BMI significantly increases risk of basal-like breast cancer. This already complex story is further complicated by the fact that other risk factors change in parallel with BMI. As a woman ages, her parity status, breastfeeding history and body size, may change. Disentangling the
Figure 1. Macrophage infiltration is shown in adipose tissue. combination of factors in relation to breast cancer requires combining carefully designed experiments with data from human populations. The study hypothesis originated from the work in the laboratory of Dr. Melissa Troester. Dr. Troester’s lab has studied how the normal tissue around breast cancers communicate with the cancer and alters the way the disease develops over time . Their work, studying how changes around the tumor, affects the aggressiveness of breast cancers led her to question whether some of the changes induced by obesity or parity might affect the development of cancer. The team aims to identify how obesity- and pregnancy-related changes in normal tissue adjacent to the tumor interact with the breast cancer. The lab will do this by studying cell-cell communication between macrophages
Photo courtesy of Dr. Melissa Troester.
Figure 2. Community Advisory Committee Established for Breast Cancer and Environment Research Program met for the first time on October 7 in Chapel Hill. and epithelial cells. Studies have suggested that the progression of basallike breast cancer may be promoted by macrophages and inflammation. Macrophages are essential players of our immune system that defend against pathogens that enter our body. Studies have shown that macrophages are recruited into adipose tissue, where they induce inflammation in the tissue and facilitate the growth of tumors and promote the development of cancer . A high level of tumor-associated macrophages has also been linked with increase in tumor aggressiveness and reduced survival of cancer patients. Understanding how cancer cells communicate with macrophages is important to understanding how breast cancers progress. Based on findings from the cell line models, the team will then determine if the tissue changes that are induced by cell-cell communication are also present in the normal tissue of women. These findings are characterized on obesity, pregnancy history and presence or absence of basallike breast cancer. These studies will lend support to relationships between obesity, pregnancy, and breast cancer. Dr. Troester will also collaborate with obesity researcher Liza Makowski in the Department of Nutrition. Dr. Makowski’s lab, in collaboration with Dr. Charles Perou in Lineberger Cancer Center, will lead studies using novel preclinical models of basal-like breast cancer to evaluate how progression of breast cancer in these models is altered by pregnancy, exposure to high-fat diet, and combinations of the two. These experimental data will lend strong support for the causal role of these factors in this aggressive breast cancer subtype. Dr. Makowski stated, “Using experimental models to examine breast carcinogenesis and comparing these data to human cell line and patient data will allow for the identification of relevant pathways or
mediators that exist across a variety of settings…a powerful indicator of biological relevance”. A unique feature of BCERP is a history of close collaboration with research stakeholder from the breast cancer community. Thus, the scientists will collaborate with the UNC Center for Environmental Health and Susceptibility’s Community and Outreach and Translation Core (COTC) to convene a Community Advisory Committee (CAC). The CAC will facilitate communication between scientists and the breast cancer community in North Carolina. This will better inform the scientists of the needs of susceptible breast cancer population and to communicate the results of the research to the public. This bi-directional communication will begin with interviews with population groups susceptible to breast cancer in regards to their understanding of their risks and knowledge about breast cancer. In response to the interviews, outreach activities will be developed to better inform women and health professionals about breast cancer based on the findings.  The project aims to develop a better sense of the relationships between basal-like breast cancer and women’s health factors after pregnancy. These proposed studies have the potential to identify the underlying mechanisms that relate obesity, pregnancy, and breast cancer to racial health disparities and may provide putative methods for diagnosis, therapeutic targets, and treatment advancements. In addition, the knowledge gained Yinmeng Yang is from the research will hopefully a Biology major translate into productive public and Chemistry and health messages that will help Medical Anthropology inform and educate the public minor. about the risks of breast cancer. References
1. Carey, L.A., et al., Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study. JAMA, 2006. 295(21): p. 2492-502. 2. Millikan, R.C., et al., Epidemiology of basal-like breast cancer. Breast Cancer Res Treat, 2008. 109(1): p. 123-39. 3. Melissa Troester.” Pregnancy, Obesogenic Environments, and Basal-Like Breast Cancer”. June 2009. Proposal for BCERP Grant. 4. Subramanian V, Ferrante AW Jr. “Obesity, m, and Macrophages” 2009. Nestle Nutr Inst Workship Ser Pediatr Program. Vol 63, 151162. 5. National Institute of Environmental Health Sciences. (2009) Concept Clearance for Key Message Development and DisseminationResearch Contract. http://www.niehs.nih.gov/about/orgstructure/boards/ naehsc/docs/dissemination-concept-clearance-may09.pdf 6. Centers for Disease Control and Prevention. (2010) U.S.Obesity Trends. http://www.cdc.gov/obesity/data/trends.html 7. World Health Organization. (2010) Obesity and Overweight. http:// www.who.int/dietphysicalactivity/publications/facts/obesity/en/ 8. Flegal, Katherin M.,et al., Prevalence and Trends in Obesity Among US Adults1999-2008.JAMA, 2010. 303(3):235-241.
