Alpha-Synuclein Aggregation and Membrane ... - Lake Forest College [PDF]

Editor's Corner Year Three: Growing Up Michelle McKinzey '07 Faculty in Focus The Translator Between Two Extremes Shaun Davis '09 Studying Biology at a Liberal Arts Institution: Dr. Lynn Westley's Approach to Undergraduate Science at Lake Forest College Elizabeth Dean '09 Dr. Pliny Smith: New Kid on the Block Lisa Jeziorny '07 The Science of Teaching Tropical Ecology: The Glories of Experiential Learning Benjamin Larsen '07 The Evolution of the Student Michelle McKinzey '07 Beyond the Classroom Clinical Shadowing: A Worthwhile Experience for all Premeds Lokesh Kukreja '08 Series of Formal Talks Launched Mithaq Vahedi '08 Lake Forest Students Present Their Research at Regional and National Symposia Michael White '07 Alumni in Focus Life After Lake Forest College: Where are they now? Michael Zorniak '07 News and Views Bone-derived Microglia Clear Amyloid Plaques Lokesh Kukreja '08 Alpha-Synuclein, and the Case of the Blocked ER-Golgi Pathway Michael White '07

Book/Film/Fine Arts Review Iris and Awakenings: Timeless Tear-Jerkers Mohammed Ejaz Ali '10 Dissecting the Ethical Brain Benjamin Bienia '10 Fact and Fantasy: The Beak of the Finch by Jonathan Weiner Michelle McKinzey '07 Healthy Marketing: The Only Solution Jason Prendergast '09 The Thin Line Between Madness and Sanity Stephanie Valtierra '08 Ferocious Beauty: All Roar and Very Little Bite Pete Wisnieff '10 Review Article History Tend to Repeat: FMR-1 Silencing in Fragile X Syndrome Joshua Haas '08 A Ride with Listeria monocytogenes: A Trojan Horse Joshua Haas '08, Krista Kusinski '08, Shruti Pore '08, Solmaz Shadman '08, Mithaq Vahedi '08 Nanotechnology May Replace Existing Treatments for Cancer Ethan Helm '07 Coal Power: Providing Energy, Asthma, Cardiovascular Disease, and Free Abortions Ethan Helm '07, Benjamin Larsen '07 Guts & Glory H. pylori: Cause of Peptic Ulcer Ashley Johnson '07, Bryan Kratz '07, Lorraine Scanlon '08, Alina Spivak '07 Evolutionary Antibiotic Resistance as Documented in Multiple Strains of Staphylococcus Michelle McKinzey '07 Alpha-Synuclein Misfolding and Aggregation in Parkinson's Disease Michael White '07 Mitochondrial Deficiencies and Oxidative Stress in Parkinson's Disease: A Slippery Slope to Cell Death Michael Zorniak '07 Grant Proposal Characterization of Membrane Permeability Alterations in Plasmodium-infected Erythrocytes: Insight into Novel Mechanisms for Malaria Chemotherapy Chloe Wormser '06

Apical Membrane Antigen 1 (AMA-1): Role in Plasmodium yoelii Infectiviey Michael Zorniak '07 Essay Some Like it Hot: Astrobiology and Extremophile Life Elizabeth Birnbaum '08 Agoutis and Seed Dispersal in Tropical Rainforests Stephanne Levin '09 Senior Thesis Reduced Sexual Attractiveness of Redundant Males in the Maintenance of Guppy Color Polymorphism Katherine Hampton '06 Calcium-stimulated Regulatory Volume Decrease in Salmo salar and Alligator mississipiensis Erythrocytes Chloe Wormser '06 Primary Article Alpha-Synuclein Aggregation and Membrane Association in a Fission Yeast Model: Implications for PD Pathogenesis Lokesh Kukreja '08 Alpha-Synuclein Causes Non-specific Toxicity in vps34∆ Yeast Mithaq Vahedi '08

Editor’s Corner

Eukaryon, Vol. 3, February 2007, Lake Forest College

Year Three: Growing Up Michelle McKinzey Department of Biology Lake Forest College Lake Forest, IL 60045 Dear Readers, The goal of any journal is to put out a product better than the last, making each issue a more difficult job than the one before. When you can actually step back and quantify that by just looking at the amount of interest there has been in the work of your peers, there’s this great feeling like you’ve done something important and worthwhile. And everyone wants to feel like their time is worth something to someone somewhere. The authors in this year’s issue of Eukaryon are very excited to have their work showcased in an asset so valuable to any college. But getting to the publication point is never easy. The volume and quality of submissions this year was higher than previous years and the review board, working with new guidelines and greater scrutiny, did their best to choose what will, hopefully, be viewed as the finest work. Sadly, you can never accept every submission and the hardest part of any reviewer’s job is always rejecting papers. This year, we have gotten better about notifying authors of reception and rejection but we are still not perfect. I hate rejection letters because I feel like the person who has to tell someone their family pet just got run over by a car. It’s just important to remember that the article does have merit or no one would have recommended it for submission in the first place. With around fifty article submissions representing at least six professors and about 55 students, Eukaryon is experiencing exponential growth. Our editorial board has dealt with this beautifully. Thanks to Michael Zorniak., the copy editing board ran smoothly in spite of being a person short. Furthermore, while we were scrambling to make the publication deadline last year, almost all of the formatting was finished in the beginning of January. More than a month before our deadline giving us more time to plan for a bigger and better 2007 inaugural ceremony. Big thanks to Lokesh for that - and to Chelsea for managing it so well. Speaking of the 2007 Ceremony, this year we are so pleased the Professor Anne Houde agreed to be our seminar speaker. Last year, we did not give a seminar before the ceremony and we are expecting a great crowd. So rarely do our mentors share their own work within our academic community and they do such wonderful work. If it weren’t for them and their support we would neither be the students nor the magazine we are today. Professor Houde has always supported Eukaryon in spite of our discrepancies (which we are in the process of fixing) and we are grateful for her patience and guidance. Our publication, though growing quickly, has still only gone from infant to toddler. This year, in addition to new review guidelines and restructuring the ceremony, we announced the creation of three new positions, amendments to the constitution, and print issues. The new positions encompass Business Manager, Records Keeper, and Rolling Editor-inChief. Though all of these positions are important to us, the Rolling Editor is especially important. He/she will serve as the understudy to the Editor-in-Chief and take over when the Editor steps down or is unable to serve. The print issues mark the transformation of Eukaryon from a caterpillar to a butterfly. We are not only a web magazine but we are a real journal that you can find various places throughout the campus. We hope you enjoy! The board will lose half of its members to graduation come May and faces a great challenge next year. Many of those who will be lost are founding members and we are sad to go. While there are some wonderful underclassmen who show immense potential, they are going to have to step up and take control - training new members and keeping everything organized. I have no doubt, however, that they will do a magnificent job with most likely more and better submissions. They are not afraid of hard work and consumptive hours. With them, Eukaryon will continue to grow and be a success. We know that we will never be Science or Nature, but maybe we can be teenagers to their parents one day. Sincerely, Michelle L. McKinzey

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Faculty in Focus

Eukaryon, Vol. 3, February 2007, Lake Forest College

The Translator between Two Extremes Outside of academia, Ann Maine is an active member of the Lake County Board. She is on the committees for Public Works and Transportation, Health and Human Services, and Forest Preserves. In this position, she again takes the role of an intermediary between the sciences and the local community. Using her knowledge of the sciences, she is able to change the scientific language into terms that an average person would understand, while voicing the concerns of the public to the scientists. Over her years at Lake Forest College, she has seen many changes. Faculty members have come and gone and schedules change constantly. One of the greatest improvements in the biology department is that, “Students are better prepared than they were in the past.” She accredits this to the fact that courses are more rigorous and have higher standards set by an excellent faculty. She did express some concern, however, with the methods of scheduling courses. When few people sign up for a course, the course gets dropped, so her schedule is constantly changing. Nonetheless, Professor Maine understands that this is part of her job, so she prepares for it. Year after year, Professor Ann Maine returns to Lake Forest College in anticipation for the academic year. “I get excited in August.” So while many students may be saying their good bye’s to their families and dreading going back to school, Professor Maine can be found sitting in her office planning courses and practicing lectures.

Shaun Davis Department of Biology Lake Forest College Lake Forest, IL 60045

While teaching at Lake Forest College for 14 years, Professor Ann Maine has certainly made a name for herself. Offering courses for both science intensive and non-science students, she has taken the position of an intermediary, teaching the two extremes of an audience. Being able to feel the passion she has for teaching makes any biology course interesting. Her expertise in a constantly changing environment allows her the freedom to teach what she loves. Professor Maine always liked a broad curriculum. For her undergraduate work, she majored in plant genetics and English. From there, she moved on to cancer research before ending up at the University of Rochester in New York to do her postdoctoral research in molecular genetics. During this time, she knew that she wanted to teach at a small, liberal arts school. In 1991, she accepted a part-time position in the biology department at Lake Forest College, and has kept that position since. At the completion of the 2006-2007 academic year, Professor Maine will have taught a total of 15 different courses. She teaches both biology majors in the Independent Research Colloquium course as well as non-science students in numerous other courses. “I end up with each end of the spectrum,” she explains. She gets to work with current research projects, for which she expressed great enthusiasm. Nevertheless, she still enjoys working with non-science major students. “I understand where they come from,” referring to their confusion towards the scientific language. To help these students understand the major biological processes, she teaches the specifics about some things, like bacteria and viruses, but with more of a broad concept, using analogies that people are familiar with. For example, when explaining the methods for cell signaling, she likes to uses the board game Mouse Trap®. This way, students can relate how one event can set off a whole set of chain reactions. No matter whom she teaches, Professor Maine always demands high standards. With her background in English, she is able to help students with their writing skills. “She wanted us to write a lot of papers, but it was in preparation for more advanced biology courses,” said junior Cory Querubin.

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

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Faculty in Focus

Eukaryon, Vol. 3, February 2007, Lake Forest College

Studying Biology at a Liberal Arts Institution: Dr. Lynn Westley’s Approach to Undergraduate Science at Lake Forest College In addition to her roles as a lecturer and internship coordinator, Dr. Westley works as the advisor of a full set of students majoring in Biology. Her connection with advisees and dedication as a professor is evident in her desire to get to her students and to help achieve their goals. “I enjoy talking to my students. And if people are interested in things that I’m interested, I know that I can help them succeed.” “What really makes her great teacher,” says Dr. Houde, “is the fact that she has an amazing feel for what students are understanding from her. She gets into students’ minds.” Outside of teaching, Dr. Westley is known for her work as co-author of a book on the ecological relationships between animals and plants. The lack of published work in the field of plant-animal interactions inspired Dr. Westley to write on this subject matter. “I was taking classes in graduate school,” she says, “and nothing in the classes was relevant to what I was interested. That’s what made me want to write the book.” Dr. Westley is also interested in the topic of allocation to reproduction in plants, and she conducts her research at a farm in central Wisconsin, where she and her family often vacation. Regardless of whether Dr. Westley is lecturing in a classroom, coordinating an internship, or conducting research, she makes evident her emphasis on the importance of experience within the field of Biology.

Elizabeth Dean Department of Biology Lake Forest College Lake Forest, IL 60045

As a graduate of Grinnell College, Dr. Lynn C. Westley, Senior Lecturer of Biology at Lake Forest College, is no stranger to the environment of a liberal arts institution. The wide-ranging fields of study and close-knit atmosphere of the liberal arts education initially drew Dr. Westley to Lake Forest College, where she has worked as a biology lecturer for nearly 15 years. Her recent appointment as Internship Liaison for the Natural Sciences brings with it even greater involvement with the college; it enables her to connect undergraduate science students with opportunities to study outside of the classroom and gain a competitive edge in the areas of research and further education. Dr. Westley’s focus lies mainly in the physiological ecology of plants. Of course, her favorite class to teach is Plant Biology, but she also enjoys teaching Ecology and Evolution “because sophomores are exciting—they’re making important decisions and are at the point at which they’re really learning how to be biologists.” Since the start of her teaching career, Dr. Westley has seen undergraduate science become a significantly more rigorous field of study. “When I started teaching science to undergraduates, it was very unusual for freshman and sophomores to be reading primary research articles,” she says. “Now, introductory-level courses require students to read this type of literature.” In terms of teaching her philosophy, Dr. Westley emphasizes research and experience over textbooks and memorization; she believes that such methods provide progressive, competitive enhancement to the Biology curriculum. Students and faculty alike take note of Dr. Westley’s emphasis on experience-based learning. In fact, Dr. Anne E. Houde, Professor of Biology at Lake Forest College, says, “if you’ve ever been in one of Dr. Westley’s classes, you know that the lectures are not the only thing that is important.”

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

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Faculty in Focus

Eukaryon, Vol. 3, February 2007, Lake Forest College

Dr. Pliny Smith: New Kid on the Block In fact, the Nobel Prize 2006 for biology was given to a scientist for work done with C. elegans and Dr. Smith could only smile. He is proud that the spotlight is on his field of study and model organism. While being a biology professor and scientist takes up most of his time, do not be shocked to see him out on a 30 mile bike ride or roaming around campus with his wife, an immunologist, and two children, Jason, 6, and Gillian, 9. Dr. Smith is eager to find out what Lake Forest College has to offer in the way of extracurriculars, so he can get involved. And because of his late move to campus, don’t be surprised to find his office and billboard undecorated. So, if you have any posters lying around, you know where to donate them.

Lisa Jeziorny Department of Biology Lake Forest College Lake Forest, IL 60045

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Dr. Pliny Smith is the “new kid on the block.” The second floor of the Johnson block that is, taking over an office previously occupied by a part-time faculty member. Dr. Smith relocated to Lake Forest College for the start of the fall 2006 semester coming from Salt Lake City, Utah. There he left two part time positions at Huntsmen Cancer Institute and Westminster College. As an undergraduate student, Dr. Smith attended Grinnell College in Iowa, a school similar to Lake Forest College. It was there he obtained his interest in Biology and decided that becoming an undergraduate professor was his calling. He reports that being able to do his own research, interacting with faculty members from different fields, and getting to know students are among the many perks of being a professor. He notes that it is rewarding to see students leave here with a new ideas and that teaching is the best way for him to continue learning. What is he bringing to the table at Lake Forest College? Dr. Smith, in addition to teaching Organismal and Developmental Biology, hopes to one day build a genetics course that can be offered to biology students. Also, in spring 2007 he is teaching a core seminar entitled The Biology of Aging. During this course, although planning is still underway, Dr. Smith hopes to start with the little concepts, like cellular based aging, and work his way up to entire biological explanations for population aging. This core class for biology majors and minors will incorporate concepts ranging from cell biology to evolution. Dr. Smith, as a professor, reports he enjoys teaching at the college level in hopes of turning “undergraduate students into scholars” by teaching them to think critically and providing them a way to apply what they learn to the big picture. In addition, he hopes to get to know a few select students really well, by employing them in his research lab. While the lab is not yet set up, Dr. Smith is already recruiting interested students to help him with his work on C. elegans and cell-fate. C. elegans have been an important organism to Dr. Smith for years and he reports it is because they are the smallest and best specimens to do genetics research.

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The Science of Teaching

Eukaryon, Vol. 3 February 2007, Lake Forest College

Tropical Ecology: The Glories of Experiential Learning college helps cover more than half the cost of the trip. While the expenses may be difficult for some students to cover, Dr. Gordon has never had a student express financial concerns to him.

Benjamin Larsen Department of Biology Lake Forest College Lake Forest, IL 60045 Spending your days in the lush tropical rainforest while studying exotic birds, plants, and insects is not typically what students expect to do during class. But then, Tropical Ecology is anything but an ordinary class. Dr. Caleb Gordon specializes in conservation biology, entomology, and ornithology, and teaches the course biannually. It includes a ten-day field trip over spring break where students conduct research projects on site.

Overall, the course provides an excellent academic and personal experience. Students are able to learn experientially in an amazing location, as well as have fun bonding with both peers and professors.

In the course’s first year, Dr. Gordon planned to take students to Bolivia. However, just weeks before the trip, violence erupted and the political tensions made the trip too dangerous. Scrambling to find another location, Dr. Gordon consulted a longtime colleague and friend who recommended the “best patch of cloud forest” in Costa Rica. Dr. Gordon contacted Savegre Lodge, located in the desired area, and hastily made arrangements over the phone. Luckily, this turned out to be “the perfect spot” and there is no reason to find a new location; transportation is easy, the Costa Rican government is stable, and Savegre has a host of amenities that cater to student needs. The lodge has a library, laboratory, restaurant, dormitories, and laundry services. Still, this class is no vacation. Tropical Ecology students are required to participate in two research projects, one independent and one class-wide project. Prior to the trip, students spend their time reading literature and preparing an independent research project. Data is collected during the spring break trip, which can be very strenuous. Students should expect long hours in the field and must be reasonably fit. Field experience is crucial to the value of the course, however. Dr. Gordon explains that hands-on experience really makes people learn. Students, including Allison Toal ‘06, spend even more time in the field than is required. She applauds Dr. Gordon for making the experience both fun and rewarding. All students from the course present their independent research projects at the annual Student Symposium at Lake Forest College. Both Lake Forest College and the Biology Department strongly emphasize a lab based and experimental curriculum. Because of this, the

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College

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The Science of Teaching

Eukaryon, Vol. 3 February 2007, Lake Forest College

The Evolution of the Student This is exactly the obstacle that Houde has to tackle with her first-year students. “I’m continually asking myself, at what point are we going into logical details that [the non-science majors] are going to dismiss,” she said. With this in mind, Houde scheduled a diverse set of trips. The second field trip was attended in conjunction with the Medical Mysteries class and a handful of upper level biology majors. A ballet entitled “Ferocious Beauty: Genome” put on by the Liz Lerman Dance Exchange at the Museum of Contemporary Art combined dance with our understanding of human genetics. “They showed concepts of Evolution in ways you wouldn’t think of,” said Campagna, an expected biology major. The final field trip of the semester tied into what scientists still face in evolutionary science today: disbelief. The class attended “Inherit the Wind” written by Jerome Lawrence and Robert Edwin Lee based on the 1925 Scopes Monkey Trial. “I really didn’t want to go at first,” said Scott Divine ’10. “But it ended up being really funny. I really enjoyed it.” Students expressed frustration at scenes where the defense was not allowed to discuss the theory of evolution and the fictional Scopes lawyer had to make his case by disproving the bible. What Scopes faced in 1925 is still being faced today. Houde’s aim is to provide information showing that Darwin’s ideas are more than just a theory through science and thought. This includes principles, simulations, and hard evidence like fossils and finches. “I thought it would be more theoretical,” said Clements. “But evolution isn’t a theory anymore, so, I guess that’s okay.” Over the semester, the students became more and more convinced that evolution is not a theory. One student used the idea of antibiotic resistance to convince the elder lady next to her on a plane that evolution is not just some quack idea. The science students agree that this class definitely helps them understand their other classes better, but it is also helpful to the non-science students. “It’s a good way to grasp concepts,” said Clements. “I like the logic and that’s a good way to think in college.”

Michelle McKinzey Department of Biology Lake Forest College Lake Forest, Illinois 60045

One the first day of class, 15 bright-eyed first-year students wonder what is in store for them. The blonde, curly-haired woman with the button nose at the front of the room introduces herself as Anne Houde, professor of biology at Lake Forest College, and welcomes them to First-Year Studies 114: Origin, Adaptation, and Evolution of Species. “The driving principle is, as a first-year studies class, to get students to think logically,” said Houde, professor of biology at Lake Forest College for 13 years. The First-Year Studies (FIYS) program at Lake Forest College is designed to help incoming students adjust to college classes and life. Its aim is to promote critical thinking and expose students to resources. Courses are offered in all disciplines. In the biology department alone, there is Evolution, Medical Mysteries and, previously, Biology of Sex and Gender. “I think [Evolution] does a good job of combining science and the implications of science,” said Houde, who is also an editor for Behavioral Ecology and reviewer for Nature magazine. The goal of FIYS 114 is to familiarize all students with the logic of Evolution or, what Darwin called, descent with modification. This idea states that all life forms on Earth are related to one another through a common ancestor. It isn’t just for biology students. One student in the class, from Kansas, could not be taught Evolution in High School. The class includes a number of English, philosophy, art, and politics majors among others. “It mixes up my schedule for sure,” said Kendall Clements ’10, an expected Spanish major. The goal of any FIYS course is to work on writing and teach students how to do their own learning as well as let them experience Chicago. FIYS 114 did the latter over trips to various events in the area. The first of these was a viewing of the Evolving Planet exhibit at the Field Museum of Natural History. Joe Campagna ’10, said that the exhibit “broke evolution down” and made it understandable through visual displays.

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

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Beyond the Classroom

Eukaryon, Vol. 3, February 2007, Lake Forest College

Series of Formal Talks Launched our bodies respond to a plethora of “stressors” like temperature and lack of nutrients, using special proteins called receptors. These receptors induce different protective responses for the varied stressful stimuli our cells experience. They can initiate mechanisms enabling the cell to survive or mechanisms to commit suicide through a systematic process known as programmed cell-death, or apoptosis. On average, our cells contain 1013 proteins! Many of these proteins functions in multiple pathways. Different proteins are also assembled into protein machines, which help carry out cellular processes. Dr. Morimoto explained that in order to make so many proteins so rapidly and with diverse functions, the cell has protein quality control machinery which makes sure that proteins are folded correctly, have the right shape, and are functioning well. Those that do not meet these requirements are degraded. When proteins are synthesized in our cells, about 10% have missense mutations, which occurs when a protein building block is misplaced in the sequence of building blocks. Thus, the most important process in the cell is error prone. Proteins with missense mutations, as a result, fold differently and may have different functions. Lightening up the mood in the auditorium, Dr. Morimoto compared our cells to a Ford motor plant, rather than a Toyota motor plant! Proteins somehow know how to fold by themselves and we are yet to discover how and why this process happens. Misfolded proteins cause “proteotoxic stress” due to their altered shape and function. These misfolded proteins, if not degraded by the cell, can be toxic, which is the major hypothesis for the cause of some neurodegenerative diseases, like Huntington’s, Alzheimer’s and Parkinson’s. To prevent misfolding from occurring, cells have a special class of proteins called chaperones, which help with correct folding. Explaining how proteins can change shape when thermally heated, Dr. Morimoto used an example of making eggs where heating up the protein (or eggs), changes its shape, form, and function. We certainly don’t want that happening in our brains! Thankfully, heat shock factors, or HSFs, help cells regulate protein’s shape in cases of stress. As we age, these HSFs do not function as efficiently, thus increasing “proteotoxic stress” in our cells and subsequent toxicity. Dr. Morimoto uses C. elegans to study HSFs. He explained that this nematode is a good model system to study diseases as each organism has only 959 somatic (non-gonadal) cells and 302 nerve cells. Further, the worms are transparent and the fate of each cell in the worm has been determined. C. elegans have about 160 chaperones. He showed data from his lab, where knocking out a heat shock factor, HSF-1 led to the formation of many aggregates in the worm nervous system. Overexpressing lots of HSF in the worms gave them a longer life span! This presentation tied in concepts from a number of biology courses, including Organismal Biology, Diseases around the Globe and Cell and Molecular Biology. The importance of protein shape and its relationship to the proteins’ function was a review for students of Organismal Biology. Students who had taken Diseases around the Globe had a clear idea of the diseases Dr. Morimoto mentioned, while Cell and Molecular Biology students could easily recall protein formation in the cell!

