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! Reviews ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! )&*%(+*$,-%./*%%01* The cardinal aim of our undergraduate enterprise, the Stony Brook Young Investigators Review, is to establish an environment in which students can interact with their colleagues and faculty members on a number of different levels, in order to receive instructive feedback on their research projects. Through print and online publications, and through symposiumâ&#x20AC;&#x2122;s, such as the Second Annual Young Investigators Symposium, we hope to attract students from a number of diverse academic and social backgrounds and to those who have not yet ventured into the world of laboratory work, offer a warm welcome to the scientific community.

As President, it has been an absolute honor to set the stage and showcase the talent and potential we have here at Stony Brook University. I hope that this journal continues to flourish and will foster an environment which will help continuously expose students to the ongoing research performed both inside and outside our Universityâ&#x20AC;&#x2122;s grounds. We hope to accomplish this goal by publishing the journal once every semester, increasing the number of copies published, expanding the scope of the material covered to the physical sciences, and to allow for more submissions. Recently, we have established an Internet presence, our website, http:// younginvestigators.com, and will be persistently updating it to

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include the most recent content. Of course, this will take a great amount of time. However, I hope that the steps we have taken so far have established a strong foundation upon which our next undergraduate leaders can build.

Our latest mission, has been to establish a network for Stony Brook University, which will allow for easier identification of labs which will suit student interest as well as will help faculty members easily identify qualified undergraduates to enroll and assist in accomplishing their research goals. We hope to satisfy our objective by creating an online network interface. We have already entered the planning phase of this project.

I would like to thank Dean Jerrold Stein, Dr. Robert Haltiwanger, Dr. Paul Bingham, Dr. Paul Bynum, Dr. Wali Karzai, Dr. Sanford Simon, the helping hands of the Student Activities Center, our writers, and our sponsors, for all of your support, for helping us transform the idea of having a large-scale symposium into a reality and for making the undergraduate collaboration you are holding in your hands a possibility. Additionally, I would like to thank Dr. Michael Lake of the Biochemistry Department, who extended the entire undergraduate research experience to his high school students.

Respectfully Yours, Isaiah P. Schuster B.S. Pharmacology (2010) President E-mail: isaiah.schuster@younginvestigators.com

The Stony Brook Young Investigators Review, Spring 2010

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2*,,*&.3&#4.,'*.5+(,#& “You are following some of the brightest minds in the world.”

Indeed, Stony Brook University has no shortage of undergraduates with academic talent and intellectual prowess; a fact that is highlighted on the back of every bus on campus. A great number of these outstanding students can be found within the biological sciences. However, within these departments, there is a large deficit of students interested in pursuing graduate study.

The vast majority of Stony Brook Biology and Biochemistry majors have one unflinching goal in common: to gain entrance to medical school. I believe this deficit in students interested in pursuing a graduate education exists due to a fundamental lack of knowledge; that is, of all that graduate school and Ph.D. programs, in particular, have to offer bright students.

The majority, if not all, Ph.D. programs in the natural and applied sciences are fully funded; meaning that tuition is paid for and a living stipend of approximately 30,000 dollars per year is typically provided along with health insurance. This means that a student will graduate without debt, unlike many professional schools. Perhaps the greatest benefit of attaining a Ph.D. is that it opens up a host of career opportunities. This includes academic research, in the form of a faculty position which includes the benefits of tenure and teaching, or a career in the biotech industry, which could include your own start-up company if you are particularly entrepreneurial in nature. Graduates of Ph.D. programs have gone on to careers in patent law, public policy, and even scientific publication. The Doctorate of Philosophy is the highest degree that an academic institution can bestow and, as Dean Lawrence Martin often relates, the word “doctor” is derived from the Latin doctoris, which means “teacher.” So professors and other holders of the Ph.D. degree are the true doctors.

So why choose a career in research? Because research is exciting; the particular scientific problem or question that you are trying to address is constantly changing and evolving. Simply put, the job is never the same from one day to the next. One important aspect of science that never seems to be emphasized enough is creativity. By conducting research you get to flex your creative muscles by designing simple, yet elegant experiments that elucidate information about the inherently complex, natural puzzle under question. Science is also rewarding. By undertaking this endeavor, you are able to expand the human knowledge base on how the natural world functions. The career itself is multifaceted; there is not only bench work in the laboratory but also traveling and presenting at scientific meetings, writing papers, and significant mentorship and teaching opportunities.

Being involved with The Stony Brook Young Investigators Review can prepare you for a career in science in a number of

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ways. Staff members often write review articles, which require the interpretation and evaluation of papers in the primary literature. Undergraduate contributors can also choose to publish their own independent research findings in our journal, which is an important step for any scientist. In fact, we have two independent research submissions in this issue alone and plan on having more in subsequent issues. The greatest benefit of working for the Young Investigators Review is that it can get you excited about science; working with like-minded individuals to produce an issue that contains articles on the most cutting-edge research.

Stony Brook certainly has a history of producing strong candidates for graduate study and many graduate school bound undergraduates have gone on to Ph.D. programs at prestigious universities; including Kevin McCarthy, class of ’08, who is currently in the Virology Program at Harvard University, JeanLuc Chaubard, class of ’09, who is currently in the Chemistry Department at the California Institute of Technology, Allison Goldberg, class of ’10, who is joining the Department of Pharmacology at Yale University in the Fall, and I myself am joining the Department of Biology at the Massachusetts Institute of Technology in September.

It is true that the pursuit of a Ph.D. may not be for everyone, as it requires a significant amount of dedication (average time to completion is about 5.5 years) and a sharp, incisive, and questioning mind. However, it is certainly a career path that promising undergraduates should consider at least once in their academic career. Conducting research and participating in our undergraduate journal of science are two great ways to gain exposure to the world of basic science and to help you, the student, decide if professional school is for you, or if maybe you have a previously undiscovered enthusiasm for science that will lead you to graduate study.

Sincerely, Kevin Knockenhauer B.S. Biochemistry (2010) Editor-in-Chief E-mail: kevin.knockenhauer@younginvestigators.com

The Stony Brook Young Investigators Review, Spring 2010

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)"??(,2 We have developed a new micropatterning technique using an inverted fluorescence microscope with programmable XY stage and layered dry-film photoresist, thermally bound to a glass slide. The method allows for flexible in-house maskless photolithography without a dedicated microfabrication facility, and is well suited for in-house fabrication of microfluidic channels, scaffold templates for protein/cell patterning or optically-guided cell encapsulation for biomedical applications.