Fall 2010, Volume III Issue I
Probing In-Vivo: In-Cell NMR Ricky Singh and Alex Krois, Staff Writers
roteins are a vital component of cells, catalyzing reactions, serving transport roles, maintaining quality control, facilitating intra- and inter-cellular communications, and providing the scaffold of the cell. A proteinâ€™s role is determined both by its location and conformational state inside the cell. Fluorescence techniques have normally been employed to study the localization of proteins; however, they do not provide adequate information about their structure, function, and dynamics . This missing link is provided by Nuclear Magnetic Resonance Spectroscopy (NMR), allowing not only conformational studies but also studies of the dynamics of proteins. Further, NMR is a non-invasive approach which permits protein analysis in their natural environment does not affect their structural and functional integrity. We can understand how NMR works by peeking at the nucleus and observing that it is spinning. This spinning, which is known as resonance, sets up a magnetic moment. When a homogenous external magnetic field is applied, all these magnetic moments align with the external magnetic field. After the pulse of resonance
is eliminated, the spins gradually wobble back to an unaligned state. As the magnetic moments decay away from the applied magnetic field, the change in magnetic field is detected and the signal is processed using the mathematical tool of a Fourier Transform. Regrettably, most NMR work is done in vitro, in buffered solutions, and has limited relevance to the inside of a cell. Incell NMR is a new technique that may overcome this problem. With in-cell NMR, the sample solution is a cell slurry, and the information gained is from proteins within their natural environment. [Figure 1]. The Pielak group specializes in in-cell NMR, which allows for high resolution NMR spectra of proteins in living cells. Their initial work with in-cell NMR demonstrated that proteins were readily visible inside cells using common NMR experiments . Unfortunately, this observation was the result of proteins leaking out of the cell and into the surrounding solution, making the results no different than in vitro experiments . Further work demonstrated that, while some proteins are visible, others are not, depending on the structure of the protein. Most intrinsically disordered proteins --
Figure 1.-2. In-cell NMR experiment .
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Carolina Scientific proteins with no secondary or tertiary structure -- are visible by 15N NMR, while most globular proteins are not . Both can be seen using 19F NMR which
Figure 3. Spectra of a globular protein (left two columns) and of an intrinsically disordered protein (right two columns), left of each section is 19F, right is 15N. Top row is in-cell, middle is supernatent of sample (to check for leaked protein), bottom is the lysate of the cell sample .