Mithaq Vahedi Department of Biology Lake Forest College Lake Forest, IL 60045 A series of exciting formal talks were held in the fall of 2006 on topics ranging from neurodegeneration to intelligent design. These presentations, held for the first time on such a regular basis throughout the semester, were launched by Eukaryon, βββ, the Biology Department, and the Center for Chicago Programs. Students were exposed to the latest research of distinguished scientists in fields such as neurodegeneration and psychology. Many students majoring in biology, psychology and chemistry attended these talks. The presentations also attracted students from the social sciences and humanities. The interactive nature of the presentations added to the rich liberal arts education at Lake Forest College and emphasized the importance of out-ofclassroom learning experiences. These presentations highlighted the mission statement of the Biology Department to help “students embark on hypothesisdriven journeys of discovery where answers are found not in textbooks, but in the lab and the field”. All of the speakers spoke in simple language, and welcomed questions from the audience, which also included professors and on occasion members of the public. A brief summary of the six talks is given below: Stress and Aging in Neurodegenerative Disease Dr. Richard Morimoto, Bill and Gayle Cook Professor of Biology, Northwestern University

Have you ever wondered if stress affects the 30 trillion cells of your body? What happens when you are stressed? Can stress increase your chances of getting neurodegenerative diseases, like Alzheimer’s disease or Parkinson’s disease? Dr. Richard Morimoto, professor of biochemistry and molecular and cell biology at Northwestern University, addressed a packed auditorium of students in the first of a series of six talks. Specifically, the particular kind of stress being spoken of was physiological stress, which includes a number of factors like temperature, viruses, genetic factors, and heavy metals to mention a few. Primarily, stress affects a diverse class of molecules called proteins, whose function depends on their natural shape or conformation. At the molecular level, cells in

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Junior Sina Vahedi, thought that the presentation was very good but he would have wanted to see more data and details about the experiments. However, Sina understood that the talk’s lack of experimental detail made it more accessible to the many non-science majors in the audience. Dr. Morimoto’s presentation was simple and easy to understand. It explained the connection between stress, protein misfolding, and neurodegenerative diseases. Dr. Morimoto’s sense of humor, calm disposition, and the tone and pace of his voice made this presentation both educational and enjoyable.

Increasing the amount of proteins in the cell yields a greater chance of getting prions. However, the fibers formed by these prions need to be broken in order for it to be given to the daughter cell. Dr. Liebman explained that a nonsense mutation is one where there is an extra stop codon in the DNA sequence. In her laboratory, when a mutation was made in the sup35 gene, the protein was still made, despite the stop mutation! Dr. Liebman’s lab also discovered that a chaperone protein that dissolved protein aggregates was required to propagate the prion. The chaperone breaks the fiber and thus helps in propagation of the protein. Inhibition of the chaperone protein by hydrochloric acid leads to decreased prion propagation. This presentation touched on many topics covered in Cell and Molecular Biology, as well as those explored in Ecology and Evolution. Why does our protein synthesis and degradation machinery differ only slightly from that of yeast? Evolutionarily speaking, how similar are we to yeast? Dr. Liebman spoke in simple language and explained cell biology terms throughout her talk. She frequently asked the audience questions. This helped almost everyone to understand the talk, and it also made the presentation an unforgettable learning experience.

Yeast as Small “Mad Cows” Demonstrate ProteinBased Inheritance Dr. Susan Liebman, Distinguished University Professor, University of Illinois-Chicago

How our Hands Help us Think Dr. Susan Goldin-Meadow, Ruml Distinguished Service Professor, University of Chicago Did you know that we have a dogma in biology? Yes, the central dogma of molecular biology says that heredity is controlled by DNA, which spells out protein formation. Dr. Liebman explained that in Mad Cow disease, a pathogen, a prion (PrP), lacks nucleic acids, yet can change a proteins original formation. There are many cousins of PrP diseases, like Creutzfeldt-Jakob disease, kuru, fatal familial insomnia, scrapie of sheep, mad cow disease of cattle, and chronic wasting syndrome of deer, all of which are known as transmissible spongiform encephalopathy’s. Dr Liebman’s talk was as exciting as it was easy to follow. She explained that proteins can exist in a normal or prion shape. Prions are infectious (selfperpetuating) proteins which form fibers that can be seen under the microscope. Comparing the DNA paradigm to the prion paradigm, Dr. Liebman pointed out that in the case of a DNA mutation, a protein can lose function or gain new function. However, in the case of prions, a normal protein can change shape and induce other molecules of that same protein to change shape as well. There can also be mutations which predispose proteins to change shape and act like a prion. Different strains of PrP cause different disease pathologies in inbred animals. These prion strain differences appear to be due to different heritable prion conformations. Showing data from her lab, Dr. Liebman pointed out that prion proteins in yeast are infectious. So why use yeast? Well, yeast contain proteins that are highly conserved. In addition, many cellular processes like DNA synthesis and repair, cellcycle progression, protein synthesis and processing, and protein transport are also highly conserved. Yeast grow by mitotic budding and propagate proteins that are in the prion shape.

Dr. Susan Goldin-Meadow presented her talk amid the excitement of the campus-wide Brain Awareness week at Lake Forest College. Her presentation was at the peak of this outreach campaign organized by the first-year studies Medical Mysteries class and Molecular Neuroscience students. She shared exciting data from her research, which studies the process of mismatch learning in children. It was discovered that gestures change when children or learners are “in transition.” Therefore gestures are associated with learning. Dr. GoldinMeadow presented data to show that a gesture is not only a reflection of human thought, but also a mechanism of learning. Using data she collected, Dr. Goldin-Meadow explained that in a child with gesturespeech match, the speech of the child about moving and the gestures show the actual movement that happened. However, in a gesture-speech mismatch, the gesture of the child describing movement does not correspond to the actual movement. Interestingly, children with gesture-speech mismatch are more likely to learn after training than children with gesture-speech match.

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Dr Goldin-Meadow found that while teaching, one strategy in speech is a lot better than two. She also discovered that gestures are powerful in their ability to shape the way we think! If the children learned only the gesture, they tended to learn much better than those who repeated only the speech. In another experiment, children were told to gesture everytime they were trying to solve a problem. Interestingly, the number of new strategies was much greater in those told to gesture. Further, she found that children who are told to gesture during a lesson remember what they learn. Also, children remember more when they gesture, in addition to coming up with new strategies to solve a problem. She pointed out that making gestures encourages experimentation and adding more ideas. Dr. Goldin-Meadow mentioned that gesturing lightens the cognitive load in the same way that writing down a problem on paper does. Another benefit of gestures is that they provide a second representational formation. Further, notions in gesture can go unchallenged. This talk attracted a great number of questions from the audience. One student requested that Dr. Goldin-Meadow replay tapes of classroom experiments involving children being taught by gesturing and non-gesturing teachers.

to a new species. Last, the only force causing evolutionary change is natural selection. Dr. Coyne went on to present data supporting the theory of evolution. He mentioned that the Archaeopteryx which has a pelvis bone, indicating that it evolved from dinosaurs. In embryology, scientists can see that dolphins develop hindlimb buds, which then regress. Further, humans develop a Lanugo (a coat of hair), which we shed. Dr. Coyne pointed that vestigial organs serve as “the senseless signs of evolutionary history,” for example the kiwi is a flightless bird. Dr. Coyne also cited the development of antibiotic resistance in bacteria as evidence for natural selection. Intelligent Design, or ID, claims that an “intelligent agent” designed some of the features of modern organisms. ID states that some features are “irreducibly complex” and could not have evolved in a stepwise fashion. They include such features as the eye, the blood clotting system, the immune response pathway and the bacterial flagellum. However, due to new fossil evidence the vertebrate jaw can now be explained. The problem with ID is that if we can’t think of a way a feature evolved, then the intelligent designer is credited with its creation. Another problem is that nothing is known or can be known about the designer’s goals and methods. Thus, claims by ID are not testable. Dr. Coyne was very careful not to downplay the important role of religion in society. He said that Bible must not be taken literally and that we can reconcile our beliefs with scientific evidence. Like a true scientist, Dr. Coyne was very comfortable with discussing evidence that would falsify or refute the theory of evolution. He mentioned that a fossil in the wrong place would be one. For example, a human fossil that is older than 10 million years old!

Feeding and Gloating for More: Intelligent Design Vs Evolution Dr. Jerry Coyne, Professor of Ecology and Evolution, University of Chicago

Alzheimer’s Disease: A Tangled Problem Dr. Lester Binder, Abbott Professor of Biology, Northwestern Feinberg School of Medicine

Do you accept evolution as a scientific theory well supported by evidence or not? Well, only 1 in 5 Americans believes in evolution. And only 12 percent of Americans think that evolution should be taught in schools. Dr. Coyne pointed out that the theory of evolution should be compared to the atomic theory of matter, which is accepted by almost 100 percent of Americans. This is because, like any other scientific theories, it makes sense of wide-ranging data that were previously unexplained, makes testable predictions and is vulnerable to falsification. However, no evidence has yet been found to falsify the theory of evolution. Dr. Coyne’s talk was reminiscent of the college’s Ecology and Evolution class! He explained that there are four parts to the theory of evolution. First, evolution occurred; that is, living species descended from a common ancestor. Second, there were very gradual changes in each descending generation . Third, speciation occurred; that is, a single ancestor gives rise

Dr. Binder’s talk on Alzheimer’s disease (AD) was the opening talk of an exciting one-day workshop on neuroscience. Dr. Binder, who studied the control elements of tau tangles found in AD patients, enlightened the audience about the culprit thought to cause the disease. Tau protein binds microtubules and stabilizes them. Tau also aggregates to form filaments that compose the neurofibrillary tangles found in brains of AD victims. Phosphorylation of this protein controls its binding to microtubules. Phosphorylated tau leads to dynamic instability which allows for plastic changes to the cell’s architecture. Hyperphosphorylation is a hallmark of AD. In addition to the tangles, plaques

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(amyloid) are also seen. The axons and dendrites of the neurons are filled with tau tangles. The density of these tangles correlates with the degree of dementia in the AD patient. Tau mutations also cause certain forms of familiar frontotemporal dementias (FTDs). In an experiment involving neurons, neurodegeneration is absent when tau is absent. Tau is known to come off the microtubules. What is not known is whether the disassociation or the aggregation of tau is the problem. Thus, the role of tangles and other tau aggregates in AD is still unknown. In his laboratory, Dr. Binder designs and conducts experiment using antibodies which recognize tau conformations, modifications, and truncations. It was found that one conformation of tau, ALZ50, was a polymer. When tau is cleaved, the rate of assembly of aggregates is increased. However, if the tail peptide is added back, the rate of assembly is inhibited! Studies in Dr. Binder’s laboratory indicated that making a tangle is protective to the cell. Other interesting data from Dr. Binder’s laboratory indicated that the N terminus of tau facilitates the assembly of full-length tau. And the deletion of a region in the N terminus of the protein decreases the rate of assembly. Findings from Dr. Binder’s laboratory have made valuable contributions to AD research and provided many targets for potential therapy. Dr. Binder’s enlightening presentation was a synthesis of concepts students had come across in Cell and Molecular Biology. Students of Molecular Neuroscience were able to appreciate Dr. Binder’s research on AD to a greater extent than the others.

pattern of drug abuse characterized by overwhelming involvement with the use of the drug (compulsive use), the securing of its supply, and high tendency to relapse after withdrawal. This pattern is thought to be “learned.” In her laboratory, Dr. Napier used rats and mice to study addiction. A drug was put at a certain place so that the rats learned to associate environmental cues with the drug. Drugs were given in repeated, intermittent doses to induce addiction. This led to the progressive enhancement of motor activity. The animals were observed visiting this location even in the absence of the drug. Dr. Napier’s laboratory also carried out research using amphetamines. Amphetamines have common mechanisms in action. These bind to receptors and are taken up, and subsequently displace the transmitters. Thus there is a great increase in transmitters. In other words, the brain is beefed up in a very big way! It was found that rats could be weaned off methamphetamine addiction by administration of the drug mirtazapine! Dr. Napier’s research and her promising results with mirtazapine generated many intelligent questions from the audience, who still seemed addicted to neuroscience after a whole day workshop! Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College.

Neuroscience in Search for a cure for drug addiction Dr. T. Celeste Napier, Professor of Pharmacology, Loyola Stritch School of Medicine

Dr. T. Celeste Napier presented the last talk in the fall series of formal seminars and the closing talk of the day for the Neurofrontiers workshop. Her presentation elicited many questions from students who thoroughly enjoyed her talk. Dr. Napier mentioned that an astonishing 9 percent of the population, or an estimated 21.6 million people aged 12 or older, can be classified with dependence or abuse on psychoactive substances (alcohol or illicit addictive drugs). Recently, there has been a large increase in ER visits for methamphetamine related cases. Methamphetamine, which is a most potent psychostimulant, is also called meth, crystal, and crank. Dr. Napier clarified that addiction refers to the pattern of self-administration. Addiction is a behavioral

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Beyond the Classroom

Eukaryon, Vol. 3 February 2007, Lake Forest College

Clinical Shadowing: A Worthwhile Experience for all Premeds “Sensing the Environment,” in which the curriculum featured a three-week section on the visual pathway. It turned out that topics like rods and cones, myopia, refraction, and modern surgeries, like LASIK, were both exciting and engaging to me.” “I am currently interning for two of the best pediatric optometrists in the Chicago land area, Dr. Mary Lou French, and her partner, Dr. Amy HansenKwilose. Patients come from all over the United States to see them, and they have been influential mentors, thus far. After spending three months working for these two doctors, I am able to say, with confidence, that this profession is perfect for me. Everyday is different. My main responsibility is using the auto-refractor to checkin new glasses, working hand-in-hand with the dispensing department. During any free time, Dr. Hansen teaches me how to view vessels and the optic nerve. I am also in the training process as a pre-tester and optician. Additionally, this internship has shed some light on pediatric optometry as a potential area of focus.”

Lokesh Kukreja Department of Biology Lake Forest College Lake Forest, Illinois 60045

Introduction

Lokesh Kukreja ‘08

Besides good grades in classes and standardized exam scores, medical schools look for students who are deeply interested in medicine. In particular, these schools are interested in students who have shadowed physicians. Shadowing is an experience in which premed students get to observe patient-physician interactions in a clinical setting. This experience should not be taken flippantly. During this time, students evaluate themselves and their desires to become doctors. If shadowing cultivates and excites their passion for medicine, the experience will definitely encourage them to pursue medicine. Shadowing is an indispensable activity for pre-med students. There are many experiences that students can lose if they dismiss the opportunity to jobshadow a physician. Medical careers, unlike other careers, involve the direct contact with sick patients. Life as a doctor is difficult to imagine unless students have clinicians in the family. This is why shadowing is essential; students gain a vicarious experience of the day-to-day activities of doctors. Careers in medicine are widespread. Sometimes, shadowing helps narrow student’s interest and also validates their pursuit of a medical career. But, what makes one initially pursue a career in medicine? It may be a clinical visit as a patient or sitting in a classroom learning biology. These are few examples of situations that inspire students to strive for a medical degree. Here is a compilation of experiences that students at Lake Forest College had while shadowing doctors. These students’ opinions are focused on how their shadowing experiences influenced their decisions of going into medical profession.

I am interested in going to medical school because I would like to have a direct major impact on people’s health. During my shadowing experiences, I wanted to do shadowing where I can see the works of many medical professionals. This is an opportunity that is hard to find. I spent a summer between my sophomore and junior year, following an anesthesiologist in the operating rooms at Rush North Shore Medical Center in Skokie, IL. Also, for a short period of time, I shadowed a gynecological surgeon at Highland Park Hospital in Highland Park, IL. So, I have been in many different operating rooms in the two hospitals. Additionally, during my shadowing experiences, I have been fortunate to be able to see the most new and effective surgeries by leading doctors. I saw my shadowing experiences to be a window of opportunity to learn more about various kinds of medical professions in person. I was always overt about my feelings of likes and dislikes on things I observed in the hospitals. I observed many surgeries: repairing abdominal aortic aneurysms endovascularly, implanting a pacemaker, performing a quadruple bypass by open-heart, laparscopic removing of a cancerous kidney, laparscopic repair of an inguinal hernia, and orthopedic surgeries of repairing rotator cuff due to a shoulder RC tear and replacement of hips and knees. When I was following the gynecologist, I observed the doctor deliver a baby by C-section. The next time I saw the same doctor, he removed unusual fibroid formations in a woman’s uterus. There are many virtues in shadowing. First of all, I really found out that I am interested in medicine, and along with my scientific research interests, I think I want to become a clinical researcher. Second of all, I have made important, close links, with doctors. This will allow me to have expert guidance in the future. Third of all, the shadowing experience has been an enriching adventure. Through talking to doctors, nurses and medical residents in the hospitals, I have learned the qualities of hard work, responsibilities, and a sense of humor, all of which are needed to become successful in a medical profession.

Lisa Jeziorny ‘07 Lisa wants to become an optometrist. Her interests generated by listening to biology class lectures about vision, but when she shadowed an optometrist, her passion for eye care became stronger. “During the spring semester of my first year at Lake Forest College, I became interested in a career in vision. I enrolled in a biology course, entitled

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Karina Nikogosian ‘07

compassionate, and collaborative relationship between doctors and patients. Especially in pediatrics, the doctor must attain the utmost trust of a child, so, the child allows the doctor to perform a physical and touch the child, in general. I simply followed the doctors as they saw their patients. The majority of the cases were monthly visits by children to get their immunizations, physicals, and other general examinations. However, there were also a lot of sick visits by both toddlers and older children. I heard doctor's recommendations on ear infections, flues, allergic reactions and other types of infections. Since I got to shadow all five doctors several times, I got an outstanding opportunity to see how different doctors talk, treat, and even examine their patients. I began to pick up on things that I liked how a doctor does, or how I would do something a little bit differently. One doctor told me, "The average time it takes for a doctor to interrupt a patient in explaining his/her symptoms is eight seconds". This doctor was tremendously patient and gave his patients as much time as necessary to explain what he/she felt as well as made sure that his diagnosis/treatment made perfect sense to the patient and the patient's parents. It was great seeing children several times—I got to see how they have grown and the way doctor's techniques change as the child ages. I also learned a little bit about the diet and proper care of children at different age groups. A tremendously valuable experience all in all. I definitely confirmed my desire to, not only become a primary care doctor, but a pediatrician.”

Karina also wants to become an optometrist. A summer of shadowing in an eye care clinic landed her a job in the same clinic when next summer came around. The shadowing experiences confirmed her interest in the field of optometry. Though, she emphasizes that during her shadowing experiences, she has made valuable connections with optometrists, these doctors, Karina believes, can help advise her so she becomes successful in this field of medicine. She explains, “two summers ago, I called multiple optometry offices in the area near my house. A doctor from one office, Johnson Eye Care, called me back and I asked if she needed a volunteer. I told her that I did not mind running errands or doing chores around the office, as long as I received exposure to the profession. One day a week for 5 hours, I sat in on eye exams and kept a notebook for questions. After the eye exams, I asked Dr. Schoepke about her choice of treatment for the patient and other eye health related issues. I also answered the phones, took out trash, and dropped off mail. I was always friendly and helpful to the patients, which is why I think Dr. Schoepke offered me a job at her office. This past summer I worked at Johnson Eye Care and got a complete exposure to what the profession in optometry entails. My experiences working at Johnson Eye Care helped me refine my career goals. Shadowing or working in the filed of interest is the best way to learn whether the profession is right for the person. Working at Johnson Eye Care made me realize that optometry best fits my personality and now I have a mentor, Dr. Schoepke, to give me advice about the application process and further information about a career in optometry.”

Chloe Wormser ‘06 Chloe wants to become a veterinarian. After graduating from Lake Forest College, she wanted to gain in-depth experience in the veterinary field. One of the most exciting things that a student like Chloe acquired during her shadowing is lots of hands-on experience. During many shadowing experiences, students solely observe. There are liability issues that prevent pre-med students to have hands-on experiences. However, the opportunities to do hands-on activity will be valued by students. This activity reflects on how students came closer to experiencing physician-patient interactions. Chloe explains, “I am interning at a small animal veterinary clinic. I work with two small animal veterinarians. The experience has been very beneficial. I have gotten a lot of hands-on experience working with animals; I help during appointments by holding animals for the veterinarians during examinations as well as assist during dental cleanings of cats and dogs. In addition, I have learned to use the laboratory equipment essential for monitoring patients and assessing animal health. For example, I have learned how to run blood analyses and how to monitor animals while they are under anesthesia using the ECG, pulse/ox, and blood pressure readings. I am planning to be a small animal veterinarian and will be entering vet school next year. Therefore, getting a head start and actually working alongside veterinarians is very valuable. Not only have I become much more comfortable working with animals, but I have really learned a lot about how to communicate with owners.”

Shruti Pore ‘08 Shruti wants to be a dentist. Critically, her shadowing experiences helped her explore and understand the depth and breadth of knowledge in the field of dentistry. “I am interested in dentistry. I have shadowed a periodontist, an orthodontist, as well as a general dentist. I spent the summer between my sophomore and junior year shadowing. I thought that it would be helpful in making up my mind. This is why I decided to shadow some dentists. The people that I shadowed were all very nice. By shadowing them I was able to understand 'a day in the life of a dentist.' Also, shadowing different kinds of dentists helped me see how, within a single profession, there are many facets to explore. I wanted to become a dentist before I ever started shadowing. Shadowing confirmed my belief that I will be happy in this particular profession.” Alina Spivak ‘07 Alina is interested in both osteopathic (DO.) and allopathic (M.D.) medicine. It is worth noting that prior to shadowing, she already developed an interest in specific area of medicine in primary care of pediatrics. Shadowing helped her cultivate her interests in the field. Alina explains, “since I am interested in pediatric medicine, I shadowed an office of five pediatric physicians who were all also hospitalists (i.e. making rounds in the hospital aside from working in private practice). I shadowed for one to three hours once a week for six months. This experience showed me the importance of forming a sincere,

Michael Zorniak ‘07 How do students know that medicine is a right career for them? When Michael shadowed a physician, he wanted to use the experience to evaluate his future career goals. He saw that more than half of the

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shadowing experience is not only the observation of a physician and patient interaction, but rather an introspection that shapes your curiosity for medicine. Michael explains, “I feel that shadowing a physician was time well spent because I was given the opportunity to objectively determine whether or not being a doctor fits my personality. This objectivity did not come with the experience, but it is something I needed to bring with me. Before contacting a physician, one must assess their own personality characteristics, traits, and values. This can be done by writing a list. Thus, when one finally contacts a physician, one can objectively evaluate his/her personality fit with the profession. Then one can ask themself, ‘Can I be a doctor?’ It is important to shadow several doctors because one does not want to limit their perspective on the field of medicine and cut themselves short. I have shadowed three different types of physicians (i.e. ophthalmologist, internal medicine clinician, and a family doctor). I have found that the scope of medicine is very different and ophthalmology best suites my interests and personality.” “Shadowing an ophthalomologist was an effective way for me to objectively evaluate my desire to become a physician. Getting personal exposure to medicine gave me an understanding of a clinician's role on a health care team. This opportunity also gave me a chance to place my feet in the shoes of a doctor. During my experience, I was allowed to record patient histories, administer basic ophthalmic examinations,

and research the pathology of different diseases of the eye. Shadowing a doctor has helped me determine the overlap of my interests with that of a physician's.” “I have personally been treated by an ophthalmologist who took immediate action to functionally prevent the decline of my vision. Dr. Brian Proctor, the ophthalmologist I have shadowed for over 6 months, immediately performed a procedure to repair my condition and prevent the development of scar tissue around the affected area. I would like to practice medicine for the chance to aid individuals in the manner that Dr. Proctor has aided me and many others. In my life, I have had a taste of this and I am resolute in gaining additional training. “ Conclusion If students are considering careers in the medical profession, shadowing is essential. The experiences will make students think more seriously and attest to their motivation for taking a path toward medicine. After reading this compilation of shadowing experiences from students at Lake Forest College, consider the great benefits of shadowing if you’re interested in pursuing medicine. Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College.