>0<=,%*-%+#'+:'>0<%&#' @1X0=-%/0' Using an approach inspired by recent work [1] in maskless photolithography, the goal was to develop a new basic photolitho;

graphic technology that is suitable for implementation in any biomedical laboratory equipped with an inverted fluorescence-enabled microscope and programmable stage.

The design involves a method for translating a desired image into programming commands to the microscope stage as well as investigating the limits of photoresist micropatterning via standard microscope objectives without additional components, e.g. shutters.

>0<=,%*-%+#U';#(92<%<'(#$' .0,:+,?(#=0'E/(9"(-%+#' We decided to use dry-film photoresist Ordyl SY 330 (ElgaEurope, Italy) because of its potential to create relatively highdepth features (30Âľm for a single layer).

Glass slides were used as a support surface and a standard office laminator was em-

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ployed to deposit layers of the photoresist.

User-provided images are transferred onto the photoresist by epi-fluorescent UV light through standard microscope objective lenses. Images are translated into microscope XY-stage movement by custom software written in C++. Specifically, we used a 40x Fluor objective lens on a Nikon Eclipse TE2000-U (Nikon, Japan) microscope equipped with a Prior OptiScanII programmable stage (Prior Scientific, UK), where UV radiation was provided by a 365nm bandpass filter (Chroma, USA) applied to a broadband xenon arc lamp light source (Cairn, UK). The illuminated area produces a negative point in the photoresist after etching and baking. In-plane features depend on the objective parameters and the encoded speed, while the height is proportional to the thickness of the photoresist layers (30Âľm each). Our results indicate feature dimensions to be commensurate with cell dimensions and acceptable for biological applications. Even though the process is serial in nature, a complex shape can be transferred within minutes. Connected patterns (images with one component) are drawn in a straightforward way with continuous movement of the stage, while disconnected patterns require further considerations (as discussed below).

The final product is a cast made from the photoresist template using biocompatible 6

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materials, e.g. polydimethylsiloxane, or a photopolymerized cell-encapsulating gel, etc. Connected Patterns: Full Aperture The high light-delivering power of the Nikon objective lens used (40x, Plan Fluor DIC M; NA = 0.75) is capable of achieving feature sizes between 250µm and 700µm.

Tuning of feature sizes is possible by careful manipulation of UV dose via neutral density filters and stage speed (Figure1A).

8+##0=-0$'.(--0,#<3'.%#5+90' ;*0,-",0 Applications requiring feature sizes between 40µm and 250µm are achieved by placing a pinhole aperture behind the objective, reducing the effective numerical aperture (NA) of the high-magnification lens while preserving its high magnification (Figure 1B).

>%<=+##0=-0$'.(--0,#< We have avoided the use of a shutter in this design for simplicity and affordability.

By taking advantage of results from computational geometry and graph theory, we can broaden the system’s use to allow for the creation of disconnected patterns. Since the photoresist shows a threshold-like response to UV radiation flux integrated over time, two exposures at one location with a dose of [(1+delta)ET]/2 will successfully create an isolated point only at this location, where ET is the exposure threshold or minimum required amount of delivered UV and delta is an arbitrarily small number to ensure exposure saturation. With careful navigation of the photoresist (via Traveling Salesperson heuristics [2,3] and 2-opt improvements [4], we prove that it is possible to use this approach to create disconnected patterns.

We have demonstrated maskless micropatterning to be possible in a biological laboratory equipped with a standard fluorescence microscope system. By controlling the numerical aperture, stage movement speed and the intensity of the delivered light, one can realize a wide range of feature sizes (40µm-700µm). Feature depth is controlled by varying the number of layers of the dry film photoresist. Although the current state of the invention is best suited for connected patterns, a shutter-based design or exposure-based traversing can create any arbitrary pattern.

;=7#+D90$&0?0#-< We acknowledge Nicolas Glade (Commissariat à l’Énergie Atomique, Grenoble, France) for assistance with the methodology for film exposure and developing and ElgaEurope for the donation of the Ordyl SY330 dry film resist. We also acknowledge Prof. Joseph Mitchell and Prof. Estie Arkin for their advice on the computational geometry and graph theory aspects of the system.

*#&#-#0(#+ 1. Breadmore MC, Guijt RM. “Maskless photolithography using UV LEDs.” The Royal Society of Chemistry 8 (2008), p.

1402 – 1404.

2. Mitchell JSB and Arkin E, Professors of Applied Mathematics and Statistics.

Stony Brook University. Stony Brook, NY. Personal communication. April 28, 2009.

3. Tucker, Alan. “Applied Combinatorics.”

San Francisco: Wiley, 2001.

4. Lin S and Kernighan BW. “An effective heuristic algorithm for the travelingsalesman problem.” Operations Research 21 (1973), p. 498-516.

The Stony Brook Young Investigators Review, Spring 2010

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;1<-,(=Every process that takes place in a cell is accomplished by innumerable intermediate steps. Chloroplast synthesis is an example of a light-mediated event. For a light-mediated process, the first step is the absorption of light. In Euglena gracilis, this initiates a co-translational pathway of protein translocation. Both nuclear and chloroplast genomes are triggered to assemble chloroplasts. The light activates a G-linked protein receptor which in turn activates the IP3 signaling pathway. IP3 signals the calcium channels in the endoplasmic reticulum (ER) to open. IP3 is then dephosphorylated and recycled back into the membrane. The ER releases calcium as well as inducing the transport of the chlorophyll binding proteins required for chlorophyll synthesis and chloroplast production. The Golgi apparatus packages the protein for transport to the growing chloroplast.

Adding the inhibitor lithium chloride arrests this process by inhibiting the enzymes which allow IP3 to be recycled back into the membrane. If the cells are washed, removing the lithium chloride, chlorophyll synthesis is resumed. Inhibiting cells already in the process of creating chlorophyll binding protein causes an accumulation of these proteins inside the ER. A protein block will lead to the ER stress response being activated. Washing the cells at various times during inhibition removes the inhibitor, allowing the cells to continue chlorophyll synthesis. There is a pronounced lag in the resumption of chlorophyll synthesis.