involves labeling aromatic amino acids; however, the usefulness of this technique is limited to looking at a very small number of enriched amino-acids [Figure 2]. More recent work from the Pielak Lab has demonstrated that this is because of the crowded environment that is the inside of a cell. NMR detection is reliant on the protein’s ability to tumble -- that is, to be able to be magnetized to a specific spin and then to decay from that specific spin. Within a crowded environment, tumbling is restricted. Intrinsically disordered proteins can exhibit regional tumbling, where specific regions of the proteins have different rotational motion than other regions. This is not the case with globular proteins -the whole structure moves and rotates as one, making rotational motion homogeneous across the protein. It is postulated that this influence of the cellular environment causes the NMR invisibility of globular proteins . A major goal of in-cell NMR is to take experiments that would normally be done in- vitro and move them into a more biologically relevant setting. A perfect example of this is a 1H- exchange experiment. This experiment is used to determine a protein’s stability through exchanging a protein’s amide protons with deuterons (a nucleus with one proton and one neutron). Deuterons are undetectable by NMR, and as deuterons replace hydrogens the signal from the protein will decrease. Measuring this decrease in signal gives information on how freely the exchange occurs, and can be used to quantify stability- the more stable the protein, the slower the exchange. Recent research within the Pielak Lab has used
this method to perform residue-level stability studies of the proteins in conditions using synthetic crowders to attempt to mimic the natural environment of a cell . Normally this experiment cannot be performed on cells, as the test proteins are typically globular and thus invisible by 15N NMR. However, initial experimental results show that by breaking the cells open and then acquiring an NMR spectra after the exchange portion of the experiment, it is possible to perform these experiments with the cell itself being the location of proton exchange. This research steps beyond the limitations of a simple test-tubes and buffered solutions, and it could shed light onto how proteins behave inside living cells. At the heart of NMR lays ability to observe changes in structures of proteins and their interactions with the environment. Changes in the cell environment due to crowding, conformational changes, binding events, or other modifications are observable on an NMR spectrum. In-cell NMR offers the chance to gain this information in an environment with Ricky Singh is a true biological relevance: sophomore Chemistry and Physics double the inside of a cell. major. References 1. Serber Z, Corsini L, Durst F, Dötsch V (2005) In Cell NMR Spectroscopy. Methods in Enzymology 394. 17-41. 2. Bryant J., Lecomte J., Lee A., Young G., Pielak G. (2005) Protein dynamics in living cells. Biochemistry 44. 9275-9279. 3. Bryant, J., Lecomte, J., Lee, A., Young, G., Pielak, G. (2007). Retraction. Biochemistry 46(27). 8206-8206. 4. Li, C., Wang, G., Wang, Y., Allen, R., Lutz, E., Scronce, H., Slade, K., Ruf, R., Mehl, R., Pielak, GJ (2010) Protein 19F NMR in Escherichia coli. Journal of American Chemical Society 132. 321-327. 5. Miklos, A., Li, C., Sharaf, N., Pielak, G (2010) Volume exclusion and soft interaction effects on protein stability under crowded conditions. Biochemistry 49(33). 69846991. 6. Pielak, G., Li, C., Miklos, A., Schlesinger, A., Slade, K., Wang, G., Zigoneanu, I (2009) Protein Nuclear Magnetic Resonance under Physiological Conditions. Biochemistry. 48. 226-234
Fall 2010, Volume III Issue I
Hybridization: A Toad’s Love Story Shannon Steel and Sonia Bhandari, Staff Writers
ybridization is typically an unfavorable from of reproduction for most species. However, Dr. Pfennig of the UNC Biology Department has conducted recent research exploring situations in which the Spadefoot toad subspecies, Spea bombifron (S. bombifron) chooses hybridization over homogenization. The subspecies Spea multiplicata (S. multiplicata) produce tadpoles that develop into frogs at a heightened rate due to the dry conditions of the species’ natural habitat. When Dr. Pfennig put S. bombifron in a tank filled with an ample amount of water, the females traveled toward the recording of the male that belonged to their own subspecies. However, when Dr. Pfennig placed the female S. Bombifron toads in a tank filled with a minimal amount of water, the female toads tended to migrate toward the sound of the S. multiplicata species. The resultant hybrid male offspring would most likely be sterile and the females would probably not produce as many eggs as a homogeneous offspring. However, they would have a better chance of completing metamorphosis during dry conditions.
Dr. Pfennig found a strong correlation between the S. bombifron females’ physical conditions and their mate choices. Females with poor health tended to migrate toward the call of the S. multiplicata male even when water levels were relatively high. When bred in deep water with males of their species, S. bombifron females in poor condition tended to produce offspring that developed at a noticeably slower rate than the offspring of a healthy female S. bombifron. This data verified the idea that hybridization of this toad species is dependant on two identified factors: the physical fitness level of the female Shannon Steel is a and the water availability of freshman Environmental Science major. the environment.