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Beyond the Classroom

Eukaryon, Vol. 3 February 2007, Lake Forest College

Life after Lake Forest College: Where are they now? coordinator for the Health Literacy and Learning Program. She and her group have published several articles on health literacy and presented their findings in conferences from Chicago to Basle, Switzerland. Although Silvia has pursued various interests, she reverts back to an experience she had in college where she mentored freshman biology students, "I really enjoy working with people in the teaching environment, and whatever career I choose, it will have to involve some kind of mentor-student relationship." She misses being a student at Lake Forest where, "the only thing I had to worry about was studying for exams." No one could have predicted the experiences Silvia has had after graduation. Despite this uncertainty, she is still resolute in pursuing a doctoral degree, yet, she maintains that, "nothing is set in stone." Allison has traveled a similarly unpredictable route. While writing a senior thesis in Dr. Kirk’s lab, she applied to graduate programs in environmental engineering and was accepted to Stanford University. By pursuing a research project in college, Allison claims she was able to, "gain admission to most of the programs I applied to, even though I wanted to switch fields." She also emphasizes that a research experience is critical for any undergraduate. She then switched gears, again, and received a master’s degree in economics from the University of California, Santa Barbara. Although her environmental engineering and economics degree don’t seem to mix, she has consolidated this education and finally pursued a doctorate in environmental science and management at UCSB. She expects to graduate in 2007. Allison believes that the "secret to life is to find the job you like" and that "sometimes it is worth taking the risks to change directions." With a plethora of academic experiences, Allison is finally "at peace" with her decision to pursue a career in environmental economics. After her unique experiences, she has gained a wealth of advice for undergraduates. She states that at a large research university "you see how the envelope of knowledge is being pushed." Yet, she cautions that "the graduate school environment is not as nurturing as undergraduate programs. There are a lot more hoops to jump through and there are a lot of competitions. And often times, you really need to expect more from yourself and less from others." Silvia and Allison have showed us how their lives have been guided simply by their interests. Both Silvia and Allison have emphasized the need to polish writing and speaking skills in college. Silvia further elaborated that these skills are the, "most necessary [abilities] in any path of life you choose." Lake Forest College has equipped these two students with skills that they will value and use for the rest of their lives, "I feel that Lake Forest College prepared me for the real world," affirms Silvia.

Michael Zorniak Department of Biology Lake Forest College Lake Forest, IL 60045

Top Left: Beth Ruedi ‘01, Top Right: Brandon Johnson ‘03, Bottom Left: Allison Huang ‘98, Bottom Right: Silvia Scripkauskas ‘04

While in college, many of you have probably wondered, "What am I going to do with my life?" Unless you were born knowing your fate, this question may be truly disheartening. After interviewing four recent biology alumni from Lake Forest College, I realized that knowing your future is not as important as knowing yourself. Writing this article as a senior has forced me to come to grips with my future. I understand that this article is supposed to feature graduates from Lake Forest College, but, being on the brink of commencement, I feel that my soon-to-be alumnus perspectives are simply an added bonus. Before talking with the alumni, I felt that graduating from college marked a fork in the road of life. I believed that by choosing one path, one would have difficulty backtracking or switching paths. Upon receiving corresponding with Allison Huang’s ’98, Silvia Skripkauskas’ ’04, Allison Huang ’98, and Brandon Johnson ’01, I realized that changing one’s mind is a natural and sometimes necessary step in life. Silvia and Allison Let’s start with Silvia, who, at the moment, is pursuing a master of arts degree in Public Policy and Administration at Northwestern University. At Lake Forest, she majored in biology and psychology while managing to pick up a minor in chemistry with medical school in mind for the future. After graduation, she tacked from one job to another, only confirming her desires for autonomy in a health-related field, "I need more say in what I do, and that can be achieved only after I get more training, whether it be the master’s degree I am currently working towards, a Ph.D., or an M.D." She first held a brief position as a research technician at Abbott laboratories in the International Pediatric Nutritional Regulatory Affairs Department. Later, Silvia obtained another research position in the Institute of Healthcare Studies at Northwestern University, where she was appointed as a project

Beth and Brandon Brandon Johnson ’03 and Beth Ruedi ’01 have progressed through life in a more linear fashion when compared to Silvia and Allison. Beth, a biology and English double major, found pleasure in studying genes and behavior when she took Dr. Houde’s Ecology and Evolution class, for which she was later a peer teacher. Following her

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interests, she entered a doctoral program at the University of Illinois in Champagne-Urbana. Beth’s goal is to become a university-level professor, "I felt that I needed to learn a great deal more about biology before I could teach it." Taking steps to further her career, Beth is employed as a teaching assistant and she is, "reinforcing the fact that this was a good career path to take." She says that although, "Lake Forest College provides students with many opportunities for thesis research and laboratory work, which is one of the key elements that can help a student get into graduate school," she was not prepared for the intense research at a Level 1 institution, "due to Lake Forest College’s comparatively relaxed atmosphere." All in all she concludes that, “graduate school is challenging and stressful. However, I can honestly say that the past five years have been the best of my life.” Brandon traveled a path no less direct than Beth’s. While at Lake Forest, he majored in biology and chemistry and wrote a thesis in Dr. DebBurman’s brand new lab. As side-effect to this, he was a permanent resident of Johnson Science Center during his junior and senior year. He became so attached to Johnson that whenever his experiments went awry he, “cursed the entirety of Johnson building.” Despite his negative sentiments toward the actual building, he says that, “the Lake Forest College experience, as a whole, really solidified my motivations towards graduate school…the opportunity to write a thesis and defend it gave me the confidence to conduct graduate research.” Now at Stanford University, Brandon is pursuing a doctorate in cell biology, which may lead him into a career in teaching where he has, “a passion for science and maintaining a high level of scientific achievement in the US.” Brandon may even want to, “reenergize scientific awareness and research,” possibly by teaching science at the high school level. He thinks that, “high school students are not receiving adequate scientific training. Right now, we are seeing reduced scientific funding in the United States and a general apathy towards basic, non-clinical scientific discovery.”

Like Allison, Brandon cautions undergraduates, “graduate school is all about learning how to conduct a long term, independent, primary research project.” Unlike Lake Forest College he says, “Classes are secondary to conducting primary research.” He faces the same challenges at Stanford as he did in Johnson, “there are many times when my project doesn’t move forward for months at a time, and brief periods where the science advances rapidly.” Even though there are ups and downs in science, Brandon looks to other successful scientists, with similar experiences, for inspiration, “I take comfort in knowing that I am on the same rollercoaster.” Additionally, Brandon is no stranger to the distress and adventure he experiences in science. He avidly surfs and hikes in California’s wilderness and has recently competed in an 11 mile relay swim. Silvia, Allison, Beth, and Brandon have all found value in immersing themselves in the academic climate at Lake Forest College. By looking inside themselves and pursuing their interests, they have become successful scholars in their respective fields. One thing they all miss is the, sometimes, daily interactions with faculty, which have strengthened their understanding of biology as well as themselves. Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College.

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Beyond the Classroom

Eukaryon, Vol. 3, February 2007, Lake Forest College

Lake Forest Students Present Their Research at Regional and National Symposia wandered around the San Diego Convention Center checking out all the latest biotechnology. Many famous scientists, such as Susan Lindquist, presented their research or participated in panel discussions involving the audience. Surprisingly, the NIH Director was in attendance and answered passionate questions on the current state of research funding. In addition to all the serious research being presented, there was an event called “Cell Slam” that was one of the most memorable parts of the trip. Participants were given several minutes to do anything they wanted in regards to cells. One scientist sang a parody of “Let it Snow” that went, “The funding situation is frightful but my grant is so insightful…Let it go, let it go, let it go”. The crowd loved it. This demonstrated that scientists loved taking a humorous attitude toward science. One afternoon during the symposium, Lokesh, Michael, and I decided to take a break from the science and travel to Tijuana, Mexico. It was a great opportunity for us to take advantage of the community surround San Diego. At the U.S.-Mexico border we took a cab into downtown Tijuana and had a drink at a local restaurant. Surprisingly, there were pharmacies on every corner. To get back into the U.S. we waited over an hour and wondered if we would ever get back to the symposium. Several hours later, we returned and had numerous stories to tell, such as the donkey painted as a zebra.

Michael White* Department of Biology Lake Forest College Lake Forest, IL 60045 During the fall of 2006, undergraduates from Lake Forest College (LFC), including myself, organized their research into posters and PowerPoint presentations to exhibit at regional and national academic symposia. Specifically, participants conducted laboratory or field research on either Parkinson’s disease, cell volume regulation, RNAi, addiction, or avian biology. Fourteen LFC students presented this great diversity of biological study at the Argonne National Laboratories Undergraduate Symposium in lecture form. Lokesh Kukreja, Michael Zorniak, and myself went a step further and traveled to San Diego, California to present two posters on our fission and budding yeast models of Parkinson’s disease at the American Society for Cell Biology. Whether presenting on the regional or national scale, young scientists from LFC demonstrated their individual research in biology and enjoyed the fellowship of other undergraduate and graduate students. The Argonne symposium consisted of undergraduate research, organized into mini-symposia consisting of several twenty-minute individual talks. Hundreds of students from the states surrounding Illinois attended. There were many presentations to choose from, and it was interesting to learn about a vast array of topics. I noticed that the structure of each talk differed significantly between colleges, indicating differences in teaching styles. Michael Zorniak said that Argonne, “strengthened my communication skills by challenging me with a diverse audience.” In the morning, a local high school physics teacher conducted a series of insightful demonstrations on the characteristics of shadows. The evening lecture was given by a chemist and, like the first, used a series of colorful chemical reactions to demonstrate the digestive functions of the stomach. Humorously, his chemists’ view of acid digesting food neglected most of the biology behind the process.

Caption: Tijuana, Mexico 2006. Michael Zorniak (left) and Michael White (right). On the final day, Michael and I presented our posted on budding yeast and Lokesh on fission yeast. Initially, I felt that the graduate students would be extremely critical of our work but once several visited and discovered we were undergraduates and our research was respectable, their comments were insightful and full of praise. A young scientist from Mayo Graduate School also worked with yeast and gave great suggestions for future experiments, one of which I am currently performing. In addition, all three of us discussed our research with others in similar fields and found these conversations very beneficial. This experience increased our confidence and demonstrated that undergraduate research can be presented alongside more advanced studies. When Michael Zorniak was asked what he liked most about attending the ASCB he said, “I was able to interact with scientists from as far as Tokyo, Japan that were interested in the same research questions. Even as an

In December, Lokesh Kukreja, Michael Zorniak, and myself traveled with our P.I Shubhik DebBurman to the American Society for Cell Biology symposium in San Diego, California for what would be one of my most memorable experiences. There were nearly 15,000 attendees, the majority of which were graduate and post-doctoral researchers. Fewer than 1,000 undergraduates attended. We presented two posters on our study of α-synuclein, the protein involved in Parkinson’s disease, on the fifth and final day of the symposium. With several days of free time, we visited a variety of lectures, poster presentations, and

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Caption: American Society for Cell Biology San Diego, California 2006. Michael White (left), Michael Zorniak (center), and Lokesh Kukreja (right). undergraduate, I was able to propose experiments to graduate students in the same field.” Together, presenting undergraduate research on both regional and international levels, provided others and myself with an indispensable asset to our intellectual growth as well as peer fellowship. Furthermore, it allows students to gather perspective on the broader research community and realize that they are a significant part of it. And of course, a trip to Tijuana adds a little more excitement to an already intriguing experience. Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

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News and Views

Eukaryon, Vol. 3 February 2007, Lake Forest College

Bone-derived microglia clear amyloid plaques Nevertheless, studying the role of microglia in AD animal model is more relevant than any study in the test tube. In AD mouse model, the outcome of clearing Aβ deposits by microglia has been questionable because Aβ deposits are abundant in the brain and form faster than they can be cleared by the microglia (Wegiel et al., 2004). Prior to the research done by Simard et al. (2006), the microglia have been shown to be inefficient at degrading Aβ deposits. Now, in the study of AD using transgenic mice model, Simard et al. (2006) show that there are other efficient microglia of blood origin which specifically phagocytose amyloid plaques. Simard and colleagues demonstrate that the monocytes pass through the blood brain barrier of CNS and differentiate into microglia. These blood-derived microglia are shown to closely associate with Aβ deposits. In the AD affected region of the hippocampus, Simard et al. found bone marrowderived miroglia to colocalize with the β-amyloid 40/42. Following the first evidence, Simard et al. (2006) test a very important question of whether blood-derived microglia are beneficial in slowing down the build up of amyloid plaques. They treat the undifferentiated blood derived cells with ganciclovir drug that impedes the cells differentiation into microglia. The scientists closely observe the changes in Aβ formation strictly when no blood-derive microglia are created. They discover that the size and the number of amyloid plaques increase with ganciclovir treatment. In addition, they see a second type of non-blood derived (resident) microglia associate with Aβ deposits but unlike the blood microglia, they are not able to clear amyloid plaques. This observation seriously suggests that the blood-derived microglia are specific species of the brain immune cells better capable of removing the amyloid plaques and possibly further preventing the plaque formations. Simard et al. (2006) observe that as Aβ deposits associate with bone marrow-derived microglia, an immune response is elicited. Astonishingly, this response happens to be concurrent with the decrease in the size and the number of amyloid plaques. As such, blood-derived microglia draws a beneficial mechanism. It is a converse of the response that resident microglia produce in which dangerous proinflammatory cytokines secrete. Past in vivo experiments have shown that resident and blood-derived microglial cells are not distinguished in their function. Usually, these studies comment on the idea that microglia are incapable of phagocytosing amyloid plaques. However, Simard et al. (2006) make an attempt to clear up the confusion that exists in explaining the role of microglia. There are two types of microglia: resident microglia and blood-derived microglia. The latter perform a beneficial mechanism for the cell by carrying out phagocytosis and protecting the central nervous system from a neurodegenerative disease. In the last decade, therapeutic methods of preventing and curing AD have failed. In the Simard et al. (2006) study, the bone marrow stem cells shine on a novel strategy of eliminating amyloid plaques to possibly treat AD patients (Figure 1). There is a strong belief now that the prospect of treating Alzheimer’s Disease will come from learning more about how the immune response plays a role in the degenerative process (Monsonego and Weiner, 2003).

Lokesh Kukreja* Department of Biology Lake Forest College Lake Forest, IL 60045 Alzheimer’s disease (AD) is an incurable neurodegenerative disease and is the most common cause of dementia that affects elderly people (Izenberg, 2000). Today as citizens are living longer, AD is reaching epidemic proportions with no cure available (Tanzi and Bertram, 2005). In America, 4 million people are affected by this disease and it is estimated that the epidemic will jump 44 percent by the year of 2025 (Medline Plus, 2006). AD patients live debilitating lives of faulty memory, judgment, and reasoning (Tanzi and Bertram, 2005). However, a promising study published in Neuron by Simard et al. (2006) suggests a future therapeutic strategy. They employ stem cells that specifically target the amyloid deposits, the toxic culprit in this disease pathology. When Alzheimer’s disease strikes the brain, it makes three main aberrant structural changes. One change is the extensive loss of neurons in the hippocampus and neocortex. The second change is the accumulation of intracellular protein deposits called neurofibrillary tangles. The third change is the accumulation of extracellular protein deposits termed amyloid (Αβ, also called senile plaques, surrounded by damaged neurites (George-Hyslop and Westaway, 1999). The build up of Aβ in the brain is considered to be a major cause toward AD pathogenesis. Aβ i s naturally produced by the breakdown of a bigger protein called βamyloid precursor protein (APP). However, in AD patients, the problem occurs when the APP is mutant. The mutation progresses the production of too much Aβ (Hardy and Selkoe, 2002). Thus far, the research on amyloid plaques has shown that they influence significant immunological changes in their cellular environment. When Aβ deposits build up, they elicit an innate immune response on the central nervous system (CNS) (Monsonego and Weiner, 2003). Specifically, they activate the microglia, which are the immune cells of brain. The inflammatory response triggers the microglia to surround the amyloid deposits. This behavior of microglia immune cells has been observed in the rodent transgenic model of AD (Malm et al., 2005). Interestingly, the scientific community is in a debate over the role of microglia in Alzheimer’s Disease. Since there is a large amount of microglia in the diseased brain, they must, undisputably, play an important pathological role. (Rogers et al., 2002). Studies show activated brain microglia to have the capacity to be either potentially neurotoxic or beneficial to the brain. In test tube studies, when cultured microglial cells encounter Aβ peptides they trigger an immune response and secrete high levels of proinflammatory cytokines. However, the secretion of cytokines in the brain would be fatal. (Rogers et al., 2002). On the other hand, test tube studies also show that the microglial cells play a positive role. They clear up cellular debris and certainly are capable of clearing Aβ deposits by phagocytosis (Wegiel et al., 2004). *This paper was written for BIOL346, taught by Dr. Shubhik DebBurman.

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Figure 1: Blood-derived microglia specifically target amyloid plaques for elimination by phagocytosis. Monsonego, A. and Weiner, H. L. (2003). Immunotherapeutic Approaches to Alzheimer’s Disease. Science 302: 834-838.

Since the activation of blood-derived microglial cells create an immune response which reduces the size and the number of amyloid plaques, these microglial cells may be the key for AD therapy.

Rogers et al. (2002). Microglia and InflammatoryMechanisms in the Clearance of Amyloid -PeptideGlia 40:260–269.

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

Simard et al. (2006). Bone Marrow-Derived Microglia Play a Critical Role in Restricting Senile Plaque Formation in Alzheimer’s DiseaseNeuron 49, 489–502. St. George-Hyslop, P. H. and Westaway D. A. (1999). Antibody clears senile plaques. Nature 400: 116-117. Tanzi, R. E. and Bertram, L. (2005). Twenty Years of the Alzheimer’s Review Disease Amyloid Hypothesis:A Genetic Perspective. Cell 120: 545–555

References

Wegiel, J., Imaki, H., Wang, K.C., and Rubenstein, R. (2004). Cells of monocyte/microglial lineage are involved in both microvessel amyloidosis and fibrillar plaque formation in APPsw tg mice. Brain Res. 1022, 19–29.

Alzheimer's Disease. (2006). MedlinePlus. U.S. Library of Medicine and NIH Accessed October 23rd , 2006. http://www.nlm.nih.gov/medlineplus/alzheimersdisease.html#ov erviews Hardy, J. and Selkoe, D. J. (2002). The Amyloid Hypothesis of Alzheimer’s Disease: Progress and Problems on the Road to Therapeutics. Science Review: Medicine 297: 353-356. Izenberg, N. (2000). Human Diseases and Conditions. Volume 1 A-D. Charles Scribner’s Sons, New York. Malm et al. (2005). Bone-marrow-derived cells contribute to the recruitment of microglial cells in response to beta-amyloid deposition in APP/PS1 double transgenic Alzheimer mice. Neurobiol. Dis. 18, 134–142. Kim, S. U. and Vellis J. de. (2005). Microglia in Health and Disease Journal of Neuroscience Research 81:302–313.

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26

Book/Film/Fine Arts Review

Eukaryon, Vol. 3, February 2007, Lake Forest College

Dissecting the Ethical Brain biomedical world have been debating. Gazzaniga initially embarks upon the notion that consciousness is the pièces de résistance of human life itself; without a brain you are unable to sustain a conscious life and therefore, undeserving of the moral status of a human (23). Through a detailed synopsis of the path to a conscious life, Gazzaniga is able to genuinely convey his belief that embryo research has validation on the basis of good intention and only during the preembryonic stage. Gazzaniga elucidates the immense apprehension that commonly follows scientific progress,especially in gene and brain enhancement, rationalizing that the notion of hyperagency is misplaced and that the extremes such as the humanzee are often something of science fiction. Gazzaniga acknowledges the possibility of negative side effects, but reminds us that “ in the end, we humans are good at adapting to what works, what is good and beneficial, and in the end, jettisoning the unwise, the intemperate, the silly and self-aggrandizing behaviors that will always be present in certain proportions in our species” (53). The Ethical Brain provides an insightful testimony for the enhancement of the human brain using precedents as well as substantiating evidence in a fluid argument that carries itself. The complex judicial system, which is based on recollection and testimony, may be forever changed from recent understanding of how the brain works. Gazzaniga relays the flaws associated with memory and suggests innovative brain scanning and brain fingerprinting as a possible alternative to incriminate or acquit a defendant. Gazzaniga makes the stunning revelation that each person is responsible for his or her actions, indicating that the insanity plea holds no value in a courtroom. He explains, “brains are automatic, rule-governed, determined devices, while people are personally responsible agents, free to make their own decisions” (90). The author exemplifies the possibility that soon the fate of a defendant may not lie in the deliberation of the courtroom but at a click of a button. Gazzaniga enthralls the reader with riveting accounts of endless possibilities the field of neuroscience has brought upon the judicial system at the turn of the twenty-first century. Distinguishing from whether or not a defendant was associated with a terrorist group or crime scene, by means of brain fingerprinting, has the reader drooling for more. The Ethical Brain controversially reassesses our position on moral beliefs, particularly on our religious beliefs. The author claims that humans react to an event, interpret it, and from their interpretation beliefs emerge about rules to live by (146). Startling evidence has shown that religious visions or “religiosity” could have an organic basis within the normally functioning brain. Gazzaniga is quick to point out that the temporal lobes are active during the perception of intense religious experience and during auditory hallucinations. He adds that disruption of this region by electrical stimulation, epilepsy, or overexcitement, might cause such out-of-body experiences (161). As a result, this new evidence introduces a gripping reality for society and possibly the way we may view religious beliefs. The Ethical Brain is a lively confrontational and thought-provoking book about the world of

Benjamin Bienia* Department of Biology Lake Forest College Lake Forest, Illinois 60045

By Michael S. Gazzaniga Dana P (2005) On September 12, 2006, Edmund D. Pellegrino, Chairman of The President’s Council on Bioethics said, “To advance human good and avoid harm, biotechnology must be used within ethical constraints. It is the task of bioethics to help society develop those constraints and bioethics, therefore, must be a concern to all of us.” Evolution through natural selection has endowed our species with the innate capacity to process information by the use of our brains. Through natural gene selection, our ability to process this information varies from individual to individual. What if the genome dictating the variation of an individual’s intellect, athletic ability, or even personality, can be enhanced by pharmaceuticals or brain therapy? How can we differentiate between an embryo and human life? When do powerful brain imaging technologies, that can literally “read” you brain, cross the abstract line of an individual’s privacy and right to self? How do we diffuse the gray cloud that surrounds ethics today? Michael S. Gazzaniga, an outspoken member of the President’s Council on Bioethics, may not have all the answers, but he provides much insight in his critically acclaimed book, The Ethical Brain. This thrilling eyeopener helps us debunk many medical ethical dilemmas our society has come to face in recent years with insightful developments in the field of neuroscience. Gazzaniga, a world-renowned neuroscientist, argues that the field of neuroethics alleviates much uncertainty about the arbitrary limitations imposed on life. He explains that through a scrupulous understanding of how the brain and its underlying mechanisms work, humans will be able to pursue a true set of universal ethics. According to Gazzaniga, “ it is the job of neuroethics to use what we know about how the brain works to help better define what it is to be a human and how we can and should interact socially”. The Ethical Brain helps define the intangibles that encompass ethical dilemmas through his exceptional understanding of the brain mechanisms in an easily digestible manner for the reader. What marks the beginning of human life? This has been the million dollar question stem cell researches, policy makers, and the rest of the * This paper was written for FIYS 106 Medical Mysteries, taught by Dr. Shubhik DebBurman.