Keeping the inhibitor in the cells for longer periods of time causes increasing lags in resumed chlorophyll production. These lags probably correlate with the steps of the ER stress response.

A#-,+$"=-%+# Communication within cells is orchestrated using second messenger systems. In a

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light-mediated system, such as chlorophyll synthesis in Euglena gracilis, the first signal is activation by light. The light initiates a chain reaction eventually leading to photosynthesis inside the chloroplast. However, in the dark Euglena do not develop their chloroplasts beyond pro-plastids [1]. In the presence of light, the pro-plastid increases its internal membrane surface area, which folds inwards to become thylakoids. The chloroplast and nuclear genomes begin transcribing chlorophyll-binding proteins as well as hundreds of other nuclear-coded chloroplast proteins. The nuclear-coded proteins will need to be transported to the growing chloroplast before it can be completed. Therefore, light initially signals the cell to complete the synthesis of the chloroplast, a process that will continue until biosynthesis is completed.

As the chloroplasts finish their development in the light, they require the chlorophyll-binding proteins for comple-

tion. Chlorophyll itself is synthesized in the chloroplast but requires the chlorophyllbinding proteins to be inserted into the chloroplast membranes [2]. These proteins are coded in the nuclear genome and, after transcription, are transported co-translationally to the growing chloroplast [2].

For this reason, the proteins are translated on ribosomes sitting on the endoplasmic reticulum (ER) and then transported to the Golgi apparatus where they are packaged and sent to the chloroplast [2] (Figure 1). As the proteins are elongated into the ER, the nascent chains are inhibited from further elongation by calreticulin, a calcium binding complex [3,4]. The release of the calreticulin from the nascent chain is brought about by another simultaneous biochemical pathway initiated by light.

In Euglena, the light receptor for chloroplast synthesis, as well as the events it activates, lies on the outer membrane of the growing chloroplast. The receptor directly activates a G-protein linked receptor, which in turn activates phospholipase C [5]. This enzyme cleaves phosphotidylinositol (PIP2) into inositol triphosphate (IP3) and diacylglycerol (DAG) [5]. IP3 moves away from the membrane and binds to a receptor on the ER, signaling the calcium channels to open. After signaling, the IP3 has its phosphate groups removed by

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The Stony Brook Young Investigators Review, Spring 2010

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fore, the nascent chains accumulate in the ER where calcium-bound calreticulin inhibits their translation. It is logical that the ER would initiate the stress response to regain equilibrium [9]. To test this hypothesis, the cells were washed at three different times after lithium chloride had been added. The expectation was that cells incubated in lithium chloride for longer amounts of time would have a longer lag period. The ER in these cells would have engaged the quality control mechanism for a longer time resulting in fewer proteins stored in the ER.

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three different enzymes before being recycled back into the membrane [5].

In the ER, calcium begins to leave through the channels. As the calcium leaves the ER, the repression caused by calciumbinding calreticulin is released. Previously repressed chlorophyll-binding proteins are sent to the Golgi where they are packaged and sent out in vesicles [6]. Upon arrival at the chloroplasts, the calcium, liberated from the ER, is required for vesicle-vesicle fusion with the outermost membrane. The â&#x20AC;&#x153;greeningâ&#x20AC;? process begins when the chlorophyll-binding protein successfully binds chlorophyll.

Inhibition of any step in the process stops chlorophyll synthesis. This can be done by the addition of lithium chloride to Euglena cells. Lithium is a competitive inhibitor of the enzymes which cleave the phosphates (Ki of binding is 1 mM) [5].

Therefore, the inositol cannot be recycled into the membrane to reform IP3 (Figure 2). When the cell has run out of signaling IP3 (roughly 1% of IP3 in membranes) the cell cannot propagate the signal any further [5]. If the cells are washed in fresh medium, removing the lithium chloride, chlorophyll synthesis can be resumed.

The washing method was originally used as proof that the cells stopped producing chlorophyll as a result of inhibition, not death. However, it was noted that after being washed the cells have a lag period

before they begin to produce chlorophyll again. The reason for this lag was hypothesized to be due to the ER quality control mechanism. Normally the ER stress response would be invoked because of excessive amounts of mis-folded proteins in the ER [7]. The ER cannot function properly with so many proteins clogging it. In this situation, the ER first sends a signal to up regulate transcription of chaperone proteins to assist in folding the new proteins [8]. However, if the proteins cannot be correctly folded, the additional chaperone proteins exacerbate the aggregation in the ER. In this case, the chaperone proteins work as a signal to have the excess proteins degraded [8]. A specific protein called BiP responds to the excessive number of unfolded proteins [7]. Under normal conditions, BiP binds to PERK keeping it in an inactive state [8]. When BiP leaves PERK to assist in protein folding, PERK becomes active [8]. In its active state, PERK sends a signal to the nucleus to down-regulate general protein translation [8]; reducing the amount of proteins coming into the ER gives the ER space to degrade or export its protein load.

The chlorophyll-binding proteins are not mis-folding in the ER, but there is evidence that they build up [1]. Although the lithium chloride stops the ER channels from being opened, chlorophyll-binding proteins are still being transcribed. There-

Euglena gracilis was grown at 31oC in enriched, sterile medium under 900 lux (illuminance). Stocks were grown in the dark. After 5 days, both the control and experimental stocks were moved into the light. This was marked as time zero. At 28 hours, 0.28 millimoles of lithium chloride was added to the experimental flask. At 31, 34, or 46 hours, the lithium chloride was washed from the cells using sterile medium and the washed cells were placed in a sterile flask with 10 mL of fresh medium.

The experiment was run for 96 hours. The amount of chlorophyll was measured by centrifuging one mL of cells and pouring off the supernatant. The pellet of cells was mixed with one mL of acetone and centrifuged again. The absorbency of the supernatant was recorded at 480, 645, 652, and 663 nanometers. Chlorophyll concentration was determined by the method of Arnon [1]. In a steady-state experiment, lithium chloride was added at 72 hours and the cells were not washed. The experiment ran to 130 hours.

*#+;8.+ When flasks are placed in the light, experimental and control flasks accumulate chlorophyll at the same rate until lithium chloride is added to the experimental flask (Figure3).At this time, chlorophyll production in the lithium flask levels off and eventually declines. When lithium chloride is washed from the cells there is a lag before chlorophyll production begins again.