Figure 1. A S. bombifrons male makes a mating call at the edge of a pond in North Eastern Arizona. The call is a rapid trill lasting less than a second .
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ate selection in animals gives rise to the diversity of animal species that exists on our planet. Studying the motivating factors behind this sexual selection and its contribution to evolutionary diversification is the primary research goal in Dr. Karen Pfennig’s evolutionary biology lab. Two particular species of Spadefoot toads found in the southwestern United States, S. multiplicata and S. bombifrons, have exemplified interesting patterns of hybridization that have sparked many researchers’ interest. However, a distinct trade-off exists for these toads between mating with organisms of their own species or of the other species. Mating between species generally creates offspring that are of lower quality than their
Figure 2. Gel Electrophoresis apparatus. pure-species counterparts; therefore, females generally wish to mate within their own species. However, through extensive field and experimental research, scientists have observed that environmental factors also have an effect on mating behaviors. While many times hybridization leads to poor quality offspring, hybridization can sometimes be beneficial for the offspring, allowing the hybrids to outperform the pure species. In Spadefoot toads, S. multiplicata females do not generally prefer hybridizing with other species because hybrid offspring generally have a lower survival and develop more slowly. However, in some instances S. bombifrons females prefer to mate with S. multiplicata males because hybrid offspring develop and undergo metamorphosis faster than pure S bombifrons offspring  Further research on the hybridization patterns between these two species is ongoing. During the Fall of 2009, I worked on a project with Dr. Pfennig’s lab in conjunction with Dr. Corbin Jones’s lab, whose work concerns evolutionary genetics. By utilizing a molecular approach, we sought to more accurately determine the difference between pure species of S. multiplicata and S. bombifrons, and hybrids. In the lab, I worked to optimize primers that could genetically discern these differences. Optimization of the primers was accomplished by using the Polymerase
Figure 3. Spadefoot male waiting in a puddle of water to mate .
Chain Reaction (PCR) process and gel electrophoreses. PCR is used to amplify a single copy of a DNA segment into millions of copies in order to produce enough DNA to be adequately tested . In Gel Electrophoresis, DNA fragments are separated by size as they move through a gel matrix, so that researchers can then identify the DNA fragments of interest along the length of the gel . Initially, the optimum conditions for the primers were to be found by altering the amount of DNA or primer in the master mix, and altering the temperature at which the PCR was run. Later on, the amount of Deoxynucleotide Triphosphate and magnesium chloride used was also altered for the PCR process. The optimum conditions were measured by studying the gels on which the PCR sample was run—making sure the primer dimerization was at a minimum, the bands were clear, and that a discrete band was shown in particular areas along the length of the gel.
Sonia Bhandari is a senior Biology and Chemistry double major, and Hindu-Urdu minor. References 1. Pfennig, K. S. 2007. Facultative Mate Choice Drives Adaptive Hybridization. Science 318: 966-967. 2. “Staining Nucleic Acids.” DNA and RNA Detection. 2005 National Diagnostics. < http://www.liu.edu/CWIS/CWP/library/workshop/citmla.htm>. 3. “Single Nucleotide Polymorphism.” 2009 Genetics Home Reference. <http://ghr.nlm.nih.gov/glossary=singlenucleotidepolym orphism>. 4. Brennan, T. C. and Rorabaugh J. “Plains Spadefoot: Spea bombifrons.” Reptiles and Amphibians of Arizona. < http://www.reptilesofaz.org/Turtle-AmphibsSubpages/h-s-bombifrons.html>. 5. “Toads mate across the species divide.” Nature News. 8 November 2007. <http://www.nature.com/news/2007/071108/full/ news.2007.231.html>.
Fall 2010, Volume III Issue I
How Influential is Your Waistline? A New Study of Obesity and Influenza Immune Response Abby Bouchon, Staff Writer
n Dr. Melinda Beck’s lab at the Gillings School of Public Health, a major study is underway that has uncovered a startling correlation between the influenza virus and obesity. The flu shot has always been recommended by the Center of Disease Control (CDC) for populations at risk for influenza-related complications, such as pregnant women, asthmatics, and young children . Are people classified as morbidly obese about to join that list?