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neuroethics and its solutions to numerous social problems. Gazzaniga illuminates scientific findings in this enjoyable read in hopes that it will write a new page in the understanding of bioethics. After reading the book, one walks away with not only academic merit but with a greater sense of self. This father of cognitive science will have you basking in his fruit of enjoyable scientific discovery and understanding. Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. References: Gazzinaga, Michael. The Ethical Brain. New York: Dana P, 2005. "The President's Council on Bioethics." Bioethics. 12 Sept. 2006

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News and Views

Eukaryon, Vol. 3 February 2007, Lake Forest College

α-Synuclein, and the Case of the Blocked ER-Golgi Pathway pathway. In order to accomplish this task, they took two approaches; one genetic and the other cellular. Together these different pathways would converge to implicate αSyn in the blocking of ER-Golgi traffic and cell death. αSyn was expressed in yeast and regulated with a galactose inducible promoter. After αSyn expression, ER stress was measured and found to be increased for cells expressing αSyn-WT and further increased for the familial mutant αSyn-A53T. Lindquist et al. (2006) hypothesized that αSyn was causing ER stress by blocking the function of endoplasmic reticulum associated degradation (ERAD). As misfolded proteins accumulate in the ER, the ERAD process functions by retrotranslocating them back into the cytoplasm for proteasomal degradation (McCracken and Bdodsky 2006). They found that out of two commonly misfolded proteins in the ER, CPY and Sec61-2p (both ERAD substrates), the rate of CPY degradation decreased even though proteasomal function was unaltered. Interestingly, Caldwell et al. (2001) demonstrated that ERAD degradation of CPY required transport through the Golgi. Because the failure of the ERAD translocation through the ER to the Golgi during αSyn expression may be an indicator of general pathway blockage, Lindquist et al. (2006) hypothesized that αSyn may be blocking ER-Golgi traffic. To determine if this was the case, they followed two proteins, CPY and ALP, through the ER-Golgi circuit when αSyn was expressed. Within three hours, ER-Golgi traffic was greatly reduced and at four hours nearly nonexistent. Simultaneously, cell growth inhibition also occurred. Thus, αSyn blocks ER-Golgi traffic (2006). Following their cellular approach, Lindquist et al. (2006) initiated a genetics approach aimed at determining if genes that enhance ER-Golgi transport could reduce αSyn’s ability to block the pathway. They identified the yeast protein Ypt1p as a promoter of traffic, and Gyp8P as a suppresser of traffic. This finding led Lindquist et al. (2006) to hypothesize that over-expression of the Ypt1p (yeast) or Rab1 (mammalian) in a variety of models would rescue them from αSyn toxicity. This final study yielded profound results that provided the strongest evidence, yet, that αSyn’s impairment of ER-Golgi traffic was the source for toxicity. They overexpressed Rab1 along with αSyn in Drosophila melanogaster (fruit fly), C. elegans (worm), and mammalian dopaminergic neurons to determine if Rab1 would prevent αSyn toxicity by enhancing ERGolgi traffic. In all three models, the cells were rescued from death when overexpressing Rab1. As a result of Ypt1p/Rab1 re-establishing ERGolgi traffic, it was hypothesized that αSyn interacted at the ER-Golgi junction. This was based on two lines of evidence; 1) CSP requires transport into the Golgi to be degraded and 2) Ypt1p/Rab1 functions within the ERGolgi vesicular binding pathway. Therefore, when Ypt1P/Rab1 is over-expressed, vesicular binding efficiency increases. Returning to the Gosavi et al. (2002) and Lee et al. (2005) manuscripts, the Lindquist et al. (2006) data provides two established lines of evidence (i.e. detailed previously) supporting the Gosavi et al. (2002)

Michael White* Department of Biology Lake Forest College Lake Forest, IL 60045 Summary Parkinson’s disease has long been associated with Lewy Bodies composed of the protein α-synuclein. A groundbreaking new study has demonstrated the pathological function of α-synuclein may be impairment of ER-Golgi traffic. Introduction Parkinson’s disease (PD) is a fatal neurodegenerative disorder of the brain. It affects 1 in 100 individuals over the age of 60 of which 5-10% of cases occur in individuals under 40, and another ~5-10% are familial (NPF, 2006). PD is the result of neuronal atrophy within the substantia nigra located in the brain stem. The substantia nigra is part of a complex circuit called the basal ganglia. It is responsible for the initiation of movement (Purves et al., 2004). The hallmark feature of PD is neurofibrillary inclusions, Lewy Bodies, composed primarily of the protein α-synuclein (αSyn; Spillantini et al., 1998). Familial forms of PD have been linked to the αSyn mutations A30P (Krueger et al., 1998), A53T (Polymeropoulos et al., 1997), and recently E46K (Zarranz et al., 2004). However, the reason these cells are dying in PD patients remains unknown even after more than a decade of heavily funded research! αSyn’s pathological component has often been associated with its role in Lewy Bodies. One widely accepted hypothesis is that αSyn is pathological when in a protofibrillar form that occurs between monomeric αSyn disappearance and Lewy Body appearance (Lansbury et al., 2003). However, a remarkable new manuscript, “α-Synuclein Blocks ERGolgi Traffic and Rab1 Rescues Neuron Loss in Parkinson’s Models”, by Lindquist et al. (2006) has demonstrated that the pathogenicity of αSyn may be due to the impairment of ER-Golgi traffic, resulting in a halt of critical cellular secretory processes. Prior to their research, little was known about α-Syn’s relationship with the ER-Golgi pathway. However, αSyn expression led to the fragmentation of the Golgi apparatus (Fujita et al., 2006 and Gosavi et al., 2002). Notably, Gosavi et al. (2002) found Golgi fragmentation to occur before Lewy Body formation but after the disappearance of monomeric αSyn. Contrary to the αSyn-Golgi interaction, Lee et al. (2005) revealed αSyn to be excreted from the cell via a vesicular, ERGolgi independent, exocytotic pathway. Thus, debate exists over which pathway αSyn is involved in. The Case of the Blocked ER-Golgi Pathway In the recent Lindquist et al. (2006) study, they wanted to determine the effect of αSyn on the ER-Golgi *This paper was written for BIOL 493 Independent Study taught by Dr. Shubhik K. DebBurman.

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Endoplasmic Reticulum (ER) (-)α αSyn

(+)α αSyn/(+Ypt1p/Rab1)

(+)α αSyn

Cell Death Traffic Blocked

Golgi Apparatus Figure 1: Rab1 enhances ER-Golgi vesicular binding affinity. This diagram portrays the ER-Golgi junction and the vesicular transport that occurs between the two organelles. (+) indicates the presence of the indicated protein and (-) indicates its absence. The black circles represent a vesicle full of cargo (ex. CPY), the red circles αSyn, and the blue cross Ypt1p/Rab1. αSyn blocks ERGolgi traffic and leads to cell death (far right). Overexpression of Ypt1p/Rab1 rescues cells from atrophy by increasing the affinity of the vesicle for the Golgi (middle). Vesicular transport without αSyn is shown on the left.

References

conclusion that αSyn interacts with the ER-Golgi to yield toxicity. Though αSyn is continuously being secreted through a Golgi-ER independent pathway (Lee et al., 2005), it is plausible that a defect in this excretory system may function to exacerbate toxicity, but not produce it.

Caldwell, Sabrina R., Hill, Kathryn J., and Cooper, Antony A., Degradation of Endoplasmic Reticulum (ER) Quality Control Substrates Requires Transport between the ER and Golgi, Journal of Biological Chemistry, volume 276, no. 26, pages 23296-23303, 2001. Fujita, Tukio et al., Fragmentation of Golgi apparatus of nigral neurons with α-synuclein-positive inclusions in patients with Parkinson’s disease, Acta Neuropathol, volume 112, pages 261-265, 2006.

Future Research The Lindquist et al. (2006) manuscript has provided the Parkinson’s disease community with what appears to be an opened door, leading to a whole new frontier in PD research and understanding. As with the relentless pursuit of the protofibrillar discovery by Dr. Lansbury, all methods of research must be exhausted on finding the mechanism by which αSyn is able to turn off the ERGolgi pathway. It is feasible that the same lentivirus used by Lindquist et al. (2006) to carry the Rab1 gene into mammalian neurons in their experiments could be re-configured to enter the cells of PD patients and reestablish traffic between the ER and Golgi. If this is, in fact, the reason these cells are dying, one of the most prevalent and debilitating neurodegenerative diseases could be cured.

Gosavi, Nirmal et al., Golgi Fragmentation Occurs in the Cells with Prefibrillar α-Synuclein Aggregates and Precedes the Formation of Fibrillar Inclusion, Journal of Biological Chemistry, volume 277, no. 50, pages 48984-48992, 2002. Lansbury, Peter Jr., and Volles, Michael J., Zeroing in on the Pathogenic Form of α-Synuclein and Its Mechanism of Neurotoxicity in Parkinson’s Disease, Biochemistry, volume 42, no. 26, pages 7871-7878, 2003. Lee, He-Jin et al., Intravesicular Localization and Exocytosis of α-Synuclein and its Aggregates, Journal of Neuroscience, volume 25, issue 25, pages 6016-6024, 2005. Lindquist, Susan et al., α-Synuclein Blocks ER-Golgi Traffic and Rab1 Rescues Neuron Loss in Parkinson’s Models, Science, volume 313, issue 5785, pages 324-328, Epub 2006.

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

McCracken, A., and Brodsky, J., Recognition and Delivery of ERAD Substrates to the Proteasome and Alternative Paths for Cell Survival, CTMI, volume 300, pages 17-40, 2006. PD Statistics Provided by the National Parkinson Foundation. Retrieved on 2 September 2006 from http://www.parkinson.org/site/pp.asp?c=9dJFJLPwB&b=71354. Purves, Dale et al., Neuroscience 3rd Edition, Sinauer Associates Inc., Maryland, 2004.

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Smith, Wanli et al., Endoplasmic reticulum stress and mitochondrial cell death pathways mediate A53T mutant alphasynuclein-induced toxicity, Human Molecular Genetics, volume 14, no. 24, pages 3801-3811, 2005. Spillantini, Maria G. et al., α-Synuclein in filamentous inclusions of Lewy Bodies from Parkinson’s disease and dementia with Lewy Bodies, PNAS, volume 95, pages 6469-6473, 1998

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Book/Film/Fine Arts Review

Eukaryon, Vol. 3 February 2007, Lake Forest College

Iris and Awakenings: Timeless Tear-Jerkers her plan for the future. Bygone events, such as the death of her girlfriend, don’t weigh her down. Instead, Iris, with an untarnished outlook towards life, spends every moment fulfilling one impulse after another. She has not only been unchained from the shackles that memory creates, she has been liberated from the pressures of working towards a goal. This is evident in the scene when Iris lifts the rocks she had placed on her papers, which contained the rough beginning to her novel, and lets them blow away. Activities aimed at achieving future gain forestall us from living in the present. Planners, time tables, and calendars rob us of flexibility of time that could be spent in enjoyment. Life is simply too short to waste. Iris and Awakenings playfully tackle the concept of age-appropriate behavior and suggest that life is too short to stick to formulated rules of what suits somebody at what age. Like inquisitive children, Iris repeatedly questions John Bayley to the point of frustration and Leonard, at the age of forty, also bombards Dr. Malcolm Sayer (Robin Williams) with simple questions. Leonard realizes that he has been given a second chance and he takes full advantage of it without worrying if his actions correspond with his age or not. At one point, even Iris is shown watching the Teletubbies. These scenes challenge the old notion of age-appropriate behavior and suggest that age need not restrict one to a set pattern of behavior. For example, Leonard is attracted to a girl considerably younger than him, while Iris dates men way over her age. In Awakenings, other patients, who also reawaken due to L-Dopa, challenge the concept of age-appropriate behavior as well. An old woman, after looking at herself in the mirror, cries out for hair dye. These scenes tie perception to age and suggest that you are, after all, as young as you feel. Indeed, life is too short to follow society’s rules of what is appropriate at what age. Along with the importance of time, Iris and Awakenings emphasize the significance of love, friendship and family. Dr. Malcolm Sayer, initially in the movie Awakenings, is a lonely guy with little to no social skills. He is engrossed in science to a level that leaves no room for company. For example, Dr. Sayer refuses Julie’s dinner invitation because he has to attend to an experiment that he is conducting on plants. However, after he gets to know Leonard, he comes to the conclusion: That the human spirit is more powerful than any drug - and THAT is what needs to be nourished: with work, play, friendship, family. THESE are the things that matter. This is what we've forgotten - the simplest things. (“Memorable” 2) This socially inept and shy doctor, even though hesitantly, asks Julie out. Iris too, subtly advocates that love is the language that ought to be spoken by all. However, unlike Awakenings, Iris suggests that the physical aspect of life is just as important. We need to find a balance between lust and love. When Iris and John are kissing each other’s hand, John says that as a married couple, they could be doing this all the time. Iris corrects him and says that perhaps not all but nearly all the time. The theme of balance is introduced. Throughout the film, Iris is in touch with her inner animal while John Bayley is simply a product of society. Iris has unshaved armpits and wrinkled skin. Yet she does not resort to cosmetics because she is comfortable within her own body. She is open to lesbianism and promiscuity – attitudes that show her instinctual nature. However, John Bayley is a figure of formality. Together these

Mohammed Ejaz Ali* Department of Biology Lake Forest College Lake Forest, Illinois 60045

Iris (2001 film) Directed By Richard Eyre

Awakenings (1990 film) Directed By Penny Marshall To sum up my classmates’ reactions to Iris and Awakenings: that was so cool. However, these movies provide more than just sheer visual pleasure. They supply food for thought. Provocative in several arenas, Iris and Awakenings are like potent cannonballs that shake you to your core. Moreover, the acting is simply impeccable. In fact, several actors were nominated and given Academy Awards for their heart-rending performances. Iris and Awakenings are two of those rare films that effectively balance comedy and drama. Truly, as films, they are simply successful. Iris vividly narrates the tale of the enduring love between noted British philosopher and author Iris Murdoch (Kate Winslet and Judie Dench) and college professor John Bayley (Jim Broadbent), love that surpasses even the hurdles created by Alzheimer’s. Awakenings portrays the miraculous return of Leonard Lowe (Robert De Niro) from a state of paralysis brought about by the neurodegenerative encephalitis. Despite their differences, these movies are remarkably similar and parallel. Iris and Awakenings both advocate that one should seize the day (carpe diem) while it lasts. This is explicitly conveyed by the frequent introduction of the concept of time. These movies highlight the point that one should not let one’s future plans or past rule him or her, but instead should always live in the present. As Alzheimer’s progresses, Iris Murdoch forgets her past and *

This paper was written for FIYS 106 Medical Mysteries: Neuroscience in Chicago, taught by Dr. Shubhik K. DebBurman.

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contrasting characters emphasize the importance of establishing balance. At one point in the movie, a cat is shown hissing at a fox and to Iris’s utter disappointment, the fox leaves. This scene also symbolically points out the conflict between formality and informality. Cats are tamed creatures while foxes are simply pure beasts. Metaphorically it basically suggests that society unjustly dominates us. Even in the subtle scene when John is reading a passage to Iris from Pride and Prejudice, this conflict is highlighted because the novel Pride and Prejudice by Jane Austen is a Victorian classic that exposes the sexual repression ensued by the prevalent, overbearing, straitlaced, societal etiquettes and deals with the conception of a balance between love and lust. It prompts the audience to imitate the fashion in which the characters in the book challenge, cross, and abide various dictatorial societal customs and establish a baseline for themselves. The nudity in the movie also renders the point that while taking into considerations the pressures created by the puritanical society, one must not stifle his or her wild, untamed, corporeal, earthly instincts. Iris makes one revaluate his or her priorities and ultimately settle the conflict between society and individuality. Thematically powerful, Iris and Awakenings stir one at various levels. They educate the social consequences of having a member of one’s family suffer a fatal neurodegenerative disease. Although the biological aspect of the diseases – Alzheimer’s and encephalitis – are not stressed enough, Iris and Awakenings do a fair job of highlighting the neurological and behavioral changes that accompany the diseases. Moreover, the insufficiency of biological substance is substituted by the presence of ample social consequences of the diseases. Iris runs at a much slower pace than Awakenings and the presence of elements such as homosexuality, nudity, and promiscuity make it all the more controversial while Awakenings is a simple, tender, love story with emotional ups and downs. Hence, I would recommend Awakenings to all but cautiously recommend Iris to all above the age of seventeen. Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. References Memorable Quotes from Awakenings. 2 Oct. 2006.

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Book/Film/Fine Arts Review

Eukaryon, Vol. 3 February 2007, Lake Forest College

Ferocious Beauty: All Roar and Very Little Bite grace that is sufficient to catch the attention of even the most skeptical observers. With impressive diversity, the cast performs a wide variety of dance routines, placing their dancing prowess on display for the general public to see. In the eyes of some, the fluidity of the dancer’s movements may more than make up for the performance’s lack of educational value. On the whole, I believe that the creators of the show achieved the finished product that they had aimed to achieve when in the process of producing their gift to the sophisticated world. With that said, any viewer who should happen to attend a future performance with the hopes of gaining a meaningful educational experience should think twice before dropping twenty dollars at the door. However, for the prospective viewer who aspires to lay eyes upon an impressive dance performance, the ticket is well worth its cost. When the performance had ended on the evening of Thursday, September 28, this viewer felt that there was definitely no need for an encore.

Pete Wisnieff* Department of Biology Lake Forest College Lake Forest, Illinois 60045 Liz Lerman Dance Exchange’s interpretive dance performance Ferocious Beauty: Genome has come and gone from Chicago’s Museum Of Contemporary Art. The performance was given nightly from September 28 through September 30 and opened before a large audience on its first night. A cast of dancers as diverse as the dances they perform place the role of the human genome on display from the very beginning. A unique production, the experimental piece attempts to educate its audiences about the nature of the human genome and the ethical dilemmas that loom in the near future as imminent advances in the study of genetics come with ever increasing ethical implications. In an attempt to help the average audience member attain a better knowledge of a complicated subject matter, the group decided to present the material through both short video clips and long sequences of dance. After sitting through the entire performance, I was left with the impression that the performance fell short in one of two ways. Either the piece failed to delve deep enough into the subject to make the performance worthwhile, or interpretive dance is simply too vague to successfully educate the masses about the nature of such a concrete scientific phenomenon. Despite the fact that dance was not a viable way to illuminate the nature of the genome, the short video clips were to the point and proved to be very informative. Unfortunately, they were short video clips. These abbreviated video information sessions made up only a small portion of the performance and left the viewer desiring more. When all was said and done, Ferocious Beauty: Genome provides only a very shallow view of the nature of the genome and continues to present a decidedly one-dimensional view of the ethical issues associated with it. There is a chance however, that I am wrong to jump all over this apparent shortcoming. Perhaps the presentation’s lack of depth is intentional; perhaps it is intended to leave the audience wanting more, and thus promoting continued awareness of genetic research. If this is the case, then they have succeeded and the video clips serve their purpose admirably. Either way, the amount and quality of the information provided by the video clips is sufficient and cannot be blamed for the feeling of emptiness I felt as I headed for the exit following the performance’s welcome conclusion. One thing is for certain, however, interpretive dance is not a successful vehicle for educating the public on the mysteries of the human genome. As a rule, interpretive dance is intriguing and thought provoking; but, by definition, it cannot provide concrete answers. For instance, in almost all sciences, the study of genetics is extremely concrete and requires definitive answers. This is a need that interpretive dance just cannot fulfill. The one saving grace of the whole performance is that the dancing is advertised. Throughout the show the dancers on stage move with a

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College.

*

This paper was written for FIYS 106 Medical Mysteries: Neuroscience in Chicago, taught by Dr. Shubhik K. DebBurman.

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Book/Film/Fine Arts Review

Eukaryon, Vol. 3, February 2007, Lake Forest College

Fact and Fantasy: The Beak of the Finch by Jonathan Weiner were to be repeated over many generations, we may see a new species of birds with larger beaks. This leads the reader to question the idea of speciation, or how new species arise. Why are there so many species of finches and what did they evolve from? Weiner quotes Thomas Henry Huxley saying that, “A race does not attract our attention until it has, in all probability, existed for a considerable time, and then it is too late to inquire into the conditions of its origin.” Thankfully, the Grants noticed Darwin’s finches in time to learn so much. Many may argue that Darwin never addresses this in his Origin of Species. However, Darwin does describe the idea of speciation. He never claims that natural selection is the only mechanism working in evolution to create new species. Over time, as natural selection tinkers with the variations within a population, adaptations accumulate. Eventually, after so many adaptations have built-up, whether between two separated populations or within a single population, a new species is observed. These ideas apply to all species of organisms, including plants, as well as us. Weiner uses the story of Cleopatra to demonstrate this. “If Cleopatra’s nose had been shaped a little less like the Grecian ideal, and a little more like Cleopatra’s Needle, there would have been no Alexandrine War, no sea-fight at Actium. The whole arc of the Roman Empire would have been reshaped by Cleopatra’s beak.” One day, Homo sapiens may be obsolete and some species of Homo somethingorother may be the dominant species on the earth. It is important to study both the process and its relation to our lives. Analogies like these allow a reader, who may not be adept in the sciences, to clearly comprehend the process of evolution. Weiner’s method of alternating the Grant’s story and Darwin’s principles of evolution, with non-science philosophies, succeed in driving his ideas home. While some readers may find certain ideas familiar, they will find them juxtaposed with an exceptionally engaging story. Time and time again, Weiner revisits Daphne Major with the Grants and their colleagues. Every time, slightly more of the island and the puzzle are revealed. One drought, one rainy season, hybrids, beak variations, adaptations, the list continues today as more people study Darwin’s finches. Each occasion the Grants visit the island, Weiner makes it seem as though instead of being on scientific study, they are there for pure adventure. We forget that the finches are the exemplar of Darwin’s theories and begin to think of them as a child thinks of the peacock at the zoo. They are not a spectacle of science, just a spectacle. Weiner is quick to remind us, though, that they are indeed spectacles of science. And extraordinary ones at that; the finches show us that evolution is not unidirectional as previously thought, but bi-directional and capable of reversing itself. An adaptation that is beneficial to one generation may be a hindrance to the next. In turn, the variation that led to the most survivors in the previously generation, such as large beak size, may be the downfall of the next generation. This shows that life itself is more evolutionary than we thought. Weiner says, “The closer you look at life, the more rapid and

Michelle McKinzey* Department of Biology Lake Forest College Lake Forest, Illinois 60045

By Jonathan Weiner Vintage (May 30, 1995) The sun sets on a small island off the coast of South America. Thirteen species of Galapagos finches settle in their various homes for the evening. Peter and Rosemary Grant along with their crew settle in, as well, and you feel like you are right there with them thanks to Jonathan Weiner. Weiner presents the story of Darwin’s finches and the Grants as if it were just that: a fabulous story. It’s no wonder, however, that Weiner’s book is a Pulitzer Prize winner. Along with the Grants’ tale, Weiner ties in Darwin’s theories of Evolution and Natural Selection, examples outside of the finches, and even philosophy making the book both a key source of public understanding and an entertaining read. Weiner reconstructs the research of the Grants’ twenty years after they first discover their amazing finches and follows them back and forth through time in his interviews. While the Grants and their colleagues struggle to discern exactly what the finches mean, we struggle along with them. Bit by bit, Darwin’s finches reveal to the Grants that evolution is not a slow process, in so doing; other common misconceptions about evolution are cleared up. Weiner presents the argument that perhaps the finches, because they can still interbreed and produce viable offspring, are in a sort of transitional phase. They are separate species but are recently diverged from a common ancestor and are constantly evolving. (This was first seen by Darwin, which is why they are termed Darwin’s finches.) Had it not been for the periods of drought and excessive rain, we may not have realized that evolution, the process of change in organisms, need not be slow. The gene pool of the finches varies from generation to generation depending upon their environment. Each successive generation is consequently better suited for their environment. For instance, in periods of drought, birds with larger beaks are better able to survive and reproduce, thus the next generation has, on average, a larger beak size and a better chance of survival. If this * This paper was written for an independent study with Dr. Anne Houde.