As the amount of time between lithium addition and washing increases, the lag time increases as well (Figure 4). In another ex-

The Stony Brook Young Investigators Review, Spring 2010

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! Articles ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! periment, both the control and experimental flask were allowed to â&#x20AC;&#x153;greenâ&#x20AC;? to a state when chlorophyll is made at a relatively constant rate. When lithium was added at this time the cells showed the same declining response in chlorophyll production (Figure 5).

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decreased production of chlorothe lithium-inhibited cells is deon the binding affinity of lithium to the three phosphatases that

cleave IP3. Since not every enzyme is bound to a molecule of lithium at every moment, small amounts of chlorophyll are still made. Increasing the concentration of lithium would decrease the amount of chlorophyll produced.

The lithium-inhibited cells do not immediately drop in chlorophyll synthesis because at the onset of lithium addition some IP3 is attached to the pro-plastid membrane. The remaining signaling IP3 leaves this membrane and opens the calcium channel even in the presence of lithium.

Therefore, chlorophyll levels continue to

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The Stony Brook Young Investigators Review, Spring 2010

increase until the cells runs out of signaling IP3. The chlorophyll level plateaus when the protein product is being made but no new signal is created. Chlorophyll levels only begin to drop when no more new protein can be made.

The lag between when chlorophyll amounts plateau and when chlorophyll production decreases can be attributed to the ER stress response. The increasing lag times between 3, 6 and 18 hour washes may be due to the stages of the response. When the cells were washed 3 hours after lithium was added the ER had sent a signal to the nucleus to down-regulate transcription of chlorophyll binding protein as well as upregulate chaperone proteins [8]. Therefore, when the lithium chloride was washed out the cell had only to transport the chlorophyll binding proteins already in the ER to the Golgi and then the chloroplast. The cells washed at 6 hours appear to be a similar situation within the ER. However, the cells washed at 18 hours have a considerably longer lag than the other two. This would be due to the degradation of proteins in an effort to promote the ER response.

When the cells were washed there was a limited amount of chlorophyll binding protein already in the ER. Transcription had to be up-regulated in the nucleus and new proteins made from mRNA before new chlorophyll was seen in the cells.

Another consideration for the lag in chlorophyll production is fate of the signaling IP3. Highly active IP3 sitting in the cytoplasm will be degraded before the cells are washed. After lithium is removed, the cells must synthesize more signaling IP3. It should also be noted that after activation the IP3 receptor on the ER is often marked for degradation [10]. It is possible for IP3 to be active in the cell without an available ER receptor due to degradation. The cycle of this system could also affect the kinetics of chlorophyll-binding protein synthesis.

The final experiment in which lithium chloride was added after the cells had reached a steady state of chlorophyll synthesis displays the dynamic equilibrium of chloroplast production. Although the level of chlorophyll remains more constant at this point, adding inhibitor still results in decreased chlorophyll production. Chlorophyll has a limited lifetime, after which the function is compromised and the chlorophyll is degraded. A constant flow of

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chlorophyll binding protein is therefore required as long as the cell is undergoing photosynthesis. The increased lag times are not due to a natural decline in chlorophyll biosynthesis but to the active down-regulation and degradation of the protein.

8+#=9"<%+# The addition of lithium chloride to Euglena gracilis successfully inhibits chlorophyll synthesis, while washing cells reverses the inhibition. The lag in chlorophyll synthesis after the inhibitor is removed is lengthened based on the amount of time chloroplast synthesis is inhibited. The lags seen probably constitute steps in the endoplasmic reticulum stress response.

*#&#-#0(#+ 1.Lyman, Harvard, Masahiro Mitsuboshi, Asami Endo, Kazuyuki Tanaka, and Tetsuaki Osafune. The Regulation of Chloroplast Synthesis by a Light Mediated Phosphoinositide System. Bull. of Nippon Sport Sci. Univ. 30 (2000): 141-52.

2.Sulli, Chidananda, ZhiWei Fang, Umesh Muchhal, Steven D Swartchbach. Topology of Euglena Chloroplast Protein Precursors within Endoplasmic Reticulum to Golgi to Chloroplast Transport Vesicles.

The Journal of Biological Chemistry 274 (1998): 457-463.

3.Jia, Xiao-Yun, Li-Heng He, Rui-Lian Jing, Run-Zhi Li. Calreticulin: conserved protein and diverse functions in plants.

Physiologia Plantarum 136 (2009):127-

Articles

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138.

4.Navazio, Lorella, Maria C. Nardi, Simonetta Pancaldi, Paoglo Dainese, Barbara Baldan, Anne-Catherine Pitchette-Laine, Loic Faye, Flavio Meggio, William Martin, Paola Mariani. Functional Conservation of Calreticulin in Euglena gracilis. Journal of Eukaroyotic Microbiology 45 (1998): 307313.

5.Berridge, Michael J. Inositol Triphosphate and Diaclyglycerol: Two Interacting Second Messengers. Annual Review of Biochemistry 56 (1987): 159-93.

6.Van Doreen, Geil G., Steven D. Schwartzbach, Tetsuaki Osafune, Geoffrey I. McFadden. Traslocation of proteins across the multiple membranes of complex plastids. Biochimica et Biophysica Acta 1541 (2001): 34-53.

7.Ellgaard, Lars, and Ari Helenius. Quality Control in the Endoplasmic Reticulum.

Nature Reviews 4 (2003).

8.Ron, David. Translational control in the endoplasmic reticulum stress response. J. Clin. Invest. 2002; 110(10):1383 9.Ma, Yanjun, and Linda Hendershot.

ER Chaperone Functions during normal and stress conditions. Journal of Chemical Neuroanatomy (2003).

10.Pearce, Margaret M. P., Yuan Wanq, Grant G. Kelley, Richard J. H. Wojcikiewicz. SPFH2 Mediates the Endoplasmic Reticulum-associated Degradation of Inositol 1,4,5-Trisphosphate Receptors and Other Substrates in Mammalian Cells. The Journal of Biological Chemistry 28 (2007): 20104-20115 The Stony Brook Young Investigators Review, Spring 2010

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! Reviews ! ! ! ! ! ! ! ! ! ! !!!!!!!!!!