Dr. Melinda Beck, Associate Chair in the Department of Nutrition at the Gillings School of Public Health
Every year, scientists work hard to try and “scientifically guess” what the flu virus will look like for the upcoming winter based on current influenza activity in the Southern Hemisphere and previous flu strains. A vaccine works by introducing a killed microbe into the body that stimulates an immune response. As a result, scientist change the flu shot each year, in hope that the vaccination defends individuals from the predominant strain circulating . Theoretically, the flu shot should induce protective antibodies or immune cells against the influenza virus so when exposed to a secondary live infection, the vaccinated host will be protected. By receiving a primary infection in the form of a flu shot, scientists hope that recipients have the proper immune defense to withstand a secondary infection . When an infection attacks the immune system, it is important to have a robust and speedy immune
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response that kills the infection and protects nearby cells. T cells, which mature in the thymus, are a type of white blood cell that is activated in response to an infection or pathogen attacking the immune system . “In a healthy individual, memory T cells would be produced during the initial influenza infection,” Dr. Beck said. “Those cells help protect the individual from a second infection. The memory T cells target internal proteins common to all strains of the virus. But if the body can’t produce these T cells during a primary infection, then the individual has decreased protection from a second infection if the antibody response is not targeted towards the infecting strain” . However, Dr. Beck noticed a startling trend in obese mice infected with a secondary influenza virus after recovering from a primary influenza infection. She and Dr. Erik Karlsson recently published an article in the Journal of Immunology which showed that after receiving a primary influenza infection, diet-induced obese mice had difficulty developing memory T cells for the influenza virus. When re-infeted with influenza virus, twenty-five percent of the obese mice died from influenza, while lean mice suffered no casualties (Karlsson, Beck: Diet Induced). Dr. Beck hypothesizes that “this increased severity may be due to obesity decreasing memory cell-mediated defenses against heterosubtypic influenza infection.” She believes that the lack of influenza-specific T cells may be correlated to the high mortality rate in obese mice . This study has huge implications for the American population, for which an estimated two out of every three adults are classified as overweight or obese. For the first time, the Center for Disease Control is suggesting that obese individuals may be at a greater risk of mortality from the influenza, and are listed as a population “at a high risk of developing influenza” . Dr. Beck recently received funding from
Figure 1. Getting a flu shot is recommended by the CDC.
the National Institute of Health to begin conducting a large clinical trial with the UNC Department of Family Medicine. The study will focus on adult patients who are interested in getting a flu shot can enroll in the study, which involves taking blood samples before and after receiving the vaccination. The samples are then tested for antibody response and T cell response, which measure the immune system’s response to the primary infection of the vaccine. Dr. Beck thinks that comparing how individuals respond to the influenza vaccine will correlate to their body weight. The study is ongoing and hopes to register a
total of 1000 patients over a two-year time period . So, what is Dr. Beck’s advice for the American population? “Watch your diet, lose weight, exercise and get your flu shot” . Special thanks to Dr. Melinda Beck for her generous time and resources.
Abby Bouchon is a sophomore Pre-Nutrition major.
1. CDC Influenza Homepage, 2010. 2. E. Nester, in Microbiology: A Human Perspective (McGraw Hill, 2009). 3. Interview with Melinda A. Beck, PhD. 9/24/10 4. E.A. Karlsson, M.A. Beck. Journal of Immunology. 2010. 184, 3127-3133. 5. F.A. Murphy, CDC Public Health Image Library
Figure 2. Several Influenza virions .
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Flying High With Drosophila Frank Mu, Staff Writer
he Drosophila melanogaster is a small fruit fly about 3mm long, similar to the ones you see attracted to your bananas and other fruit. Though they may be an annoying pest, they also serve as an important model organism for the studies involving every aspect of eukaryotic biology. Research on Drosophila has helped uncover many basic principles of eukaryotic genetics and continues to provide important insights into understanding similar phenomena in other species. For instance, many genes required for embryonic development in Drosophila have similar corresponding genes, called homologs, in other mammals, including mice and humans (Figure 1). One of the critical genes the Peifer lab studies encodes for the adenomatous polyposis coli (APC) protein. APC plays an important role in the well studied Wnt-signaling pathway [2,3]. For humans, proper regulation of the Wnt-signaling pathway is necessary for the maintenance of intestinal cells ; in Drosophila, disruptions of the Wnt pathway result in wingless flies . Normally in the absence of the
Figure 1. Example of APC homologs among different species .