33

intense the rate of evolutionary change. The further back in time you stand, the less you see.” It’s easy to see that Weiner researched this subject very well. Nonetheless, the book can feel a little drawn out. Sometimes, the reader may wonder at the connections between a sub-topic and Darwin, but with a little further reading and brainpower, the haziness soon vanishes. The timeline can seem a little muddled at times, but may be for clarity and aesthetic purposes. Despite these few minor qualms, Weiner does prove, in essence, his statement that, “The beak of the finch is an icon of evolution the way the Bohr atom is an icon of modern physics, and the study of either one shows us more primal energy and internal change than our minds are built to take in.” It’s clear that Weiner’s aim is to further impress the knowledge of evolution as well as entertain the reader. He succeeds. For those of us who already know the outcome, we are immersed in a wonderful story. For those concerned with understanding evolution, there is much to be learned from this book. Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

34

Book/Film/Fine Arts Review

Eukaryon, Vol. 3, February 2007, Lake Forest College

Healthy Marketing: The Only Solution he felt well by any stretch of the imagination. Morgan’s cholesterol went from 165 mg/dL at his first weigh-in to 225 mg/dL after the third weigh-in; one of the doctors said that anyone would say that Morgan is sick. The doctors concurred that Morgan was at risk of liver failure, which usually is a product of alcoholism, not a high-fat diet. By day 21, Morgan woke up in a hot sweat with difficulty in breathing. One of the physicians stated that Morgan needed to stop, because his liver resembled that of an alcoholic. Morgan continued despite pleas from his girlfriend and physicians to stop the, “McDiet.” In his final assessments, Morgan weighed 210 lbs–24.5 lbs heavier–and increased his body fat from 11% to 18%. When Morgan returned to a normal diet and exercise it took him 5 months to lose 20 lbs and 9 months to lose 4.5 lbs. This one-month test of a McDonald’s diet proved that McDonald’s food definitely has adverse effects on health. In the American society, we seem to be having a difficult time digesting the fact that our own family members are overly obese. We look for escape routes when we are pinned in the corner of being labeled overweight from proclaiming that the Body Mass Index is a faulty measuring tool, to trying every fat burning pill on the market. In an interview in Super Size Me, Dr. David Sattch stated, “We live in an environment that makes us sick, a toxic environment” (Sattch 2004). The environment that Dr. Sattch is referring to is the trend of replacing home cooked meals to on-the-go fast food that is pumping our arteries full of saturated fat. The marketing teams of fast food and junk food manufacturers are undoubtedly the cause of this drastic transgression. There are over 10,000 advertisements a year that are targeted at the youth of America. Since the early days of Michael Jordan endorsing Big Macs, the healthy food industry has been matched to an unfair fight with the fast food and junk food industries. To show the considerable variation in available capitol between marketing of healthy foods and fast food, consider that McDonalds spent 1.4 billion dollars on advertising in 2005, while healthy marketers were able to dig into their pockets for a meager 2 million dollars. Such disparity is evident in the way that fast food faces like Ronald McDonald, the playpens in fast food establishments, and Happy Meal toys are all unfair ways in which the fast food industry gains the upper hand in parental food choices and overcomes the small healthy food market. These marketing tactics have allowed being overweight a socially acceptable idea. The fact is that pressuring someone to quit smoking is socially permissible, but telling someone to lose weight is completely out of the question. Maybe it should be considered impolite to demand another to lose weight, because according to nationwide statistics, obesity is about to take over smoking as the number one cause of preventable deaths in the United States. The obesity epidemic is unquestionably an intense dilemma for the youth of the nation, and it all starts with healthy eating habits at home and school. Rarely are school lunches made from fresh ingredients, rather they are reheated packages with disturbing amounts of preservatives. Food for the majority of schools is government-issued, which poses an up-hill challenge in the attempt to offer children affordable, healthy lunches. This government task of providing

Jason Prendergast* Department of Biology Lake Forest College Lake Forest, Illinois 60045

Hart Sharp Video (2004) Little Jonathon scurries down the aisle in search of his usual supermarket purchase. As he impatiently tugs at his mother’s arm, she drops boring food items like oatmeal and fresh fruit into the cart. After quite some time, Jonathon’s mother gives him permission to go to the “fun food aisle.” There, Jonathon drops a cereal box into the cart with Sponge Bob on the front, and a bag of chips with his favorite basketball players’ (i.e. LeBron James) picture on it. These unsafe advertising strategies are raking in millions of dollars for cereal and junk food manufacturers. As Morgan Spurlock points out in his documentary Super Size Me, when young children are raised to idealize cartoons and athletes that market unhealthy food, it should be no shock that the United States is the fattest country in the world. Morgan’s unprecedented documentary shines light on the unknown effects of regular diets based solely on the American fast food industry. In his first-hand analysis, Morgan attempts to tackle an unimaginable task: eat only McDonald’s for three meals a day, eat everything that McDonald’s offers on their menu, and super-size his meal whenever offered the option to upgrade. In order to affirm that his high fat “Mac diet” was not going to cause any serious health concerns, he consulted cardiologists and nutritionists to evaluate his health and to predict any health risks or side effects of the diet. The main predictions that the team of doctors decided was that Morgan would increase his blood pressure based on high cholesterol and sodium intakes, gain overall weight, and feel sluggish and miserable. Such forecasts by the team of doctors seemed to be relatively accurate when analyzing Morgan’s weekly results. After just five days of eating nothing but McDonald’s food, Morgan gained 10 lbs, he started feeling pressure on his chest, he felt depressed, and he could not seem to quell the intense hunger attacks that he experienced shortly after eating a meal. Morgan’s second weigh-in put him at 203 lbs with no apparent side effects. The doctors hypothesized that his body was beginning to adapt to the intense increase in fat-intake. Although his health seemed to be adequate after the second weigh-in, the same could not be said for the time between the second and third weigh-ins. When Morgan showed-up for his third health update, he weighed 202 lbs, but that did not mean that * This paper was written for BIOL106 Food , taught by Dr. Nicole Sleiter.

35

healthy lunches is a long-term predicament that will take years to sort out. For that reason, America needs to achieve short-term success in the obesity epidemic by turning our attention to snack food marketing. Shortterm incentives for marketing healthy foods seems to be a step towards slowly changing the nutritional habits of our youth. Frito Lays has a healthy marketing scheme that marks snacks that are low in calories and fat, known as “Smart Choices Made Easy.” Such a conversion between product concept and healthy marketing is the only way in which healthy foods have a chance to compete with the evil empires of the junk food and fast food industries. While cartoon characters and sports heroes continue to be brandished on junk food packages, moms like Jonathan’s will have to struggle to quickly pass by the unfair marketing strategies of junk and fast food companies, whether pushing a cart or driving a car. Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

36

Book/Film/Fine Arts Review

Eukaryon, Vol. 3 February 2007, Lake Forest College

The Thin Line Between Madness and Sanity loss of countless relationships. As a patient, Jamison’s story is one of a personal struggle to carry on, a personal struggle to love and to live. As the doctor, part of Jamison’s struggle was to keep her illness a secret from those whom she was supposed to treat, those who just like her, were ill. With this, several questions arise. Was she capable of helping others who were ill when she had been, up to that point, unable to help herself? Dr. Jamison faced struggles every day of her life, in many dimensions. As a woman in the field of psychiatry, she had to prove herself to others. As a manic-depressive she had to prove to herself that a life without invigorating highs was better than no life at all. Dr. Jamison had to convince herself to take the medications. She had to prove to herself that just as she was capable of helping others, she could also help herself. Would others be understanding? Dr. Jamison was very conscious of the fact that this illness could have meant the revocation of her clinical license and the end of her career. Her illness represented a threat to everything she had accomplished until then. This meant that she would have to fight to stay at the top of her field and she was not willing to lose this fight. Dr. Jamison had a daily battle just to live normally. She found her sense of normalcy professionally, by treating others with the disease that she dealt with, but also found her sense of normalcy in love. A strong emphasis is made on the need for acceptance and love. Love from her family, her husband and later her partners allowed Jamison to be able to value her life and gave her a reason to want to get better. Through her stories of love we get the message of acceptance and understanding that is needed by those ill. Jamison describes to the reader her struggle with a disease which eventually affected her personal life, her relationships, her family, friends, and her professional life with coworkers and patients. This book gives us an insight as to how it feels to be on both sides of the desk, the healer and the healed. As the disease and its consequences are described in detail, one feels almost as if she is speaking directly to you. Jamison’s story allows us to see the sick person as that, a human being. This captivating book is written in a way that anybody, even those with no scientific background, can pick it up, enjoy it and most importantly, be educated by it. Along with learning about the ups and downs of manic depression and becoming informed individuals, we begin to partially understand what manic depression is like. We begin to understand where the line between madness and sanity lies and why in some cases it gets blurred. With Dr. Jamison’s story, our image of the mentally ill is changed from one of pity and fear to one of understanding.

Stephanie Valtierra* Department of Biology Lake Forest College Lake Forest, IL 60045

By K. R. Jamison Vintage Books (1996) As we go about our daily lives we experience many emotions. We are annoyed by traffic, saddened by bad news and angered by delays. While most of us quickly get over what has angered us or saddened us and continue to go about our day, many others live a very different life. What would it feel like to go through rapid mood swings, where one can go from a debilitating depression to a time filled with invigorating highs? What would it feel like knowing that for you, there is a thin line between madness and sanity? Dr. Kay Redfield Jamison has had a life full of these highs and lows and was able to tell us her first-hand experience with disorder that affects millions. Dr. Jamison tells us the story of her struggle with manic-depressive disorder, also known as bipolar disorder, in her memoir, An Unquiet Mind: A Memoir of the Moods and Madness. Jamison gives us the perspectives both of the patient and the healer. The author was born into a good, middle class American family and she was an intelligent, bright young woman with aspiration of going to medical school. Mood swings started plaguing her life as a teen. Jamison did not see these mood changes or the persistent depressions as serious problems. She believed that these mood changes were just another part of her, something by which she could be defined. The mood swings not only began to define her, but also connected her to her father and sister, who also suffered from these mood swings. The symptoms soon turned into advanced manias, depressions and suicide attempts. Medication was an option and could make her life “normal,” but this meant that she would not experience the invigorating highs and the accompanying devastating lows. This highly educated doctor became just another patient. The many highs and lows of manic depression did not only affect Jamison, but also affected those around her. A strong emphasis is placed of the affects that this disease has on those who live around and with the ill. The disease lead to the destruction of her marriage, loss of friendships, and the

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

* This paper was written for BIOL346, taught by Dr. Shubhik K. DebBurman.

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Review Article

Eukaryon, Vol. 3, February 2007, Lake Forest College

History Tends to Repeat: FMR-1 Silencing in Fragile X Syndrome Joshua G. Haas* Department of Biology Lake Forest College Lake Forest, Illinois 60045

of the FMR1 gene with binding sites and the CpG island is depicted in Figure 1. Studies have revealed a significant decrease in the expression of FMR1 mRNA in fragile X cases as a result of the hypermethylation of the CpG island (35). An important feature of repressing of this particular gene is the presence of FMR1in fetal tissue. The varying levels of expression between normal and fragile X fetal tissue reflects a very important role in development. This data also suggests early acquisition of the methylation, probably during embryogenesis (38,40). Early acquisition of methylation points out the importance of the loss of FMR1 expression. The human FMR1 gene, being highly conserved among species (16), has been shown to display alternative splicing properties suggesting the presence of many isoforms (4). Mouse models have been developed to study fragile X syndrome due to the allelic similarities (16). Three different levels of repeat within the gene have been reported: normal (7-50 repeats), permutation (50-200 repeats), and full mutation (> 200 repeats). The class of permutation is interesting because it only becomes affective in successive generations when the repeat region has a chance to expand leading to Shermans’s paradox. The permutation allele is unstable when transmitted from generation to generation. All males with the full mutation display the fragile X phenotype where only approximately 50% of females with the mutation display the fragile X phenotype (4, 40). Taken together with data from our more recent studies this proves that the germ line is susceptible to full expansion (32). There seem to be no new mutations appearing to cause fragile X syndrome. The only known mutation of resulting in fragile X syndrome interestingly shows increases in the transcription of FMR1 have been proportionally linked to the CGG tri-nucleotide repeat length and the repeat number approaches 200 in permutations. As the number of repeats increases the number of FMR1 mRNA levels increase. Unexpectedly, the FMR1 protein products decrease in relation to the CGG repeat length in permutation (25). This finding shows the development of the disease at various points of repeat length during the permutation stage of fragile X syndrome. Upon extending from the permutation to the full mutation, the FMR1 gene is silenced. The silencing of the FMR1 gene is interesting because it remains unclear as to how the tri-nucleotide repeat expansion occurs in an organism. Moreover, questions still remain as to how the loss of the FMR1 protein, FMRP can have such broad effects as those observed in fragile X syndrome (21, 40). Studies have started to reveal the role and importance of FMRP in patient and mouse models uncovering mRNA binding properties (5,29,17) Within the past few years we have uncovered information linking FMRP to protein synthesis (30) and a micro-RNA dependent translational suppression pathway (23). In this review we will focus on explaining the neuronal functions and importance of FMRP in model systems. We will also propose a mechanism for both translational and transcriptional repression through a micro-RNA pathway.

[Role Playing: Steven Warren Howard Hughes Medical Institute and Department of Biochemistry and Pediatrics, Emory University School of Medicine, Atlanta GA 30322] Abstract Fragile X syndrome is the most common form of inherited mental retardation, and afflicts 1 in 1250 males and 1 in 2500 females. The symptoms include connective tissue displasia, mental retardation, and macroorchidism (enlarged testis). My lab discovered that the most common forms of this disorder are caused by the expansion of CGG tri-nucleotide repeats on the X chromosome at the FMR-1 gene locus; an excess of 200 repeats in diseased individuals suppresses the translation of FMR-1. The CGG repeat expansion leads to hypermethylation of a CpG island distal to the repeat, leading to transcriptional repression of FMR-1. This gene silencing is also aided by RNA interference and de-acetylation of histones H3 and H4. The FMR1 gene codes for fragile X mental retardation protein (FMRP), which plays a functional role in protein translation in neurons. FMRP selectively binds to specific mRNAs essential to development of the brain and other parts of the body, and plays a major role in shuttling its ligands from the nucleoplasm to the dendritic cytoplasm. FMRP knockout mice models demonstrate abnormal dendritic spine growth, suggesting altered synaptic plasticity, which may be responsible for the fragile X phenotype. Elucidating the fragile X mechanism of pathogenesis can aid the development of possible treatments to the world’s leading cause of mental retardation. Introduction Fragile X syndrome is one of the most prevalent forms of mental retardation affecting approximately 1 in 4,000 males and 1 in 8,000 females. FMR1 has been identified as the gene associated with fragile X syndrome (1, 2, 6). This gene was mapped revealing a CGG tri-nucleotide repeat in the 5’ non-coding region of the gene. This region was noted to expand in repeat length in fragile X syndrome (1). The expansions of the CGG repeat to levels of 200 repeats or greater is responsible for the instability of the fragile site on the X chromosome. Normal cases posses an average of 3050 repeats of the CGG region (27). This instability results in the hypermethylation of a CpG island distal to the tri-nucleotide repeat (6,1,26,35). Examination of fragile X patients has consistently shown that the hypermethylation of the CpG island is the primary factor implementing the fragile X phenotype being that methylation is responsible for approximately 99% of known fragile X phenotypes (35, 41). A representation *This paper was written for BIOL 346 Molecular Neuroscience taught by Dr. Shubhick DebBurman.

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Figure 1. Molecular Basis of Fragile X Syndrome: A model of the normal FMR1 gene (top) and the fragile X FMR1 gene (bottom) with the defining difference in CCG repeat number between the two labeled. In the case of repeats >200 (fragile X syndrome), the CpG island is hypermethylated, histone 3 and 4 experience changes in acetylation, or RNA interference can all inhibit transcription of the FRM1 gene which codes for FMRP.

Functions of FMRP

X syndrome, a functional role of FMRP was investigated (38). FMRP was found to be associated with ribosomes in the dendritic structures of neurons elucidating a possible role of the proteins related to dendritic structure and neuronal plasticity (18). The association of FRMP with polyribosomes was eliminated in I304N mutations of extreme fragile X syndrome (19). The tissue specific expression of FMR1 connects the protein to important developmental structures in the brain such as the hippocampus (21). A set of FMR1 knockout mouse models were used to understand the relationship between drendritic formation and FMRP. Knock out mice were found to posses irregular dendritic spines (14). The importance of altered neuronal formation is discussed later. The next important link connecting neuronal alterations and the function of FMRP is the evidence pointing to negative regulation of mRNA translation. By removing the binding site of FMRP we found a lack of translational inhibition (30, 31). Specifically, FMRP has been shown to regulate the production of MAP1B, a protein. In Futsch mutant mice show inverse regulation of MAP1B and altered synaptic growth (31, 45). This shows that FMRP possibly plays a major role in development of neuronal structure throughout the body.

In fetal tissue, FMRP is found to be most abundantly and universally expressed in the testis and the brain (21). The differentiation of neuronal stems cells related to the fragile X phenotype has shown alteration in fragile X patients linking the disease to development (10). FMRP was discovered to assert properties of RNA-binding proteins in areas of expression (40). The protein itself encompasses three RNA binding domains, two K domains and an RGG box (40, 15). Importantly, the FMRP protein maintains both a nuclear location signal (NLS) at the amino terminus end of the FMRP and a nuclear export signal (NES) encoded by exon 14 of the FMRP. These signals help to confirm RNA binding properties of FMRP by hinting at the possible translocation of various mRNAs via the FMRP pathway (17). In order to bind mRNAs, FMRP forms an RNP complex containing FMRP, FRX1P, FRX2P, nucleolin, and three other proteins. The particles making up human RNPs are conserved in mice as well. (11). This RNP binds mRNA selectively by associating the G-quartet structure of the mRNA to the RGG box of the FMRP (5, 15). The G-quartet structure acts as a target for FMRP explaining the selective binding properties and the importance of transcriptional regulation via FMRP of the targeted mRNAs (15). FMRP binds directly to mRNAs via this interaction (9). This direct interaction with mRNA allows FMRP to act as a translational control factor for many of these targeted mRNAs. Evidence for this was found because a large majority of FMRP is associated with ribosomes, translational machinery (38). Because the absence of the FMR1 gene was associated with fragile

mRNA ligands associated with FMRP Because FMRP is shown to play significant roles in both mRNA transportation and translation, the characteristics of the specific mRNAs associated with FMRP have been explored. A variety of mRNAs are associated with FRMP. One of our recent studies examined 13 potential candidate mRNAs associated with FMRP and found that at least ten encode proteins

42

involved in synaptic plasticity or neuronal development. This data helped to identify some of the specific mRNA translation that is affected in mental retardation (15, 33). One specific mRNA was the mRNA coding for MAP1B. MAP1B is negatively regulated in the Drosophila nervous system. In the absence of FMRP, MAP1B experiences no inhibition and altered dendrite and axon development is noted (45). We also interestingly found that FMRP associates with the non-translatable BC1 RNA. In addition to binding directly to FMRP, BC1 can associate with the FMRP target mRNAs in the absence of FMRP (43, 44, 45). This data suggests that BC1 is involved in specificity of FMRP to mRNAs and BC1 helps to inhibit translation of some mRNAs by blocking the initiation codon (43, 44). Large numbers of mRNAs have been recently associated with FMRP. Of these mRNAs, many have been found to differ in expression and distribution among wild type and FMR1 knockout mice (31). These recent studies identifying specific mRNAs associated with FMRP and their coded proteins have shown the importance of FMRP in neuronal development. Altered neuronal Syndrome

development

in

Fragile

phosphorylated where the FMRP associated with actively translating polyribosomes was consistently dephorphorylated in both brian cells and cultured cells (12). This is possibly the mechanism by which FMRP releases its inhibitory effect on targeted mRNAs. Recent models have associated the release of translational inhibition with the presence of mRNA granules and mGluR-induced translation (3). FMRP normally inhibits mRNA translation, but induction of translation has been associated with glutamate receptors. In fragile X syndrome mGluR-induced translation is heightened due to the lack of inhibition normally provided by FMRP. The decrease in mRNA granules in knockout mice supports this finding. Furthermore, upon excitation with an mGluR5 agonist increased granule levels were observed (3). The lack of rapid protein translation in sites mediated by neurotransmitters such as glutamate has been proposed to cause several abnormalities observed in fragile X syndrome (41). The micro-RNA silencing of FMR1 The FMR1 gene is silenced by methylation induced by the CGG tri-nucleotide repeat expansion alone, but also by interaction with micro-RNAs (23, 24). FMRP interacts with Argonaute (AGO; 33) and components of the micro-RNA pathway such as Dicer (33, 24). In Drosophila melanogaster models the AGO ortholog was found to be suppressed in the presence of FMRP. When FMRP was removed the models experienced a loss of AGO suppression leading to a rough eye phenotype. Upon induced suppression of AGO, the rough eye phenotype was significantly reduced to levels of almost normal (24). AGO was found to be important to the biological functions of synapses, but not totally dependent on FMRP. FMRP interacts with many molecules like AGO to influence translation. FMRP also has recently been associated with translational regulation through a micro-RNA pathway. Transcripts are produced from the expanded fragile X allele at some point early in development before complete methylation of the CpG island (23, 24, 34). These transcripts form structures referred to as hairpins that are cleaved by the enzyme Dicer (23) resulting in small mRNAs approximately 20 nucleotides in length. These small mRNAs communicate with the RITS complex, a transcriptional silencer of genes. The small RNAs direct the RITS complex to homologous mRNAs through complementary base pairing. The RITS complex recruits methylation machinery eventually leading to the suppression of the FMR1 gene as observed in fragile X syndrome (24). The RITS complex also may change the acetylation of specific histones (7, 24). The FMR1 promoting region has shown to possess qualities of varying chromatin conformations linked to altered histones (20). When FMR1 is inactive as in fragile X cells the chromatin was displayed uniform conformation. In normal cells the interactions are far lesser than in fragile X cells (20). Histones acetylation is associated with normally expressed FMR1, but not the absence of FMR1 as in fragile X syndrome (7, 8, 20). Recent studies have treated fragile X cells with 5-aza-2-deoxycytidine and observed re-acetylation of histones 3 and 4. The re-acetlyation of these histones resulted in FMR1 transcriptional reactivation (8). This finding presents an alley of treatment investigation for fragile X patients in the future.