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The words “Lyme disease” bring to mind pictures of the red rings of a bull’s eye rash - a classic sign of early infection. However, there is much more to this disease than meets the eye. First identified in 1975 after an outbreak of juvenile rheumatoid arthritis in the town of Lyme, Connecticut, Lyme disease has been slowly revealed to be an extremely complicated and treacherous infection.

In fact, it is now known to be the most common tick-borne disease in the Northern Hemisphere [1]. In 1982, Dr. Willy Burgdorfer discovered the culprit from studying deer ticks - a bacterium, named B. burgdorferi in honor of the discoverer. B. burgdorferi is a spirochete, a spiral-shaped bacterium, that lives in the gut of Ixodus ticks and is successfully transmitted to humans by both nymph and adult ticks about 48 hours after the bite [2,3]. There are multiple species of Borrelia bacteria - in North America, for example, Lyme disease is caused by B. burgdorferi, but in Europe it can also be caused by its cousins, B. afzelii and B. garinii [4]. In the United States, endemic areas include Connecticut, Delaware, Maryland, Massachusetts (especially Cape Cod), Minnesota, New Jersey, New York (especially Long Island), Pennsylvania, Rhode Island and Wisconsin [5]. Ticks are most common at ground level, in grass and low shrubbery. It should be a habit, therefore, for hikers and campers to wear light colored, long-sleeved clothing and check themselves for ticks at the end of the day.

The major clinical symptoms of Lyme borreliosis are numerous, often worsening in cyclical intervals. They include flu-like symptoms, such as muscle and joint pain, fever, fatigue, nausea and fibromyalgia, along with more serious neurological and psychiatric symptoms such as memory loss, myocarditis, palpitations, headaches and neck stiffness. The bull’s eye rash - erythema migrans - occurs in about 50% of patients. The B. burgdorferi infection can be successfully cured in the majority of cases with promptly administered antibiotics such as doxycycline, one of the macrolides (erythromycin and its derivatives), ceftriaxone, and/or amoxicillin

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(in children) [3]. Untreated or inadequately treated Lyme disease can lead to severe neurological, cardiac, and/or arthritic complications. Bell’s palsy, which is paralysis of the facial nerve, is an example of a neurological manifestation of borreliosis. Lyme disease, however, has been seen to manifest itself differently around the world - in Europe, patients most frequently exhibit neurological symptoms, or neuroborreliosis, whereas in North America arthritis prevails [2]. Furthermore, it should be kept in mind that Lyme borreliosis is only one disease out of many that are caused by other tick-borne viruses and bacteria, such as spotted fever rickettsiae, babesia, ehrlichia, bartonella, anaplasma and mycoplasma [6].

People displaying persistent symptoms of Lyme disease are often found to have co-infections of one or more of these other bacteria. Diagnosis is based on clinical symptoms and laboratory tests, such as Western blots and Enzyme-linked immunosorbant assays (ELISA), although reaching a definite diagnosis is often extremely complicated [3]. In addition, the laboratory tests have been described by some physicians as unreliable, and a seronegative patient may still be suffering from Lyme disease [3,7].

The B. burgdorferi spirochete contains over 1,500 genes and 21 plasmids, the highest number found in any known bacterium, which allow B. burgdorferi to adapt rapidly to changes in the environment [8]. It can penetrate into the mammalian central nervous system and invade cells such as fibroblasts, synovial cells, endothelial cells, and macrophages.

In addition, the bacteria can become resistant to treatment by assuming a non-replicating cyst-form, which can become active months after the initial infection [8]. Traces of the bacteria have been found in the ligaments of patients who had been suffering from Lyme disease for years [9]. PCR results have confirmed the presence of B. burgdorferi DNA in cerebrospinal fluid, urine, blood, skin, and synovial fluid and tissue as well as in the muscles of patients with persistent Lyme disease [10]. In order to type the bacteria, two markers have been used by researchers - OspC, an outer surface protein, and ribosomal RNA spacer typing (RST) that finds a unique region in RNA specific to the bacteria. OspC

The Stony Brook Young Investigators Review, Spring 2010

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typing divides the bacteria into 21 types, while RST divides them into just 3 groups. The RST1 strain was, in addition, found most frequently in joint fluid of patients who did not respond to antibiotic treatment [11].

Over the past years, there has been debate in the scientific and medical fields on proper treatment of the disease. The disagreement centers on the usefulness of prolonged antibiotic treatment for patients whose symptoms, such as fatigue, cognitive impairment, and arthritis, do not go away after 14-21 days of treatment.

Persistent symptoms were found in 53% of cases in a study that was done with serologically positive patients who were evaluated three years after infection [12]. However, there have been very few antibiotic therapy trials, and the ones that do exist have shown contradicting results. For example, Fallon et al. showed that yes, prolonged antibiotic treatment results in better cognitive scores unless it is discontinued [13,14], whereas two other studies showed improvement in fatigue, but not cognition [15,16]. Each of the clinical trials, however, was performed under different parameters and none of the trials noted the duration of infection and symptoms in the participants [17]. This problem highlights the need for more controlled clinical trials to be conducted before a final decision is reached on the usefulness of long-term treatment.

Along with clinical assessments, over twenty years of research have begun to resolve the multiple perplexities of B. burgdorferi. Published results have shown that Borrelia spirochetes can be found in patients even after treatment, with evidence that the bacteria can change into cyst forms that are not affected by certain types of antibiotics [18]. Experiments on adult Macaca mulatta primates revealed that B. burgdorferi in the central nervous system localized to nerve roots, leptomeninges (the innermost layers that surround the brain) and dorsal root ganglia, which relay sensory information into the spinal cord from the rest of the body [19]. An additional primate model that evaluated B. burgdorferi-infected rhesus monkeys six months after infection revealed the presence of Borrelia antigens identified by Western blot analysis and PCR, with observed arthritis of the knee and elbow joints, joint structural changes, demyelinization and other signs of peripheral nervous

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system damage [20].