signaling molecule Wnt, APC interacts with several other proteins to form a protein destruction complex that targets the protein β-catenin for degradation (Figure 2). However, mutations in the APC gene can prevent formation of the destruction complex and degradation of β-catenin. As a result, β-catenin is stabilized in the cytoplasm and eventually moves into the nucleus, where it interacts with transcription factors to abnormally turn on Wnt target genes . In humans, lifelong accumulation of mutations in the APC gene is the largest contributor to non-hereditary or sporadic colon cancers . In addition to helping regulate the Wnt-signaling pathway, APC also participates in a range of other processes that involve interactions with cytoskeletal proteins . Recent evidence indicates Figure 2. The activation of Wnt pathway when Wnt protein binds to membrane that interaction of APC receptor. Mutations can cause the pathway to continuously be active, though Wnt with the cytoskeleton protein is absent . might also contribute to
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Figure 3. The Syncytial stage of (a) wildtype and (b) mutant APC Drosophila embryos (3).
tumor initiation and progression . The loss of APC can lead to changes in cell architecture and ultimately affect cell migration and cell division, which are vital processes for normal maintenance of gut tissue . The Peifer lab examines APC’s cytoskeletal roles by observing the syncytial stage of Drosophila embryos (Figure 3). At this syncytial stage, the Drosophila embryo contains a large number of nuclei that are not separated into individual cells. Division occurs rapidly as four stages of mitosis occur within 50 minutes. In order to maintain genomic integrity and to prevent interactions between spindles and hundreds of adjacent dividing nuclei (red in Figure 4), the plasma membrane forms actin-rich extensions (green in Figure 4), called pseudoclevage furrows, to separate nuclei. These furrows serve as a point of attachment for microtubule spindles. The overall effect of APC on microtubules is an increase in their stability. In APC mutants, the spindle is believed to not correctly anchor to the furrows, resulting in the loss of the nucleus (Figure 5). Each nucleus divides after attaching to the surrounding pseudoclevage furrows . APC2 mutants show obvious defects in syncytial development. In APC2 mutants, nuclei migrate to the cortex normally, but then many nuclei are lost from the cortex into the internal cytoplasm . Since β-catenin is involved in spindle tethering in the syncytial embryo, the role of APC2 in the cytoskeleton has been proposed to Figure 4. Cross-sectional involve APC2 bindview of a wildtype Drosophila ing of β-catenin that embryo in the syncytial stage together help link .
spindle microtubules to pseudoclevage actin [4,5]. However, many questions remain about APC2’s function. The loss of functional APC may only subtly affect individual cytoskeletal processes, but the combined effects within the cell result in detrimental consequences. Understanding how APC loss affects the mitotic spindle should help determine APC’s involvement in cytoskeletal regulation, which may have important implicaFigure 5. Nuclear fallout tions in carcinogenis observed in APC mutant esis . Drosophila embryos .
Frank Mu is a junior Biology and PWAD double-major.
1. I. Näthke. Nature Reviews. 2006, 6, 967-974. 2. R. Fodde. Eur. J. Cancer. 2002, 38, 867–871. 3. B. M. McCartney et al. Nature Cell Bio. 2001, 3, 934-938. 4. D. Pinto et. al. Exp. Cell Res. 2005, 306, 357-363. 5. Y. M. Yamashita et al. Science. 2003, 301, 1547-1550.
Fall 2010, Volume III Issue I
“Men love to wonder, and that is the seed of science.” - Ralph Waldo Emerson
Carolina Scientific Fall 2010 Front Cover: The Large Hadron Collider, Credit: European Organization for Nuclear Research (CERN ©) This publication was funded at least in part by Student Fees which were appropriated 40 Government at UNC-Chapel Hill. Fall 2010, Volume III Issue I and dispersed by the Student