X

FMRP regulates the translation of many mRNAs which code for proteins involved in neuronal development. FMRP has been specifically found to be highly expressed in neurons. Areas controlling cognition are commonly affected in fragile X syndrome. A mouse model study shows deficient amygdale and hippocampal functions in FMR1 knockout mice during fear and conditioning tests (36). Specifically, FMRP is highly expressed in the dendritic formations of nonfragile X organisms (18). The dendritic functions encoded by the target mRNAs of FMRP are deficient in fragile X patients thus leading to altered synaptic function as observed in the fragile X phenotype (18). Further proving the neuronal effects of FMR1 silencing mouse models have elucidated interesting findings in the area of altered synaptic plasticity related to fragile X mental retardation (22). Long term depression (LTD) dependent on glutamate receptors was found to be significantly altered in the hippocampus cells of knockout mice. By using DHPG to induce the glutamate dependent LTD, enhanced results were found in the knockout mice (22). This supports the earlier hypothesis that FMRP is important in regulating protein production in the synapse. In mice lacking the expression of the FMR1 gene, irregular dendritic spines were observed (26) connecting the FMRP protein with synaptic growth. Dendritic spines in the visual cortex were compared among FMR1 knock out mice and wild type mice to explore the specific differences in dendritic growth. The dendrites of the knockout mice show a high incidence of long thin dendritic spines as well as dendritic spines of higher density (13). This data combined with the mRNAs targeted by FMRP that regulate neuronal structure connect fragile X syndrome with altered synaptic plasticity. FMRP influences synaptic growth by normally inhibiting translation of functional mRNAs in synaptosomes (18). Two methods of the release of inhibition in normal models have been discovered: the first is dephosphorylation (12) and the second is excitation by glutamate receptors (3, 42). FMRP associated with stalled polyribosomes was consistently

43

2 proposes an over all view of the FMRP interactions in a normal neuron. The alterations observed in fragile X syndrome are due to the loss of translational regulation via the various pathways that lead to transcriptional silencing of FMR1 (methylation of the CpG-island, miroRNA interactions, and histone deacetlyation). Altered synaptic plasticity leads to dysfunctional communication between neurons. This altered communication leads to the fragile X phenotype observed in humans. The new insights into the AGO (in fly models, Drosophila melangaster) and the micro-RNA pathway provide insight into a more specific cause of FMR1 silencing with possible drug treatment options. The mGluR induced translation provides the same exciting knowledge about translational regulation and drug treatment. Knowledge pertaining to further functions of FMRP still remains elusive along with the cause for the CGG tri-nucleotide repeat expansion which is thought to be the central cause behind fragile X syndrome. Interesting studies have also shown the influence of environmental factors on fragile X phenotypes. When knockout mouse models were raised in enriched environments there were found to show increased dendrite branching, length, and dendrite spine density to levels near normal (39). This

Discussion FMRP has shown tissue specific expression in areas of the brain and other parts of the body associated with the observed phenotype of fragile X syndrome such as the hippocampus and the testis. Specific binding of FMRP to various mRNAs has been elucidated also linking FMRP to an inhibitory role in the translation of respective mRNAs. Translation inhibition and mRNA targeting may be influence by non-translatable mRNAs such as BC1. The silencing of the FMR1 gene is the central link in fragile X syndrome, however, recent studies have shown gene silencing by the binding of transcription factors to the promoter regions of FMR1 (28). This is data another example of the mechanism by which methylation silences the FMR1 gene. The specific mRNAs associated with FMRP have proven to reveal a wealth of information explaining the gap of knowledge between the silencing of the FMR1 gene and the fragile X phenotype. MAP1B is an example of one such mRNA. MAP1B encodes for microtubule structural functions, thereby influencing physical features in the development of neurons. The absence of FMRPs regulatory influence leads to severely altered neurons in fragile X syndrome. Figure

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8. Bradford, Coffee, Fuping Zhang, Stephen T. Warren, and Daniel Reines. "Acetylated Histones are Associated with the FMR1 in Normal But Not Fragile X-Syndrome Cells." Nature Genetics 22 (1999): 98-101.

provides non-invasive treatment options to counter the silencing of the fragile X gene, FMR1. Transgenic genes have also been explored as a possible means of treatment. Because mouse models display some human-like symptoms in knockouts, a transgenic line of mice with yeast artificial chromosomes were generated. The study found behavioral and morphological changes. Also, overexpressing the gene did not have the opposite effect being a important consideration in treatment (37).

9. Brown, Victoria, Kersten Small, Lisa Lakkis, Yue Feng, Chris Gunter, Keith D. Wilkinson, and Stephen T. Warren. "Purified 10. Recombinant Fmrp Exhibits Selective RNA Binding as an Intrinsic Property of the Fragile X Mental Retardation Protein." The Journal of Biological Chemistry 273 (1998): 1521-15527. 10. Castren, Maija, Topi Tervonen, Virve Karkkainen, Seppo Heinonen, Ero Castren, Kim Larsson, Cathy E. Bakker, Ben Oostra, and Karl Akerman. "Altered Differentiation of Neuronal Stem Cells in Fragile X Syndrome." PNAS 102 (2005): 1783417839.

Acknowledgements I would like to thank Jenny Riddle for all of the time and dedication as a mentor through out the process of developing my project. I would also like to thank my peers for their support. Lastly, I would like to thank Dr. DebBurman for his constant advice, aid, and faith.

11. Ceman, Stehpanie, Victoria Brown, and Stephen T. Warren. "Isolation of an FMRP-Associated Messenger Ribonucleoprotein Particle and Identification of Nucleolin and the Fragile X-Related Proteins as Components of the Complex." Molecular and Cellular Biology 19 (1999): 79257932.

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.

12. Ceman, Stephen, William T. O'donnell, Matt Reed, Stephana Patton, Jan Pohl, and Stephen T. Warren. "Phosphorylation Influences the Translation State of FMRPAssociated Polyribosomes." Human Molecular Genetics 12 (2003): 3295-3305. 13. Comery, Thomas A., Jennifer B. Harris, Patrick J Willems, Ben A. Oostra, Scott A. Irwin, Ivan Jeanne Weiler, and William T. Greenough. “Abnormal dedritic spines in fragile X knockout mice: Maturation and pruning deficits,” PNAS 94 (1997): 5401-5404.

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6. Bell, M V., M C. Hirst, Y Nakahori, R N. Mackinnon, A Roche, T J. Flint, P A. Jacobs, N Tommerup, L Tranebjaerg, U Froster-Iskenuis, B Kerr, G Turner, R H. Lindenbaum, R Winter, M Pembrey, S Thibodeau, and K E. Davies. "Physical Mapping Across the Fragile X: Hypermethylation and Clinical Expression of the Fragile X Syndrome." Cell 64 (1991): 861-866.

21. Hinds, Heather L., Claude T. Ashley, James S. Sutcliffe, David L. Nelson, Stephen T. Warren, David E. Housman, and Martin Schalling. "Tissure Spedific Expression of FMR-1 Provides Evidence for a Functional Role in Fragile X Syndrome." Nature Genetics 3 (1993): 36-43.

7. Bradford, Coffee, Fuping Zhang, Stephenie Ceman, Stephen T. Warren, and Daniel Reines. "Histone Modifications Depict and Aberrantly Heterochromatinized FMR1 Gene in Fragile X Syndrome." American Journal of Human Genetics 71 (2002): 923-932.

22. Huber, Kimberly M., Sean M. Gallagher, Stephen T. Warren, and Mark F. Bear. "Altered Synaptic Plasticity in a Mouse Model of Fragile X Mental Retardation." PNAS 99 (2002): 7746-7750.

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23. Jin, Peng, Daniela C. Zarnescu, Stephanie Ceman, Mike Nakamoto, Julie Mowrey, Thomas A. Jongens, David L. Nelson, Kevin Moses, and Stephen T. Warren. "Biochemical and Genetic Interaction Between the Fragile X Mental Retardation Protein and the MicroRNA Pathway." Nature Neuroscience 7 (2004): 113-117.

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25. Kenneson, Aileen, Fuping Zhang, Curt H. Hagedorn, and Stephen T. Warren. "Reduced FMRP and Increased FMR1 Transcription is Proportionally Associated with CGG Repeat Number in Intermediate-Length and Premutation Carriers." Human Molecular Genetics 10 (2001): 1449-1454.

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27. Kremer, E J., M Pritchard, M Lynch, S Yu, K Holman, E Baker, S T. Warren, D Schlessinger, G R. Sutherland, and R I. Richards. "Mapping of DNA Istability At the Fragile X to a Trinucleotide Repeat Sequence P(CGG)N." Science 252 (1991): 1711-1714.

40. Siomi, Haruhiko, Mikiko C. Siomi, Robert L. Nussbaum, and Gideon Dreyfuss. "The Protein Product of Fragile X Gene, FMR1, Has Characteristics of an RNA-Binding Protein." Cell 74 (1993): 291-298.

28. Kumari, Daman, and Karen Usdin. "Interaction of the Transcription Factors USF1 USF2, and a-Pal/Nrf-1 with the 28. 29. Kunst, Catherine B., and Stephen T. Warren. "Cryptic and Polar Variation of the Fragile X Repear Could Result in Predisposing Normal Alleles." Cell 77 (1994): 853-861.

41. Sutcliffe, James S., David L. Nelson, Fuping Zhang, Maura Pieretti, C Thomas, Debra Saxe, and Stephen T. Warren. "DNA Methylation Represses FMR-1 Transcription in Fragile X Syndrome." Human Molecular Genetics 1 (1992): 397-400.

30. Li, Zhenzhong, Youyi Zhang, Keith D. Wilkinson, Stephen T. Warren, and Yue Feng. "The Fragile X Mental Retardation Protein Inhibits Translation Via Interacting with MRNA." Nucleic Acids Research 29 (2001): 2276-2283.

42. Christine K. Base, and William T. Greenough. "Fragile X Mental Retardation Protein is Necessary for NeurotransmitterActivated Protein Translation At Synapses." PNAS 101 (2004): 17504-17509.

31. Lu, Robert, Houping Wang, Zhe Liang, Li Ku, William T. O'donnell, Wen Li, Stephen T. Warren, and Yue Feng. "The Fragile X Protein Controls Microtubule-Associated Protein 1B Translation and Microtubule Stability in Brain Neuron Development." PNAS 101 (2004): 15201-15206.

43. Zalfa, Francesa, Marcello Glorgi, Beatrice Primerano, Annamaria Moro, Alessandra Di Penta, Surya Reis, Ben Oostra, and Claudia Bagni. "The Fragile X Syndrome Protein FMRP Associates with BC1 RNA and Regulates the Translation of Specific MRNAs At Synapses." Cell 112 (2003): 317-327.

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Review Article

Eukaryon, Vol. 3, February 2007, Lake Forest College

A Ride with Listeria monocytogenes: A Trojan Horse Joshua Haas*, Krista Kusinski*, Shruti Pore*, Solmaz Shadman* and Mithaq Vahedi* Department of Biology Lake Forest College Lake Forest, Illinois 60045

extensively in murine macrophages in the past 40-50 years. The study of the life cycle of LM in this experimental model has contributed significantly to the understanding of the immune response to intracellular pathogens (Portnoy et al., 2002). Listeriosis manifests itself through flu-like symptoms and can lead to diarrhea, meningitis, encephalitis, meningoencephalitis and stillbirths. In humans, it primarily infects immunocompromised individuals like pregnant women, neonates, and the elderly. (Portnoy et al., 2002, Vázquez-Boland et al., 2001, and Dyer et al., 2002). Listeriosis is more dominant in females because of differential production of the immunosuppressive cytokine IL-10 (Pasche et al., 2005). Immunocompetent individuals usually survive the infection, whereas those with debilitating diseases often die (mean mortality rate of 30-40%) (Vázquez-Boland et al., 2001). LM can cross the placental and blood brain barriers; however, in order to do so, it needs to pass the intestinal barrier and survive the harsh environment of the stomach. The primary method of entry into endothelial cells is believed to be via a zipper-like mechanism (Alberts et. al., 2003) Invasion proteins on the surface of the bacteria, like Internalin A, and Internalin B, and P60, help the bacterium bind to host surface receptors (Drevets et al., 2004). This binding induces phagocytosis of the bacteria into the host cell. Another mechanism by which LM is internalized is phagocytosis by macrophages. Once inside the host cells, the bacteria secrete a pore forming hemolysin, known as Listeriolysin O (LLO) and two distinct phospholipases, PI-PLC and PC-PLC. These hemolysins, along with the phospholipases mediate the degradation of the phagolysosome and the escape of the bacteria from the vacuole into the intracytoplasmic environment of the host cell. Once LM is in the cytosol, the ActA protein recruits host cell Arp2/3 complexes to enable efficient actin-based motility. Actin-based motility enables the bacteria to form filopods (pseudopod-like extensions) and propels it into neighboring cells, resulting in spread of the infection (Portnoy et al., 2002 and Vázquez-Boland et al., 2001). The immune response to L. monocytogenes is entirely cell mediated. CD8+ T-cells recognize and lyse infected cells. The NF-κB pathway is used to activate immune response genes and in the subsequent production of interleukins (IL’s) (Portnoy et al., 2002 and Vázquez-Boland et al., 2001). Current treatments of Listeriosis are intravenous administration of ampicillin and gentamicin. Treatment can last for 10 days, but depends on the body’s ability to fight the infection (www.kidshealth.org). This paper will review the current understanding of mechanisms underlying the internalization of Listeria monocytogenes, its subsequent escape from the phagolysosome, replication in the host cytosol and its actin-based motility (Fig 1). Currently available treatments will be discussed along with future experiments which could lead to more effective therapies.

Summary Listeriosis, a disease caused by Listeria monocytogenesa facultative, intracellular bacterium, spreads through contaminated food. It affects epithelial cells and macrophages and has a mortality rate of about 30%. The bacterium can cross the blood brain barrier, causing meningitis, and the placental barrier, causing abortion. Some mechanisms for entry into cells include the InlA- Ecadherin adhesion and InlB-Met pathway. hly, one of the many genes activated during infection, leads to the production of Listeriolysin O (LLO). LLO and two distinct phospholipases are indispensable to the spread of Listeria. Phosphatidylinositol-specific phospholipase C (PI-PLC) activates a host protein kinase C (PKC), which facilitates the escape of the bacterium from the primary vacuole, along with LLO. Once inside the cell’s cytoplasm, Listeria replicates. At this point, both the original bacterium and the daughter cells use Act A protein to exploit the cell’s machinery to polymerize actin. Actinbased motility propels the Listeria throughout the cell and facilitates its intercellular spread. Current curative methods include ampicillin, gentamicin, and chloramphenicol, reserved for life threatening infections. Treatment via plant extracts of Pluchea quitoc is in the experimental stage. This review focuses on tracking the progression of the L. monocytogenes bacterium from its entry to spread. The story of the Trojan horse: Introduction Listeria monocytogenes (LM) is a ubiquitous, facultative intracellular bacterium which can thrive in a variety of environments and hosts. It is a Gram-positive bacterium and spreads primarily through contaminated foods (Portnoy et al., 2002). Foods that are most commonly contaminated by Listeria are meats, milk, soft cheese, dairy products and industrially-produced refrigerated food products (Vázquez-Boland et al., 2001). LM can survive and proliferate in acidic environments, high salt concentrations and even at very low temperatures (www.textbookofbacteriology.net, Vázquez-Boland et al., 2001). Listeria microorganisms were first discovered in 1924 by E.G.D. Murray, R.A. Webb, and M. B.R. Swann, as the microorganisms causing a septicemic disease in rabbits and guinea pigs in their laboratory in England. However, the first case of the disease was reported in humans in Denmark in 1929 (VázquezBoland et al., 2001). An outbreak of Listeriosis, the infection caused by L. monocytogenes, in California, in 1985, claimed the lives of 18 adults and 30 fetuses, outlining the high mortality of this disease (www.textbookofbacteriology.net). LM has been studied

The horse looks like a present, but…: Listeria monocytogenes entry mechanisms

* This paper was written in BIO221 Cellular and Molecular Biology, taught by Dr. Shubhick DebBurman

LM being an intracellular bacterium, it is very important for this pathogen to gain entry into a cell in order to

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Figure 1: Model showing the entry and spread of Listeria monocytogenes The bacterium is phagocytosed by the host cell, using cell adhesion proteins. It escapes the vacuole by secreting a pore-forming toxin, Listeriolysin, and the action of phospholipases. Subsequent to its escape from the vacuole, LM acquires actin based motility, which enables it to form filopods and spread from cell to cell (Reproduced from http://textbookofbacteriology.net/Listeria.html).

the “N-terminal cap” and at the C terminus there is a conserved sequence known as the IR or Inter repeat region (Marrino et al., 2000). The LRR regions are structurally and functionally important to the internalization of LM (Dussurget et al., 2004).

replicate and thus cause Listeriosis. LM has many mechanisms by which it can gain entry into a cell. A widely studied pathway involves proteins of the Internalin family. Internalin (InlA) and B (InlB) are involved in two distinct pathways by which they allow the bacterium to enter the cell.

Bacterial Internalin (InlA) is a ligand for E-Cadherin receptors

Internalin (InlA) and Internalin B (InlB) dependent internalization of LM

InlA is involved primarily in the infection of epithelial cells. InlA, specifically, is anchored to the cell wall of the bacteria through a LPXTG motif in its carboxyl terminal region (Lecuit et al., 1997). The receptor for InlA is ECadherin. E-cadherin is a part of the cadherin superfamily of transmembrane glycoproteins that act as adhesion molecules. It is located at the adherens’ junctions and allows for the Ca2+ dependent adhesion of two cells (Dussurget et al., 2004). In addition, not all E-cadherins are receptors for InlA. Rat and mouse Ecadherins cannot bind InlA. This may be due to the absence of a proline at position 16 of the rat and mouse E-cadherins that allows for such specificity (Lecuit et al., 2001). In species that do present a favorable Ecadherin receptor, InlA interacts with the first two ectodomains(protein domains outside the cell) of Ecadherin. Specifically, the amino terminal region of InlA that contains the LRR and IR regions interacts with Ecadherin and is necessary and sufficient to promote the internalization of LM. Furthermore, it is the LRR region that directly interacts with the E-cadherin ectodomains, whereas the IR region is important in the folding of the LRR region (Lecuit et al., 1997). The carboxyl terminal of E-cadherin directly interacts with the intracellular β-catenin. α-catenin, in

The Internalin family of Listeria proteins is very large. It is composed of at least seven members including InlC, InlC2, InlD, InlE, InlF, InlG, and InlH (Marrino et al., 2000). The two members of this family that are relevant to entry of LM into the host cell’s are InlA and InlB. These proteins are found on the cell surface of LM and are involved in its internalization. Internalins bind specific receptors on the host cells surface and thus trigger phagocytosis of LM. Through these mechanisms, LM can induce phagocytosis in nonphagocytic cells in vitro. This phagocytosis is attributed to the reorganization of the actin cytoskeleton, which, in turn, leads to membrane folding (Marrino et al., 2000). These two proteins as well as other proteins that belong to this family have some structural features in common. Both proteins have a Leucine Rich Repeat region (LRR) and a β repeat region, as well as an Inter repeat region between the two (IR) that is extremely conserved (Lecuit et al., 1997). The LRR motif is involved in protein-protein interaction and is repeated in a highly regular fashion (Marrino et al., 2000). The LRR regions are flanked by highly-conserved sequences on either side that may play a role in the stability of the protein. At the N terminus there is a hydrophilic cap known as

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phagocytosis and membrane ruffling (Bierne et al., 2002). Gene Expression in the Intracellular Life Cycle of Listeria monocytogenes After the bacterium has been internalized, its gene expression changes with respect to its new environment. The virulence genes (prfA, plcA, hly ,mpl, actA, and plcB) located in the major virulence gene cluster are strongly regulated during intracellular growth (Chatterjee, et al., 2006). The PrfA gene is an autoregulatory positive regulatory factor required for the regulation of the virulence genes, in addition to expression of other genes elsewhere on the chromosome, like inlA. PrfA is regulated by the general stress-response alternative sigma factor σB, which plays a crucial role in the invasion of cells, but not the systemic spread (Garner et al., 2004). Genes important for the escape of bacteria from the phagolysosome to the intracytoplasmic environment are hly and plcA. hly encodes for the poreforming toxin listeriolysin O (LLO), while the plcA gene is responsible for the production of phosphatidylinositol phospholipase C. Both of these proteins are essential for the escape of the pathogen from the primary vacuole (phagolysosome) into the cytoplasm of the host cell. In a study conducted using murine macrophages, it was shown that ∆hly and ∆plcA double-deletion mutant Listeria are rapidly killed (Chatterjee, et al., 2006). It was also shown that these genes are up-regulated during the intracellular phase of growth. In an intracellular milieu, LM changes its normal sugar metabolism. Genes encoding enzymes in the second part of glycolysis were reduced during infection. Further, it was found that an operon encoding glycerol kinase and the glycerol uptake facilitator (lmo 1538 to lmo 1539) and glycerol-3-phosphate dehydrogenase (lmo 1293) were upregulated, indicating that glycerol was being used as the additional carbon source for intracellular growth. This could be a mechanism whereby the bacteria do not affect the host cell’s energy source and are able to spread more rapidly, rather than killing the host cell. Glucose also inhibits the expression of prfA, and hence the expression of the major virulence gene cluster (Chatterjee et al., 2006). Genes required for the normal replication of LM were downregulated. The ftsZ and ftsA genes, which are the major bacterial cell division determinants, were downregulated, suggesting lowered cell division. This lowered cell division activity is probably a result of the host cell’s defense mechanism to keep bacterial multiplication in check. Another important gene that is altered during the intracellular phase of LM’s growth is lmo0593, which is a nitrite transporter gene. The increased transport of nitrite by the bacteria suggests that nitrite is used in place of oxygen as the final electron acceptor in the electron transport chain. This mechanism allows the bacteria to survive under oxygen deprived conditions in the host cell (Chatterjee et al., 2006).

Fig 2: Model for InlA-dependent entry of LM into epithelial cells. Proteins known to play a role in entry are indicated, including Ecadherin, α and ß-catenins, vezatin, myosin VIIA, and actin. This model highlights how myosin VIIA could help the membrane rearrangements during LM entry. (Modified from Sousa et al., 2003)

turn, binds to β-catenin and interacts with actin (Lecuit et al., 2000) This interaction leads to the formation of a fusion molecule consisting of the ectodomains of the Ecadherin and the actin binding site of the α-catenin which eventually leads to LM entry (Dussurget et al., 2004). Furthermore, myosin VIIA and its ligand vezatin together function as the molecular motor in the internalization of Listeria. When myosin VIIA binds vezatin, coupled with an actin polymerization process, it provides the tension necessary for bacterial internalization (Fig. 2) (Sousa et al., 2003). InlB as a virulence factor for hepatocytes and other non-epithelial cells during Listeriosis InlB is a bacterial protein that is anchored to the cell wall of LM through a series of GW repeats (Lecuit et al., 1997). InlB has an elongated structure and its main receptor for LM invasion is the hepatocyte growth factor receptor, also known as Met (Dussurget et al., 2004). It is interesting to note that InlB does not strictly mimic the hepatocyte growth factor (HGF), in that HGF and InlB do not share sequence similarities. Once Met has been activated by InlB, it autophosphorylates two tyrosine kinase residues and recruits Gab 1, Sch and Cbl as well as PI 3-kinase. PI 3- kinase, which is known to be involved in control of the actin cytoskeleton, activates PLC-γ1 (Vazquez-Boland et al., 2001). The activation of Met is enhanced in the presence of glycosaminoglycans (GAG’s) (Bierne et al., 2002). These are normally involved in the oligomerization as well as storage and protection from extracellular proteases (Dussurget et al., 2004). Another receptor for InlB is some form of the surface associated gC1q-R. However, the specific mechanism by which InlB binds and interacts with gC1q-R remains to be determined. Since this protein lacks a transmembrane domain, it may act as a signaling co-receptor (Vazquez-Boland et al., 2001). In addition, the partial inhibition of InlB-mediated signaling pathway due to gC1q-R antibodies supports this hypothesis. InlB-Met signaling leads to both

There was no door, so how did they get out? : Escaping into the cell cytoplasm Once inside the host cell, the bacterium is enclosed by the phagosomal membrane. LM must have a way to escape the vacuole because this is where the bacteria replicate. It is here that LM uses the host cell

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(A)

(B)

Figure 3: Transmission Electron Micrographs of Listeria monocytogenes (A) Wild type Listeria monocytogenes free in the cytosol of a macrophage (size bar = 2µm). (B) A secondary macrophage with LM in a double membrane vacuole (size bar = 0.5µm) (Gedde et al, 1999).