A great deal of research has been done at different universities on the mechanisms of borreliosis and the resulting cellular inflammatory response. At Stony Brook’s Center for Infectious Diseases, Borrelia burgorferi has been studied for almost two decades in the laboratory of Dr. Jorge Benach. Dr. Benach, chair of the Department of Molecular Genetics and Microbiology, has been studying the mechanisms of antibody function on B. burgdorferi. The group discovered that one of the bacteria’s outer-surface proteins, OspB, is recognized by one of the body’s bactericidal antibodies, CB2, which then lyses the bacteria through pore formation [21]. Dr. Benach’s research has elucidated the mechanism by which CB2 recognizes OspB through a Lys-residue on the carboxy terminus of the protein. This is a unique and important result because very little is known about the mechanism of bactericidal antibodies, and their defensive role may carry potential for developing future treatments.

Along with Dr. Benach’s work, the laboratory of Professor Martha B. Furie has published on the role of interferon-gamma (IFN-gamma), a cell-signaling molecule of the immune system, in the endothelial tissue of a genetically engineered mouse model that was infected with B. burgdorferi. “IFN-gamma is like a molecular switch that turns on chronic inflammation,” explained Dr. Furie.

When the bacteria disseminate throughout the body after the tick bite, they activate the endothelium and begin the inflammatory process by attracting T lymphocytes that secrete IFN-gamma [22].

The inflammation was found to be due, in large part, to a synergistic effect of B. burgdorferi and IFN-gamma, which together activate the transcription of a series of genes in endothelial cells. These genes encode chemokines, or chemoattractants, specific for T lymphocytes. Interestingly, there seemed to be selection for those T lymphocytes that secreted more IFN-gamma, and the result was a positive feedback loop that generated more and more IFN-gamma, leading to a state of chronic inflammation in the tissue. The damage to human tissues is likely caused by the body’s reaction to the bacteria, not the bacteria themselves [22].

Today, immunologic studies have joined forces with neurologic and clinical assessments of both groups of infected patients and animal models to study the twists and turns of B. burgdorferi. The spirochete remains both a serious infectious threat to the public and a tantalizing puzzle to scientists. The development of a successful vaccine would be an immense accomplishment. So far, a failed vaccine called LYMErix was removed from the market in 2002 due to patient concern over side effects [23]. Various research has been done on other options - just recently, scientists at Yale University showed that antiserum to Salp15, a protein found in tick saliva that shields the tick from mammalian immune response, successfully protected mice from B. burgdorferi infection [24]. Thus, there is hope that with such promising research and continued collaboration within the scientific community, a much needed solution to Lyme disease will be developed in the not so distant future.

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The Stony Brook Young Investigators Review, Spring 2010

*#

! Reviews ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! *#&#-#0(#+ 1. Vojdani A, Hebroni F, Raphael Y, et al. Novel Diagnosis of Lyme Disease: Potential for CAM Intervention. 2009. Evid Based Complement Alternat Med. 6(3):283-95.

2. Rosner, Bryan. The Top 10 Lyme Disease Treatments. 2007.

BioMed Publishing Group. p316.

3. Donta ST, MD. Late and Chronic Lyme Disease. 2002. Medical Clinics of North America. Vol 86:(2):341-9, vii.

4. Steere AC, Gross D, Meyer A et al. Autoimmune Mechanisms in Antibiotic Treatment-Resistant Lyme Arthritis. 2001. J Autoimm. 16, 263-268.

5. Map: National Lyme disease risk map. Apr 28, 2004. www.cdc. gov.

6. Hamlen, R. Lyme borreliosis: perspective of a scientist-patient.

2004. The Lancet: Infectious Diseases. Oct, Vol. 4: 10 (603-604).

7. Dattwyler RJ, Volkman DJ, Luft BJ et al. Seronegative Lyme disease. Dissociation of specific T- and B- lymphocyte responses to Borrelia burgdorferi. 1988. N Engl J Med. 319:1441-6.

8. Phillips SE, Harris NS, Horowitz Ret al. Lyme disease: scratching the surface. 2005. The Lancet. Vol 366.

9. Häupl T, Hahn G, Rittig M et al. Persistence of borrelia burgdorferi in ligamentous tissue from a patient with chronic lyme borreliosis. 2005. Arthritis & Rheumatism. 36(11); 1621-1626.

10. Frey M, Sibilia J, Piemont Y et al. Detection of B.burgdorferi DNA in Muscle of Patients with Chronic Myalgia Related to Lyme Disease. Amer J Med. 104(6);591-594.

11. Fiore, K. Strain Differences Associated with Refractory Lyme Arthritis (Review of Allen C. Steere’s article in July on Arthritis and Rheumatism). 2009. Medpage Today.

12. Asch ES, Bujak DI, Weiss M et al. 1994. Lyme disease: an infectious and postinfectious syndrome. J Rheumatol; 21(3):454-61.

13. Fallon BA, Keilp JG, Corbera KM et al. A randomized, placebo-controlled trial of repeated IV antibiotic therapy for Lyme encephalopathy. 2008. Neurology. 25;70(13):992-1003.

14. Cameron, D. Severity of Lyme disease with persistent symptoms. Insights from a double-blind placebo-controlled clinical trial. 2008. Minerva Med; 99(5):489-96.

15. Krupp LB, Hyman LG, Grimson R et al. Study and treatment of post Lyme disease (STOP-LD): a randomized double masked clinical trial. 2003. Neurology;60:1923-30.

16. Kaplan RF, Trevino RP, Johnson GP, et al. Cognitive function in post-treatment Lyme disease: do additional antibiotics help?

2003. Neurology;60:1916-22.

17. Cameron DJ. Generalizability in two clinical trials of Lyme disease. 2006. Epidemiol Perspect Innov. 17;3:12.

18. Mursic VP, Wanner G, Reinhardt S et al. Formation and cultivation of Borrelia burgdorferi spheroplast-L-form variants. Infection. 1996 May-Jun;24(3):218-26.

19. Cadavid D, O’Neill T, Schaefer H, Pachner AR. Localization of Borrelia burgdorferi in the nervous system and other organs in a nonhuman primate model of lyme disease. 2000. Lab Invest;80(7):1043-54.

20. Roberts ED, Bohm RP Jr, Lowrie RC Jr. et al. Pathogenesis of Lyme neuroborreliosis in the rhesus monkey: the early disseminated and chronic phases of disease in the peripheral nervous system.1998. J Infect Dis;178(3):722-32.

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21. Anderton JM, Tokarz R, Thill CD et al. “Whole Genome DNA Array Analysis of the Response of Borrelia burgdorferi to a Bactericidal Monoclonal Antibody.” 2004. Infect and Immunity.

72(4): 2035-2044.

22. Dame TM, Orenzoff BL, Palmer LE, Furie MB. IFN-y Alters the Response of Borrelia burgdorferi-Activated Endothelium to Favor Chronic Inflammation. 2007. J Immun. 178:1172-1179.

23. Abbott A. Lyme disease: Uphill Struggle. 2006. Nature 439, 524-525.| doi:10.1038/439524a 24. Dai J, Wang P, Adusumilli S, Booth CJ et al. Antibodies against a tick protein, Salp15, protect mice from the Lyme disease agent.

2009. Cell Host Microbe. 6(5):482-92.

The Stony Brook Young Investigators Review, Spring 2010

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Reviews

ceptors, a reduction in DA release and transmission, as a re:-+%*3)$@11*+2*-3#$ sult of natural stimuli, has been seen in drug abusers [5]. This lessening of natural dopamine transmission and decreased number of D2 receptors contribute to the desire for users to D31),&2%31*34$*2&$ re-administer cocaine after it has worn off, as without the natural stimuli cannot elicit the same level of transmis56%,>%+-(-4*+%($E%&*&$ drug, sion as they do in a non drug addicted individual [1]. This decrease in dopaminergic transmission due to natural stimuli is a major factor in why addicts have such trouble quitting. %31$5&<+6-(-4*+%($ 450'C0",+1%+9+&2'605%#$'8+=(%#0';$$%=-%+# F99)+2&

Dopamine (DA) is a neurotransmitter within the Central Nervous System which plays many roles. Its transmission can affect behavior and cognition. Voluntary movement, mood, sleep, working memory, and punishment and reward learning are just some of the processes that are regulated by dopaminergic transmission [1].

Dopamine is released by pre-synaptic neurons into the synaptic cleft and then bind to dopamine receptors on postsynaptic neurons [2]. The performance of tasks associated with dopaminergic transmission rely on proper maintenance of dopamine levels present in the synaptic cleft. Cocaine disrupts this maintenance of DA levels in the synaptic cleft by inhibiting dopamine transporters (DAT), which normally function as dopamine re-uptake transporters. DAT moves DA from the synaptic cleft back into the pre-synaptic neuron where it can no longer interact with post-synaptic receptors and elicit responses associated with dopaminergic signal transmission. Cocaine binds to the transporter DAT1 and the complex can no longer function to uptake DA. The high associated with taking cocaine is a result of this major increase in dopaminergic transmission due increased levels of dopamine in the synaptic cleft interacting with post-synaptic receptors [3].

A group of researchers at Brookhaven National Laboratory (BNL) have been focusing on the neurobiological basis for the psychological symptoms of cocaine addicted individuals. It has previously been found that the orbitofrontal cortex (OFC) and cingulated gyrus (CG) are involved in the regulation of motivation and drive. Enhanced activation of the OFC and CG occurs with drug-induced DA stimulation.

This enhanced stimulation of regions known to regulate motivation and drive could lead to an increased drive for cocaine addicts to self-administer the drug [6]. Additionally, the OFC has been found to be involved with stimulus-reward reinforcement learning. Its hyper-activation in drug addicted subjects suggests it is a neural pathway of reward prediction, which contributes to drug cravings in drug addicted individuals [1]. In addition to the development of cravings, cocaine addicted individuals have been found to give disproportionate value to their drug while disregarding other stimuli which are seen as rewarding by non-drug addicted individuals. This was seen in a study which found more than half of cocaine abusers rated a $10 reward equally valuable to a $1000 reward [7]. This assignment of greater value to the drug than to non-drug stimuli and lack of ability to recognize a gradient of reward among non-drug stimuli may play a role in leading cocaine addicts to make decisions which are recognized as disadvantageous by normal individuals. An example given by Dr. Goldstein at BNL: â&#x20AC;&#x153;trading a car for cocaineâ&#x20AC;? [8].

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Tolerance is a common issue that evolves with continued use of a drug. Habitual cocaine use results in recurrent situations of excess dopamine in the synaptic cleft, which then bind to post-synaptic receptors. In response to this continual excess of dopamine in the synaptic cleft, a down-regulation of dopamine receptors occurs. This leads to fewer dopamine D2 receptors on post-synaptic neurons. As a result, it is more difficult to achieve the same level of dopaminergic transmission originally experienced with cocaine use. Additionally, this leads to disruption of normal dopaminergic transmission (without cocaine), as the amount of DA maintained in the synaptic cleft is no longer adequate to elicit the same transmission that occurred when a greater number of receptors were present [4].

In addition to the down-regulation of dopamine D2 re-

Drug addicts have always displayed abnormal behaviors, but it is only with current research on the neurobiology behind the psychological changes in addicts that we are beginning to understand the basis of their condition and the importance of drug addiction as a scientific issue and not just a societal disturbance. Based on the behavioral and fMRI studies conducted in Dr. Goldsteinâ&#x20AC;&#x2122;s laboratory at BNL and in laboratories at Stony Brook University and the National Institute on Drug Abuse, it is becoming clear that neurological pathways in cocaine addicted individuals become altered from that of normal individuals [5]. These alterations in neurotransmission contribute to addiction and the psychological symptoms seen in drug addicted individuals. It is this finding which serves as the basis for continued research into the neurobiology of the development of addiction and

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The Stony Brook Young Investigators Review, Spring 2010

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! Reviews ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! the neuronal alterations caused by drug addiction, which will lead researchers to better treatments for drug addiction.

*#&#-#0(#+ 1. Volkow ND, Fowler JS, Wang GJ, Goldstein RZ (2002) Role of dopamine, the frontal cortex and memory circuits in drug addiction: insight from imaging studies. Neurobiol Learn Mem 78: 610–624.

2. Landes, NM. “How Does Cocaine Alter Neurotransmission.” National Institutes of Health, National Institute on Drug Abuse. Bethesda, MD. 2000. Video. <http://science.education.nih.gov/supplements/nih2/ addiction/activities/lesson3_cocaine.htm>.

3. Berrios, Miguel. “Adrenergic Drugs.” BCP 401: Principles of Pharmacology. Department of Pharmacology, Stony Brook University Medical Center, Stony Brook, NY. 11 Nov. 2009. Lecture.