In a study by Lety et al, it was shown that a PEST-like motif in LLO is required for the escape of LM from the vacuole and for causing virulence. The removal of this PEST-like sequence results in a strain that is extremely toxic to host cells and is 10,000 times less virulent in mice macrophages (Portnoy et al., 2002). In contrast to LLO and PFO, streptolysin O (SLO) was found to have a 10-fold lower activity. B. subtilis expressing SLO could not grow in the cytoplasm of host cells efficiently, presumably because they were unable to escape the phagolysosome. It is possible that SLO is less stable in an acidic environment and is not able to lyse the vacuole (Portnoy et al., 1992). Gedde et al used a genetic approach to investigate the role of LLO in intracellular growth and cell-to-cell spread. SixHis-tagged LLO (HisLLO), noncovalently bonded to the surface of nickel-treated LLO lacking LM, enabled some of these cells to escape the host vacuole and replicate in the cytoplasm. Both LLO lacking LM and wild type LM were able to replicate in the cytosol of the host cell (Fig 3A). LLO lacking LM could also spread to adjacent cells, however, these LM were trapped in doublemembrane vacuoles (Fig. 3B). Surprisingly, phospholipase C (PC-PLC) and PI-PLC were also not required in the spread of LLO negative LM into secondary cells.

machinery. In order for LM to enter the cytosol it first needs to escape the phagosome. Both LLO and two types of PLCs play a key role in mediating this escape. Virulence Factors: Listeriolysin O LM is one of the many bacteria that produce hemolysins. Listeria secretes Listeriolysin O (LLO), which, along with a phospholipase, PlcA, plays a very important role in the escape of the pathogen from a vacuolar compartment (Portnoy et al., 1992). Listeriolysin is a member of the thiol-activated cytolysins, which include perfringolysin O (PFO), streptolysin O (SLO), and pneumolysin, among others. These hemolysins are inhibited by free cholesterol and cysteine; free cholesterol is the common hemolysin receptor, and cysteine oxidation causes reversible protein inactivation (Portnoy et al., 1992). It has been shown that the expression of listeriolysin in an extracellular non-pathogenic soil bacterium, Bacillus subtilis, enables the bacterium to grow in the cytoplasm of mammalian cells. In a subsequent study, it was shown that LLO is not the only cytolysin that allows bacteria to proliferate in the cytoplasm of host cells. When B. subtilis expresses PFO, it is also able to escape the phagolysosome and replicate in the cytoplasm. However, unlike LLO, PFO causes damage to host cells (Portnoy et al.,1992,1994). It has also been shown that LM expressing PFO instead of LLO is much less virulent (Portnoy et al., 1994). This result is consistent with the observation that LLO works best in an acidic pH, whereas PFO functions in both acidic and neutral environments. The increased efficiency of LLO at a pH of 5.5 is a mechanism by which LM compartmentalizes the activity of LLO to escape the vacuole and subsequently replicates in the cytoplasm without causing damage to the host cell (Portnoy et al., 1992).

Phospholipases LM also uses phospholipases C to aid in the escape from the vacuole. Two specific phospholipases (PLCS) are used. One is the phosphatidylinostiolspecific PLC (PI-PLC), and the other is more general, phosphatidylinostiol-specific (PC-PLC) (Portony, et al., 2002). LLO has been studied in great detail as the major factor for the permeation which leads to phagosomal escape. Studies have also attributed PCPLC to the escape even in the absence of LLO (Sibelius et al., 1992).

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The role of the PI-PLC secreted by LM is to catalyze the production of inositol phosphate and diacylglycerol (DAG) through cleavage of the membrane lipid PI. DAG then has the ability to activate protein kinase C (PKC). There are four types of PKCs, but the PKC β of the host is shown to be linked with the PI-PLC signaling cascade. The PKC β has been shown to facilitate the permeation of the phagosomal membrane before the bacteria escape (Poussin et al., 2005). PI-PLC has another component which allows for the escape of the bacterium into the cytosol. It is known that LM has a weaker effect on the GPIanchored proteins than most bacteria do. This has been found to be the result of the PI-PLCs of LM differing from those of other bacteria. There is a Vb β-strand which is found in other bacterial PI-PLCs which is absent from L. monocytogenes. The Vb β-strand is known to give a contact for the glycan linker of GPIanchored protein. This contact enhances the ability of the cell to cleave the GPI anchors. When Vb β-strand was absent in LM, the cell’s ability for the bacteria to escape the phagosome and be released into the cytosol of the host cell was increased. It is through these observations, as well previous knowledge of Vb β-strand, and PI-PLCs that it can be hypothesized that LM have evolved this absence or loss in order to promote growth inside the host cell (Wei et al., 2005). PI-PLC and PC-PLC have also been shown to have an activating effect on immune response during LM infection. NF-κB is a transcription factor which can be used by a number of genes. Some of these genes are activated during infection. One which is activated and is specific to LM is Listeriosis biphasic NF-κB activation. This phase of NF-κB is regulated by IκBβ. When IκBβ is degraded NF-κB becomes active. This activation can be seen as a result of the degradation of IκBβ by lipoteichoic acid (LTA), but also through the listerial PI-PLCs and PC-PLCs which is in correlation with the IκBβ degradation. The NF-κB transcription factor can then be used by LM to enter the host cell. LM can then use the host cell machinery for its own replication. The nuclear NF-κB complexes which are formed can therefore be attributed to the effect of the Listerial PLCs on the host cell (Hauf et al., 1997). In summary, the secretion of PLCs during listerial infection has several effects on the host cell. One of these effects has been shown to increase the permeation of phagosomal membrane. The activation of PKC β through PI-PLC facilitates the escape of the bacterium. The decreased affinity of L. monocytogenes for the glycan linker of the GPI-anchored protein due to the lack or absence of the Vb β-strand also increases the ability for the bacterium to escape during infection. The degradation of IκBβ by listerial phospholipases PIPLC and PC-PLC leads to the activation of NF-κB, which allows the bacteria to exploit the host cell machinery.

(Portnoy et al., 2002). The ActA protein activates the Arp2/3 complex by mimicking proteins of the WASP family. Research is underway to understand the nature of the binding between ActA and the Arp2/3 complex. In order to determine what is most important in actin polymerization, many different proteins and their functions have been examined in past studies. The Apr2/3 complex has been found to organize signals of actin cytoskeletons and initiate actin assembly because of its specific function. The Arp2/3 complex is comprised of the actin related proteins Arp2 and Arp3, along with p41-Arc, p34-Arc, p21-Arc, and p16-Arc. Arp2 and Arp3 both have insertions in loops that are exposed to the cytosol, and Arp2 contains a profiling binding site. However, neither Arp2 nor Arp3 are capable of the polymerization of actin alone. The Arp2/3 complex nucleates actin filaments, elongating the barbed ends. Also, the complex binds to other actin filaments and produces a branching formation when present at a filament pointed end. Once the polar actin tails become long enough, LM is propelled inter/intracellularly with actin-based motility (Fig 4). Why Only Act A Is Needed WASP family proteins become activated by interactions with Cdc42 and PIP2 region and other proteins. This interaction opens the given protein exposing the C-terminal region. The Arp2/3 complex and the C-terminal ends of these proteins interact. Arp2/3 binds to the CA-like region which all of the WASP family proteins share. Equivalent regions of the Act protein are also capable of activating the Arp2/3 complex in this way. Because of this unique ability to activate the complex, LM can by-pass the machinery for polymerization and directly interact with the complex. The complex attracts G-actin or F-actin to further actin polymerization. In addition to the Arp2/3 complex, other host cell proteins are involved in actin polymerization. Research has found that VASP proteins bind to the proline-rich region of ActA at the EVH1 domain. This was proven by mutating VASP proteins, which resulted in aslower rate bacterial locomotion. Many different ligands exist for the ActA protein. Therefore, ActA has more than one mechanism for polymerizing actin and eventually for locomotion. Listeria monocytogenes Infects Neighboring Cells After polymerizing actin, Listeria combines other mechanisms in order to spread to neighboring cells. In the areas of newly polymerized actin, two molecules have been found to concentrate. Phosphatidylinositol 3,4,5-biphosphate (PtdIns(4,5)P3) and phosphatidylinositol3,4,5-biphosphate (PtdIns(4,5)P2) play essential roles in the actin-based motility of LM. Recent studies have found that reducing the amount of PtdIns(4,5)P3 and PtdIns(4,5)P2 with AktPH-GFP and PCLδ-PH-GFP, respectively, significantly slows the movement of actin within a cell. As expected, this also inhibits the filopod formation process. When a PI 3-Kinase was used to allow the concentration of PtdIns(4,5)P3 by degrading Akt-PH GFP, but not PCLδ-PH-GFP, actin based motility was completely inhibited. When this PI 3-kinase was removed, full recovery of actin based motility and filopod formation was observed. The specific kinase used was LY294002. These results imply that

Charge! The Main Act: ActA Protein Fills the Role Quickly after Listeria enters the host cell’s cytosol, the protein ActA interacts with the cell’s proteins to mediate actin-based motility. ActA is a 610amino-acid protein containing a charged N-terminal end, proline rich repeats, and a C-terminal end to anchor the protein to the bacteria surface (Cossart et al., 2000). Specifically, ActA directly activates the Arp2/3 complex, followed by other proteins that exploit the host cell’s machinery for actin polymerization

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Figure 4: Activation of the Arp2/3 complex by the ActA protein Recruitment of the Arp2/3 complex by the ActA protein followed by nucleation and elongation of the actin tail. Activation of the Arp2/3 complex results in propulsion of LM.

PtdIns(4,5)P2 is the substrate for formation of PtdIns(4,5)P3. Thus, the newly formed actin polymers (formed through the recruitment and activation of the Arp2/3 complex by Act A proteins) dissociate when PtdIns(4,5)P2 and PtdIns(4,5)P3 are not present. This charged actin filament polymerization allows for the directional force, actin-based motility, through the cells’ cytoplasm. Eventually the bacteria are propelled to the peripheral membrane of the host cell. Enough force is present to actually push the membrane outward, forming distinct filopods on the host cell using polymerized actin as support (Fig 5). The filopods are then ingested by adjacent cells, and the cell cycle continues to repeat. Filopod formation and the survival of newly polymerized actin is directly correlated with the presence of PtdIns(4,5)P2 and PtdIns(4,5)P3 (Vingjevic et al., 2003). Also, the PI 3-Kinase activity can play a major inhibitory role in the actin-based motility of LM and formation of filopods (Sidhu et al., 2005). The formation of filopods, as well as the specific interactions of the proteins involved, is still being researched.

bacteriostatic, for L. monocytogenes (Taege et al., 1999). Often ampicillin is combined with gentamicin for synergy (Kamath et al., 2002 and Taege et al., 1999). With the interaction of these two agents, the overall effect will be greater than the sum of their individual effects.While ampicillin targets the cell wall; gentamicin hampers metabolic actives in the bacteria. Gentamicin operates by binding to a site located on the bacterial ribosome, which results in the misreading of the genetic code. For patients who are allergic to ampicillin, an alternative treatment option including Trimethoprim and sulfamethoxazole may be used to treat Listeriosis. Trimethoprim interferes with the action of bacterial dihydrofolate reductase to prevent the synthesis of folic acid, which is an essential precursor in the new synthesis of the DNA nucleosides thymidine and uridine. When the bacterium is unable to take up folic acid from the environment, enzyme inhibition starves the bacteria of two bases necessary for DNA replication and transcription (Taege et al., 1999). These two antibiotics are used in a combination known as cotrimoxazole. Co-trimoxazole works by inhibiting the successive steps in folate synthesis (http://www.pubmedcentral.nih.gov). Other treatments include chloramphenicol, which is reserved for life-threatening infections (Wei et al., 2005). This antibiotic has acute side-effects, including damage to bone marrow in humans. Chloramphenicol is used by the World Health Organization (WHO) in third world countries in the absence of cheaper alternatives. Chloramphenicol operates by stopping bacterial growth by hindering the ribosomal enzyme peptidyl transerase which assists in the formation of peptide links between amino acids during the translation process of protein biosynthesis (Wei et al., 2005).

Figure 5: Formation of Filopods and cell to cell spread. Filopod formation and entry into adjacent host cells is mediated by actin-based motility.

Current Experimental Treatments via Plants

Attacking the Listeriosis

Trojan

horse:

Treatments

Treatment via plant extracts from Pluchea quitoc is in the experimental stage, studying the effects it has on Listeria infection. Pluchea quitoc extract is a well known remedy used in South American traditional medicine for the treatment of digestive diseases. In research, it has demonstrated strong anti-inflammatory and antioxidant activities. An experiment to study the effects of the P. quitoc extracts determined that it is helpful in defending the host against L. moncytogenes infection by increasing the number of leukocytes in the model mice (Queiroz et al., 2000). Results indicated that the administration of P. quitoc increased hematopoietic recovery in the mice infected with LM. The increase in the number of.

for

To diagnose Listeriosis, blood or cerebrospinal fluid cultures are used to establish bacteterium growth, and characterization of the bacterium (Taege et al., 1999).The most common and current curative method of treating Listeriosis includes the use of ampicillin. Ampicillin works by targeting the cell wall of the bacterium, inhibiting the third and final stage of bacterial cell wall synthesis, which ultimately causes the cell to lyse. However, ampicillin is only

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Figure 6: Model showing current understanding of Listeria monocytogenes infection in host cells. Bacterial entry into the cell is facilitated via the internalin adhesion proteins. Listeriolysin O (LLO) and PI-PLC’s mediate lysis of vacuoles and escape of the bacteria into the cytoplasm. Recruitment of actin filaments aids in bacterial movement and spread (Reproduced with modifications from Vaquez-Boland et al, 2001).

leukocytes enhances the host’s ability to defend itself against the infection. It is still unclear what mechanism improves the survival of mice that are treated with P. quitoc, however, when this mechanism is discovered, further studies could make these extracts a useful remedy for humans.

and should be cited as such only with the consent of the author. References Alberts, Johnson, Lewis, Raff, Robers, and Walter. Molecular Biology of the Cell. Garland Signs: New York. 2002: 1446-1448. Bierne, Helene, and Pascale Cossart. "InIB, a Furface Protein of Listeria Monocytogenes That Behaves as an Invasin and a Growth Factory." Journal of Cell Science 115 (2002): 3357-3367.

Recapping the story of the Trojan Horse: Summary Figure 6 illustrates the pathway and specific components through which LM enters, replicates and moves in mammalian cells. The main mechanisms for entry that have been described are the E-cadherin mediates Internalin pathway and the Met mediated InlB pathway. Furthermore, LLO and PC- PLC play the major role in the escape of LM from the vacuole. Once inside the cytosol, LM uses actin polymerization to gain motility and spread from cell to cell. Ampicillin, gentamicin, chloramphenicol and co-trimoxazole are currently used to treat listerial infection.

Chatterjee, Som Subhra, Hamid Hossain, Sonja Otten, Carsten Kuenne, Katja Kuchmina, Silke Machata, Eugen Domann, Trinad Chakraborty, and Torsten Hain. "Intracellular Gene Expression Profile of Listeria Monocytogenes." Infection and Immunity 74 (2006): 1323-1338. Cronan, Kate. "Listeria Infection." Nov. 2005. . Drevets, A.D., Leenen, P. J. M., Greenfield, R. A. "Invasion of the Central Nervous System by Intracellular bacteria." Clinical Microbiology Reviews 17(2004): 323-347. Dussurget, Oliver, Javier Pizarro-Cerda, and Pascale Cossart. "Molecular Determinants of Listeria Monogytogenes Virulence." Annual Review of Microbiology 58 (2004): 587-610. Dyer, Neil W., and Charles L. Stoltenow. "Listeriosis." Public Health Watch:Focus on Agreculture. Feb. 2002. 10 Apr. 2006 .

Acknowledgments We would like to thank Dr. Shubhik K. DebBurman for his guidance and support throughout this endeavor. We are grateful to Katrina Brandis for reviewing our paper and for her invaluable comments. We would also like to thank Michael Zorniak, Michael Wollar and Jenny Riddle for their assistance in writing this review article. Our thanks also go to Nelka Fernando, David Piper, Sina Vahedi and Lokesh Kukreja.

Garner, M R., B L. Njaaa, M Wiedmann, and K J. Boor. "Sigma B Contributes to Listeria Monocytogenes Gastrointestinal Infection But Not to Systemic SPread in the Guinea Pig Infection Model." Infection and Immunity 74 (2006): 876-886. Gedde, Margaret M., Darren E. Higgins, Lewis G. Tilney, and Daniel A. Portony. "Role of Listeriolysin O in Cell-to-Cell Spread of Listeria Monocytogenes." Infection and Immunity 68 (2000): 999-1003. Hauf, Nadja, Werner Goebel, Franz Fiedler, Zeljka Sokolovic, and Michael Kuhn. "Listeria Monocytogenes Infection of P388D Macrophages Results in a Biphsic NF-KB (RelA/P50) Activation Induced by Lipoteichoic Acid and Bacterial Phospholipases and Mediated by IKBalpha and IkBbeta Degradation." The National Academy of Sciences 94 (1997): 9394-9399.

Note: Eukaryon is published by students at Lake Forest College, who are solely responsible for its content. The views expressed in Eukaryon do not necessarily reflect those of the College. Articles published within Eukaryon should not be cited in bibliographies. Material contained herein should be treated as personal communication

Jones, Sian, and Daniel A. Portnoy. "Characterization of Listeria Monocytogenes Pathogenesis in a STrain Expressing Perfringolysin O in Place of Listeriolysin O." Infection and Immunity 62 (1994): 5608-5613.

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Kamath, Binita M., Petar Mamula, Robert N. Baldassano, and Jonathan E. Markowitz. "Listeria Meningitis After Treatment with Infliximab." Journal of Pediatric Gastroenterology of Nutrition 34 (2001): 410-413.

Poussin, Mathilde A., and Howard Goldfine. "Involvement of Listeria Monocytogenes Phosphatidylinositol-Specific Phospholipase C and Host Protein Kinase C in Permeabilization of the Macrophage Phagosome." Infection and Immunity 73 (2005): 4410-4413.

Lecuit, Marc, Helene Ohayon, Laurence Braun, Jerome Megaud, and Pascale Cossart. "Internalin of Listeria Monogytogenes with an Intact Leucine-Rich Repeat Region is Sufficient to Promote Internalization." Infection and Immunity 65 (1997): 5309-6319.

Queiroz, Mary L. S., Giselle Z. Justo, Fatima R. R. Pereira-Da-Silva, Adolfo H. Muller, and Giselle M. S. P. Guilhon. "Stimulatory Action of Pluchea Quitoc Extract on the Hematopoietic Response During Murine Listeriosis." Immunopharmacology and Immunotoxicology 22 (2000): 721740.

Lecuit, Marc, Reinin Hurme, Javier Pizzarro-Cerda, Helene Ohayon, Benjamin Geiger, and Pascale Cossart. "A Role for Alpha- and BetaCatenins in Bacterial Uptake." The National Academy of Science 97 (2000): 10008-10013.

Sibelius, Ulf, Eva-Cathrin Schulz, Frank Rose, Katja Hattar, Thomas Jacobs, Siegfried Weiss, Trinad Chakraborty, Werner Seeger, and Friedrich Grimminger. "Role of Listeria Monocytogenes Exotoxins Listeriolysin and Phosphatidylinositol-Specific Phospholipase C in Activation of Human Neutrophils." Infection and Immunity 67 (1999): 11251130.

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Review Article

Eukaryon, Vol. 3, February 2007, Lake Forest College

Nanotechnology may replace existing treatments for cancer The cell cycle can be perpetuated through two types of genetic mutations: oncogenes and tumor suppressor genes (Kruh et al., 2000). Tumor suppressor genes normally are involved with the repair of damaged DNA. Thus, whenever these genes are inactivated, damaged DNA is not properly repaired (Moossa et al., 1990). According to Ames and Gold (1991), every cell in the body experiences 105 DNA damaging events daily. Thus, the regulatory process of repairing DNA is an active and important process. Tumor suppressor genes can be broken down into two categories: caretakers and gatekeepers (Kruh et al., 2000). Gatekeepers have a direct roll in controlling cellular proliferation, while caretakers help preserve the integrity of the genome by preventing mutations from occurring. An inactivated caretaker does not lead directly to tumor initiation, but instead it causes genetic instability, which causes subsequent mutations. In contrast, inactivated gatekeepers play a more direct role in the tumorigenesis process (Kruh et al., 2000). While tumor suppressor genes are dangerous when inactivated, oncogenes are only hazardous when active, at which point they are capable of inducing cancer in normal cells (McKinnel et al., 1998). Due to this, oncogenes are highly regulated in the body. Additionally, oncogenes have a wide variety of functions. For instance, some encode for growth factors that increase the proliferation of cells, others bind to DNA and regulate transcription, and yet others code for receptors or ligands involved in the cell cycle (Kruh et al., 2000). If over expressed, however, all of them can contribute to the development of cancer by promoting cell division (McKinnel et al., 1998). Tumorigenesis, or tumor formation, is a multistep process requiring more than one active oncogene or inactive tumor suppressor gene. If a group of cells has a small number of these mutations, a benign tumor may form. These tumors lack the ability to metastasize or spread to other parts of the body. However, if the benign tumor has more mutations, it is possible for it to become malignant (McKinnel et al., 1998). The process of carcinogenesis involves four steps. The first step is initiation, in which a carcinogen reacts with DNA causing a strand break or altering a nucleotide to form an adduct (McKinnel et al., 1998). Normally, a DNA polymerase repairs this problem, however, if the DNA replicates before the repair, the error can be permanently fixed into the genome (Kruh et al., 2000). Most errors of this type have no real effect on the body, but if a tumor suppressor is inactivated or an oncogene activated, the cell has a significant growth advantage, and the next step, promotion, may begin. During promotion, a molecule called a promoter causes selective proliferation, which may lead to the formation of multiple benign tumors (Alberts et al., 2003). Through one or more additional genetic alterations, the third step, known as progression, may occur. In this step, the tumor cells develop a significant growth advantage, which is so strong that they are able to break through the blood vessel membrane and travel to other areas through the process of metastasis. This actual conversion is the last step, and is referred to as malignant conversion (McKinnel et al., 1998). This further establishes the importance of multiple mutated tumor suppressor genes and

Ethan Helm* Department of Biology Lake Forest College Lake Forest, Illinois 60045 In 2002, 23% of all deaths in the United States were caused by cancer making it the second biggest killer, only ranking behind heart disease (Jemal et al., 2005). Every year, over a half million Americans die of cancer and more than a million are diagnosed with the disease. It is also the second biggest killer among children, with nearly 12% of all childhood deaths coming from the disease (Jemal et al., 2005). Cancer is a disease in which cells proliferate uncontrollably (Campbell et al., 2002). Unlike most cells, cancerous cells do not display density dependent growth, meaning they divide with little spatial regulation (Moossa et al., 1990). Moreover, these cells have the ability to spread by breaking into blood vessels and moving to other systems (Moossa et al., 1990). Cancer can be fatal due to a combination of its properties. For instance, cancerous cells lose their ability to function normally. That is, they stop responding normally to cellular signals and therefore no longer perform their job (McKinnel et al., 1998). Not only do cancer cells cease working, they also affect neighboring cells because cell division and metabolism require nutrients and energy; eventually the cells require more nutrients than the body can provide and slowly organ systems begin to fail, a process known as cachexia (American Cancer Society, 2000). Additionally, the growths themselves can cause immense pain or death in hollow organs (such as the colon) by blocking the lumen and preventing proper function. Moreover, tumors can cause pressure on the brain which can lead to brain failure, seizures, or partial lack of function depending on the location of the tumor (McKinnel et al., 1998). The formation of cancer requires several genes to be altered through mutations, which can be caused by spontaneous errors in replication or by exposure to carcinogens that alter nucleotides or break the DNA strand. In order for a mutation to lead to cancer, it has to perpetuate the cell cycle (Kruh et al., 2000). The cell cycle is a highly regulated process that ultimately results in the division of one cell into two (Campbell et al., 2002). In somatic cells, this cycle includes four phases: G1, S, G2, and mitosis (M). During G1 phase, the cell grows as it prepares for DNA synthesis, S phase. Then in G2 phase, the cell grows in preparation for mitosis, in which the replicated DNA is equally divided into two newly formed daughter cells (Campbell et al., 2002). Errors in the cell cycle are normally corrected during specific checkpoints at G1 to S, intra-S phase, and S to M. At these points, the cell cycle is temporarily arrested while regulatory enzymes ensure that there are no errors in the DNA sequence. If an error is found, the DNA damage is either repaired or the cell is tagged by a marker protein to commit suicide through apoptosis (Alberts et al., 2003). If inhibited, the cell cannot properly identify damage, and the cell cycle continues without the appropriate regulation (Kruh et al., 2000). *This paper was as part of an independent study on Oncology.

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oncogenes in cancer development. In other words, the growth advantage brought about by one mutation is not significant enough to overcome the natural immunity of the body. Tumors of this nature are contained because they are unable to break into the blood vessels (McKinnel et al., 1998). However, through multiple mutations, the growth advantage may be increased sufficiently to break through blood vessel membranes (McKinnel et al., 1998). For many years, scientists had no clue how to deal with this growth advantage. As a result, cancer was virtually untreatable, and even today, many types have no specific treatment. Chemotherapy’s potential to treat cancer was discovered during December of 1943, when an Allied warship holding mustard gas exploded (Williams, 2000). As a response to this, the army performed autopsies on the soldiers, which showed that their bone marrow had been destroyed by the gas, thereby inhibiting the production of red blood cells, white blood cells, and platelets. Accordingly, scientists hypothesized that the chemical may be used to fight cancer. To test this hypothesis, a chemical derived from mustard gas, known as mustine, was given to Hodgkin’s disease patients and, even in some patients with late-stage Hodgkin’s, the disease responded to the drug (Williams, 2000). In fact, this drug is still a key component of the MOPP (mustine, vincristine, procarbazine, and predinisone) regimen (Rüffer et al., 1998), which is one of the two primary treatments for Hodgkin’s disease, the other being ABVD (Adriamycin, bleomycin, vinblastine, and dacarbazine) (Kennedy et al., 2003; Murphy et al., 1997). Unfortunately, the treatments commonly used for cancer (radiation and chemotherapy) are both deleterious to the health of patients, and can actually cause death themselves by weakening the immune system and making patients more susceptible to other diseases (Schnell et al., 2003). The problem with these treatments is that they are not selective. That is, they act on all rapidly dividing cells causing the most recognizable symptom of cancer treatment: loss of hair. These treatments also inhibit the production of erythrocytes and white blood cells, causing patients to become anemic and neutropenic (Schnell, 2003). Anemia, a state of insufficient O2 delivery to tissues, can cause problems with blood clotting, as well as lead to dizziness and lethargy. Neutropenia refers to a decrease in the number of neutrophils in the blood signifying a weakened immune system. When neutropenic, patients are more susceptible to secondary infections; even a common cold can be fatal. Furthermore, chemotherapy triggers neuroreceptors, such as those that bind dopamine and serotonin, which stimulate nausea and cause vomiting (Schnell, 2003). Not only are chemotherapy and radiation dangerous, they also are not completely effective. According to Dr. Frank Balis, “We attribute our inability to cure many adults with more common forms of solid tumors to the ineffectiveness of chemotherapy to these diseases” (1998). In fact, the average five year survival rate among all cancers in the United States is only 63% (Jemal et al., 2005). Thus, newer and more effective treatments are being sought by scientists and pharmaceutical companies alike. In the last few years, the field of nanotechnology has exploded as some scientists believe tiny objects known as nanoparticles may be able to help treat a variety of diseases, including cancer. By definition, nanoparticles can range in size from 1 to 100 nanometers (Cervellino et al., 2005). The

nanoparticles being studied have a variety of compositions, shapes, and sizes. The most common composition includes either a carbon backbone or the presence of an inorganic metal, such as a gold (Zharov et al., 2003). Recently, scientists have discovered that nanoparticles can easily enter cells. However, it is uncertain how this occurs. Dai et al. (2005) claims the influx of nanoparticles occurs by endocytosis. In contrast, Bianco et al. (2005) suggest the process happens through insertion and diffusion of particles through the lipid bilayer of the cell membrane. Furthermore and surprisingly, these particles can be linked to proteins, such as antibodies, and still enter cells (Dai et al., 2005). Fortunately, cancer cells express certain receptors that are not expressed by normal cells. Thus, nanoparticles attached to antibodies for these receptors can be directed to cancerous cells exclusively (Dai et al., 2005). The ability of nanoparticles to selectively enter cancer cells has duel significance. Firstly, nanoparticles can work as drug deliverers. For instance, by linking certain proteins, such as tumor necrosis factor (TNF), a protein with known antitumor activity, to the particles a new mechanism for fighting cancer can be utilized (Paciotti et al., 2004). Secondly, nanoparticles have been shown to absorb different wavelengths of light than the body, and when exposed to appropriate wavelengths nanoparticles heat up, but the body does not. This method, known as hyperthermia, can be used to selectively kill cancer cells by heating nanoparticles that are linked to antibodies (Ito et al., 2003a). The specificity of these techniques is key, because unlike the deleterious effects of chemotherapy and radiation, treatment with nanoparticles should result in no major side effects. Furthermore, in preliminary studies, hyperthermia and drug delivery have both been successful, and currently, both hyperthermia and drug delivery are being heavily investigated as treatments for cancer (Dai et al., 2005; Onishi et al., 2003). The purpose of this review is to discuss the nanoparticle techniques of hyperthermia and drug delivery and determine whether they may one day replace the current techniques of chemotherapy and radiation as a treatment for cancer. Imaging to Detect Cancer Cells Beyond having the power to treat cancer, nanoparticles may also be used to detect the disease. Moreover, some therapies hope to utilize hyperthermia in such a way that diagnosis and treatment can occur together. There are several techniques scientists are investigating to improve cancer detection and couple it with hyperthermia (Loo et al., 2004). One popular technique involves attaching bioconjugates, such as antibodies, to the nanoparticles. Loo et al. (2005) attempted to analyze this technique by utilizing the tendency of breast carcinoma cells to overexpress the HER2 biomarker. Thus, by conjugating an antibody of HER2 to a PEG linker complex, which enhances biocompatibility and blood flow, and then attaching the complex to a gold nanoshell, the particle is linked exclusively to breast cancer cells (Loo et al., 2005). Using this, Loo et al. (2005) cultured three types of cells: cells with the anti-HER2/PEG/nanoshell complex, cells with a non-cancer specific antibody, and cells without nanoshells. These cells were viewed with

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Figure 1. Imaging and hyperthermia using nanoparticles. Imaging and therapy of SKbr3 breast cancer cells using HER2 linked nanoshells. Top row: darkfield imaging of of HER2 expression based on light scattering. Bottom row: cell viability assessed through calcein staining with exposure to ~820 nm near infrared (NIR). Cell death was observed only in cells treated with anti-HER2 nanoshell take from (Loo et al., 2005).

a darkfield microscope sensitive to scattered light, and only the Anti-HER2 cells showed much light scattering (Figure 1). In contrast, the cells with the non-specific antibody showed some light scattering, but it was not as dense. This illustrates that the Anti-HER2 treated cells attached exclusively to cancer cells, and exposure of light identified cancer cells. Furthermore, when treated with near-infrared (NIR) light of around 800 nm, cytotoxicity was observed only in the presence of the cells treated with Anti-HER2 nanoshells (Figure 1) (Loo et al., 2005). Thus, the hyperthermia treatment was successful, but only with the Anti-HER2 treated cells.

protein 70 (HSP70), in conjunction with hyperthermia with MCLs. Expression of this protein protects cells from heat-induced apoptosis (Mosser et al., 2000), but recently, it has also been shown to be a key component in immune reactions (Srivastava et al., 1998). To analyze HSP70 gene therapy combined with hyperthermia, Ito et al (2003a) analyzed how mice with malignant melanoma reacted to tumors that had been given a plasmid containing human-inducible hsp70 complimentary DNA. The primary finding was that hsp70 gene transfer successfully boosted the immune system during hyperthermia (Ito et al., 2003a). They determined this by comparing tumor size after exposure to hsp70 containing plasmid, hyperthermia, and the combined treatment. Both treatments alone showed improvement, but in each case, additional treatments would be required because the tumors began to grow again at around the tenth day. The combined therapy, however, completely eradicated cancer in 3 of the 10 mice with only one treatment. Because hyperthermia can be used multiple times without any negative effects, it is believed that the cancer could have been eradicated in the other mice with subsequent treatments. Moreover, tumors with the combined therapy were 16 times smaller than the hyperthermia only treated tumors after thirty days, and 24 times smaller than the tumors given hsp70 (Ito et al., 2003a).

Hyperthermia to Kill Cancer Cells As mentioned earlier, hyperthermia is the killing of cells through the heating of nanoparticles. One of the problems of hyperthermia is containing the heat in such a way that it does not affect other cells. To combat this, scientists use specific types of nanoparticles for hyperthermia, such as magnetite cationic liposomes (MCLs) (Kobayashi et al., 2005). These spherical particles contain a positively charged phospholipid exterior that interacts with the negatively charged cell surface, easily entering cells. The inside of the MCLs is a 10 nm magnetite nanoparticle (Kobayashi et al., 2005). Additionally, these particles have maintained the ability to bind to antibodies and can provide tumorspecific contrast enhancement.

Hyperthermia with Dendritic Cell Addition The use of immune triggering proteins is not the only way to boost anti-tumor activity. For instance, mature dendritic cells (DC) are an integral part of a normal immune response, which stimulate the growth of CD4+ T cells, CD8+ cytotoxic T lympocytes, and natural killer cells (Palucka et al., 1999). Unfortunately, mature DCs cannot take up antigen, and thus addition of these cells would not result in the proper immune response. Injection of immature DCs, however, has been reported to cause antitumor activity (Celluzzi et al., 1998). Tanaka et al. (2005) decided to go straight to the source by actually adding additional dendritic cells (DC) after mouse EL4 T- lymphoma tumors were treated with hyperthermia. While only 1 in 8 of the mice

Gene Therapy/Hyperthermia Combination Hyperthermia appears to be effective in some cases by itself, however, in advanced stages of several types of cancer, such as melanoma, it may not be sufficient (Ito et al., 2003a). Furthermore, to treat cancer, hyperthermia requires many treatments. However, in conjunction with other processes, scientists hope to find a way to use one round of hyperthermia to eradicate the disease. The combination therapies revolved around the use of substances to boost anti-tumor immunity. Thus, in addition to hyperthermia, the cancer cells will be assaulted by a revamped immune system (Ito et al., 2003b). Ito et al. (2003a) have been analyzing the use of one such protein, heat shock

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treated with hyperthermia alone had complete tumor regression, 6 in 8 of the mice treated with hyperthermia and immature DCs had complete tumor regression. Based on this, it appears the tumor cells killed by hyperthermia release antigen proteins which the immature DCs take up and are then presented to T cells via MHC class I and/or II antigens (Tanaka et al., 2005).

the drugs not only have increased cytotoxic activity, but also adverse side effects are limited (Alberts et al., 1985). Doxorubicin Doxorubicin hydrochloride (Dox), also known as adriamycin, is a cytotoxic anthracycline that is an essential component of chemotherapeutic regimens used to treat acute lymphoblastic leukemia, breast carcinoma, Hodgkin’s and Non-Hodgkin’s lymphoma (Murphy et al., 1997). The drug works by halting DNA replication, and thereby preventing further proliferation of the disease (Reddy et al., 2004a). Fortunately, Dox’s anti-tumor activity has been widely documented, and there is no reason to think it would behave differently if attached to a nanoparticle. At the same time, intravenous treatment of Dox causes systemic toxicity that can cause severe diarrhea, neutropenia, anemia, hair loss, and heart damage. Thus, scientists are investigating the use of different types of nanoparticles that can be used to deliver Dox directly to cancer cells, ultimately preventing systemic toxicity (Wilkes et al., 2000). Reddy and Murthy (2004a) investigated this by analyzing two different polymerization techniques for making polybutyl cyanoacrylate (PRC) nanoparticles: dispersion polymerization (DP) and emulsion polymerization (EP). The result of each polymerization technique produced structurally similar molecules. The difference, however, was that the EP nanoparticles were smaller. Therefore, Reddy and Murthy (2004a) sought to find out whether the size difference of the PRCs affected the nanoparticles’ ability to deliver Dox. They found that EP particles provided a longer half-life of Dox in the blood and a lower tissue distribution, which is consistent with their previous finding that EP nanoparticles have enhanced permeability and retention effects (Murthy and Harivardhan, 2003). Conversely, DP nanoparticles were quickly cleared into the RES. Both techniques demonstrated a significant increase in bioavailability of Dox compared to intravenous injection of Dox solution (Reddy and Murthy., 2004a). Together, the experiment identified the EP nanoparticles as a potential method of improving Dox therapy by reducing systemic toxicity (Reddy and Murthy, 2004a). Following the polymerization study, Reddy et al. (2004b) examined the affect of Doxorubican loaded poly(butyl cyanoacrylate) (DPBC) nanoparticles on Dalton’s lymphoma. They found that the DPBC nanoparticles sequestered in the tumor after subcutaneous injection much better than did free Dox. Additionally, they noted that there was a low amount of Dox found in the heart from the DPBC nanoparticles, and confirmed that Dox delivered by DPBC nanoparticles has an increased retention time within tumors. This confirms the results of the previous experiment, and also shows that cardiac toxicity may be limited through this technique. Ma et al. (2004) developed another type of nanoparticle to be used for Dox delivery to tumor cells. The particles, known as carbon magnetic nanoparticles (CMNP), were created using a new technology known as dense medium plasma (DMP) technology. The particles consist of a carbon-based host structure with iron and iron oxide particles evenly dispersed (Ma et al., 2004). The CMNP-Dox and intravenous free Dox were applied to osteosarcoma cells to test antiproliferative activity. The results showed that at the highest dose,

Drug Delivery Using Nanoparticles Drug delivery is the carrying of drugs using nanoparticles specifically to the cells causing the disorder. In the case of cancer, these drugs are frequently known chemotherapeutic agents. Intravenously, these drugs cause a variety of side effects. However, by linking them to nanoparticles the drugs go directly to the source and do not affect healthy cells (Paciotti et al., 2004). As is the case with hyperthermia, certain types of nanoparticles are better adapted for drug delivery than others. For instance, nanoparticles composed of colloidal gold easily attach various drugs. Colloidal gold is a dispersed solution of nanoparticles of Au0 (Paciotti et al., 2004). Additionally, polybutyl cyanoacrylate (PCB) nanoparticles attach drugs, protect them against enzymatic degradation, reduce their toxic effects, and limit distribution of the drug outside the target area (Reddy et al., 2004a). Tumor Necrosis Factor and Colloidal Gold Tumor necrosis factor (TNF) is a cytokine that affects coagulation, lipid metabolism, insulin resistance, and proper function of endothelial cells (Paciotti et al., 2004). It is produced during immune response primarily by monocytes and macrophages and has the ability to induce death in tumor cells (Elliott et al., 1994). Unfortunately, TNF causes systemic toxicities that have prevented it from being used as an anti-cancer drug (Furman et al., 1993). This toxicity can be attributed to rapid uptake of TNF by the reticuloendothelial system (RES) (Paciotti et al., 2004). Through the use of colloidal gold nanoparticles, Paciotti et al. (2004) were able to construct a vector which can avoid detection and clearance by the RES. Thus, the nanoparticles (PT-cAu) delivered TNF specifically to tumor cells, eliminating the associated systemic toxicity. Next, Paciotti et al. (2004) compared treatment using native TNF and PT-cAu-TNF which showed both reduced tumor size in a concentration dependent manner. However, mice given 12 µg native TNF suffered 25% fatality and all given 24 µg native TNF died whereas none of the mice treated with PTcAu-TNF perished. Furthermore, Figure 2b illustrates that while 15µg of Native TNF has approximately the same affect on tumor size as PT-cAu-TNF through 16 days, the survival rate using the native form is 40% lower. Thus, without the colloidal gold nanoparticles, TNF is extremely toxic. These nanoparticles help TNF circumvent the RES and enter selectively into cancer cells, which ultimately causes tumor cells to die (Paciotti et al., 2004). Localized Chemotherapy As mentioned earlier, the main problem with chemotherapy is that it is not tumor specific. Thus, chemotherapy drugs tend to act on all rapidly dividing cells. Through the use of nanoparticles, however, the same drugs can be linked specifically to cancer cells at higher concentrations for longer periods of time. Thus,

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Figure 2: TNF effect on tumor volume in mice MC-38 colon carcinoma tumors. a.) Antitumor efficacy of native TNF and the cAu-TNF vector. Mice with MC-38 colon carcinoma tumors were intravenously injected with increasing concentrations of native TNF of cAu-TNF vector (n=4/group/dose). Tumors were measured 10 days after treatment using three dimensional measurements (L x W x H). b.) Antitumor efficacy of native TNF and PT-cAu-TNF vector using one group as a control. Two groups with either 7.5 or 15 g of intravenously injected PT-cAu-TNF. Another two groups were intravenously injected with 7.5 or 15 µg of native TNF. The size of tumors were then measured on various days (Paciotti et al., 2004).

free Dox had no significant effect on the tumor cells compared to CMNP-Dox, which completely stopped proliferation at 120 µg/ml Dox. Interestingly, at 240 µg/ml, CMNP-Dox had a reduced effect, believed to be because of steric hindrance caused by excess nanoparticles (Ma et al., 2004). One of the chief advantages of this system, however, is that it can be made in one step under atmospheric pressure using inexpensive chemicals, such as benzene and acetonitrile, making it both effective and cost efficient (Ma et al., 2004).

urothelium utilized gelatin nanoparticles loaded with the drug. These nanoparticles are hydrophilic and thus uptake fluid rapidly allowing for paclitaxel to be released easily. This is important because the quicker the drug is released, the longer its exposure to cancer cells before urination. The concentration of paclitaxel in the urine, which was collected during treatment, was 2.6x that of the cremophor/EtOH formula. Additionally, 87% of the drug was released in two hours (Wientjes, et al., 2004), compared to only 45% after 3 days for paclitaxel-loaded poly(ethylene oxide)poly (lactide/glycolide) nanospheres used to regulate smooth muscle cell regulation (Suh et al., 1998). In summary, paclitaxel loaded gelatin nanoparticles were able to penetrate the urothelium of the bladder and rapidly release the drug, making them a promising treatment for bladder cancer (Wientjes et al., 2004).

Paclitaxel Paclitaxel is a chemotherapy drug that can be used to treat Kaposi’s sarcoma and metastatic breast, ovarian, and bladder cancer (Wilkes et al., 2000). It is an antimicrotubule compound that prevents continuation of the cell cycle and thus proliferation (Wientjes et al., 2004). In the case of bladder cancer, doxorubicin and mitomycin C are ineffective treatment options due to their inability to pass through the transitional epithelium in the wall of the bladder known as the urothelium. Since paclitaxel is lipophilic, however, it can freely pass through the urothelium (Wientjes et al., 2004). The FDA approved formulation for paclitaxel includes the solvent Cremophor. Cremaphor causes paclitaxel to become entrapped in the micelles of the bladder, which lowers the drugs ability to penetrate the urothelium (Knemeyer et al., 1999). To combat this, Wientjes (2003) used DMSO as a surface-active agent that disrupted Cremaphore micelles and enabled paclitaxel to be delivered to the tumors; however, this technique caused increased urine production and associated drug removal. Consequently, with less time in contact with the cancerous cells, paclitaxel was less effective. Wientjes’s et al. (2004) second attempt to facilitate the transfer of paclitaxel through the

Gene Delivery using Nanoparticles Nanoparticles can deliver proteins with anti-tumor activity into tumor cells and additionally, they can be used to deliver chemotherapeutic drugs directly to tumors, avoiding systematic toxicity. The versatility of these small particles also allows them to transport plasmid DNA with tumor suppressor genes to tumor cells. This causes a tumor suppressing protein to be produced which induces tumor cell apoptosis, effectively fighting the cancer (Ramesh et al., 2004). MDA-7 First identified in human melanoma cells (Jiang et al., 1995), the human melanoma differentiation associated gene 7 (mda-7 or IL-24) is a tumor suppressor gene. In late stage human melanoma, MDA-7 protein is absent, whereas in early stage melanoma it is present. Accordingly, this gene

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product is likely involved with progression of the disease (Ellerhorst et al., 2002). Furthermore, the protein is absent in a variety of human tumors including lung, breast, and colorectal carcinomas and sarcomas, and thus, it is believed to be involved in both the development and progression of these human cancers (Chada, et al., 2003). Previous studies have shown that through using adenoviral vectors, expression of MDA-7/IL-24 triggers cytotoxic related cell death and growth suppression in several human cancer cells (Ramesh et al., 2004). Moreover, normal cells are not affected by exposure to mda-7gene, making it a potentially strong anti-tumor therapy. In 2003, Chada et al. used an adenoviral receptor to deliver mda-7 to tumors in the lungs. The results were promising, because this procedure caused expression of MDA-7 induced apoptosis in the tumors. Unfortunately, the adenovirus vector can cause an immune response and liver toxicity (Vlachaki et al., 2002). Therefore, a new vector for mda-7 delivery to disseminated cancers is needed. Ito et al. (2003c) demonstrated that DOTAP: cholesterol nanoparticles can transport tumor suppressor genes to tumors in the lungs and increase the transgrene expression of these genes. Based on this, Ramesh et al. (2004) tested the use of cationic DOTAP: cholesterol (Chol) nanoparticles as a vector for delivery of mda-7 gene. They found that cells treated with the DOTAP/mda-7 gene showed significantly fewer tumors (Figure 3). Additionally, they found no resistance to multiple treatments with this therapy, as well as no systematic toxicity. Furthermore, the treatment was still successful in immunodeficient and immunocompetent organisms. Thus, using DOTAP: Chol nanoparticles as a vector for the mda-7 gene is a novel approach for cancer therapy that shows much promise (Ramesh et al., 2004).

Firstly, neither of these treatments causes systematic toxicity. In fact, both hyperthermia and drug delivery can be directed specifically to cancer cells. Ultimately, this is advantageous because it greatly reduces the physically and psychologically demanding side effects of chemotherapy and radiation, which include, but are not limited to anemia, neutropenia, hair loss, diarrhea, sterility, and nausea. These side effects are thought to be worthwhile because of chemotherapy’s effect on cancer, but all cancer cells are not responsive to chemotherapy. Furthermore, some cancers develop resistance to chemotherapeutic drugs (Gottesman, 2002). There are several reasons for this. As mentioned in the beginning, tumor cells have a variety of mutations, and all tumor cells do not have the same mutation. Some mutations allow cells to randomly develop resistance to drugs because they no longer express the protein receptors to which the drug interacts. Thus, the cells without the receptor have a growth advantage, and if another drug is not used, these cells will proliferate rapidly (Gottesman, 2002). Additionally, tumor cells may produce more target proteins than the drugs can bind. Since chemotherapeutic agents are not specific, the concentration of the drugs cannot be raised, as other systems of the body would be effected as well (Gottesman, 2004). Furthermore, enhanced amplification of the MDR1 (Multiple Drug Resistance) gene results in the encoding of a large transmembrane protein which can stop certain drugs from entering a cell and also eject drugs already in it (Bredel et al., 2002). With chemotherapy, any form of resistance requires another type of drug; however, nanoparticles may hold the key to circumventing such resistance. Early trials with hyperthermia and gene delivery show that each technique may be used multiple times. Hyperthemia, for instance, does not work on hindering processes inside the cell, but instead, it heats the cell up to such high temperatures that it denatures proteins and DNA (Dai et al., 2005). Heat shock proteins that stabilize proteins to prevent denaturing are themselves denatured when exposed to heat of this magnitude (Ito et al., 2003). Thus, hyperthermia can be done

Discussion In this paper, I have chronicled three promising techniques for treatment of cancer using nanoparticles: hyperthermia, drug delivery, and gene therapy. These techniques each have several advantages over the current treatments of radiation and chemotherapy.

Figure 3- Mice treated with mda-7 exhibit a lower number of tumors. Mice with A549 and UV2237m lung tumors were treated daily for a total of six doses (50 g/dose) with phosphate-buffered saline (PBS), DOTAP:Chol-chloramphenicol acetyl transferace (CAT) nanoparticles, or DOTAP: Chol-mda-7 nanoparticles. Tumor growth was only inhibited by DOTAP:Chol-mda-7 nanoparticles (P