4. Berrios, Miguel. “CNS Pharmacology.” BCP 401: Principles of Pharmacology. Department of Pharmacology, Stony Brook University Medical Center, Stony Brook, NY. 9 Nov. 2009. Lecture.

5. Goldstein RZ, Volkow ND (2002). Drug addiction and its underlying neurobiological basis: Neuroimaging evidence for the involvement of the frontal cortex. Am J Psychiatry 159: 1642–1652.

6. Tucker, DM, Luu, P, & Pribram, KH. (1995). Social and emotional self-regulation. Annals of the New York Academy of Sciences, 769, 213–239.

7. Goldstein RZ, Tomasi D, Alia-Klein N, Cottone LA, Zhang L, Telang F et al (2007b). Subjective sensitivity to monetary gradients is associated with frontolimbic activation to reward in cocaine abusers. Drug Alcohol Depend 87: 233–240.

8. Brookhaven National Laboratory. Medical Research Department. Altered Perception of Reward in Human Cocaine Addiction. Laboratory News. Department of Energy, 15 Oct. 2006. Web. 12 Mar. 2010. <http://www.bnl.gov/ medical/personnel/Rita-Goldstein/files/Altered Perception of Reward in Human Cocaine Addiction.pdf>.

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The Stony Brook Young Investigators Review, Spring 2010

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A#-,+$"=-%+# Stem cells are unspecialized cells that are responsible for producing all of the organs and tissues in eukaryotes. They are formed from the onset of fertilization, first appearing in the morula stage, which possesses totipotent stem cells (capable of forming an entire organism). In the blastula stage, stem cells are found in the inner cell mass. These cells are pluripotent and can differentiate into nearly all of the body’s cells, including the three germ layers [1].

Embryonic stem (ES) cells are derived during this phase of development. Adult stem cells, which are multipotent (have the potential to form multiple tissue lineages), develop from the germ layers [2]. Cell fate is determined by growth factors, such as SONIC Hedgehog and Activin, released by surrounding cells [3].

Stem cells possess two unique features: ability to self-renew and the ability to differentiate. Self-renewal allows stem cell progeny to retain their stemness capacity. When stem cells differentiate, they lose their stemness and gain a specialization [4]. For instance, hematopoetic stem cells are multipotent blood stem cells which can self-renew and give rise to all of the daughter cells of blood, including B-cells, T-cells, and red blood cells, due to signals from the bone marrow microenvironment [5].

Induced pluripotent stem (iPS) cells were first discovered by Shinya Yamanaka in 2006 when he transformed mouse somatic cells into pluripotent cells throughout transfection by retroviruses of four transcription factors that code for stemness— octamer binding transcription factor 3/4 (Oct 3/4), Sry-related HMG-box transcription factor (Sox2), cellular myelocytomatosis oncogene (cMyc), and Kruppel-like factor 4 (Klf4). This groundbreaking study redefined the stem cell field because it introduced the potential of cellular reprogramming [6]. Initially, these cells exhibited DNA methylation errors; error frequency was greatly reduced the following year when Yamanaka replaced the detection marker, Fbx15, with Nanog [7]. In the same study, Yamanaka found a number of the offspring containing c-Myc developed tumors. Due to cMyc’s promotion of cancer, Yamanaka developed a method of iPS creation which ecluded the use of this gene [7]. In 2007, adult iPS cells were created through a viral vector transfection system, but in 2008, Yamanaka showed that plasmids could be used to transfect these genes instead [8], reducing the risk of teratoma development (which are cancer-like tumors derived from the three germ layers), even though iPS efficiency was lower. Yamanaka was awarded the 2009 Albert Lasker Basic Medical Research Prize for his discoveries of nuclear reprogramming that led to the creation of iPS cells.

In 2009, scientists from the Scripps Research Institute in California devised a method that utilized a poly-arginine transduction domain and would allow the transcription factors (the original four used by Yamanaka) to be inserted directly into mouse somatic cells via protein channels [9]. This method is safer than either plasmid or viral transfections because it does not result in genome modifi-

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cation of the host organism’s cells.

Although ES cells, adult stem cells, and iPS cells are all stem cells, they are different with respect to cellular features and potentials. ES cells and iPS cells possess the most potential in forming all of the cell types within the body, while adult stem cells are organ-specific with a limited cell-type differentiation capability. This is due to ES cells giving rise to adult stem cells. ES cells are immortal, while adult stem cells possess a limited lifespan. This is due to adult stem cells differentiating into body cells, such as blood, while losing their stemness in the process [3]. Examples of adult stem cells include mesenchymal stem cells, blood stem cells, muscle satellite stem cells, and heart resident stem cells. Therapies with adult stem cells utilize the patient’s own cells and therefore, there is little potential of immune rejection. ES cells possess a greater therapeutic potential, but their progress is confronted with ethical dilemmas, including how they are obtained. Additionally, patients face the possibility of immune rejection. iPS cells resolve both of these problems, but they possess their own limits. iPS cells are currently predominantly created through viral vectors. This presents a problem because the viral genetic material can potentially be inscribed into the genome of patients. In addition, iPS differentiation is not a stable process because these cells have the potential to differentiate into the wrong cell types. Recent studies have also shown that iPS cells possess quicker rates of apoptosis as compared to ES cells [10]. These cells also proliferate quickly, which is ideal for harnessing a substantial cell population for experimental studies, but can also lead to cancer if their growth becomes uncontrolled.

Although all three cell types possess their respective deficiencies, scientists are heavily researching them in an attempt to overcome these deficiencies. Much progress has recently been made in stem cell research in the categories of aging, blood regeneration, diabetes, cardiology, organogenesis, and neurology.

;&%#& Body tissues differ in their cell turnover and regenerative potential. For instance, blood cells, epithelium and epidermis possess a high turnover and regenerative potential, while the heart, brain, and spinal cord possesses low cellular turnover and regenerative potentials [11]. This is correlated with the extent to which stem cells are responsible for homeostasis and repair—where regions with lower cellular turnover and regenerative potential have a limited repair capability. Throughout the life of an organism, cells in regions with low turnover rates and regenerative potentials are replaced less frequently.

Skeletal muscles, which possess a low turnover rate and high regenerative potential, experience an impairment of the Notch signaling pathway with age. This pathway is vital for the first skeletal muscle cells in the embryo and regulates proliferation and differentiation of muscle satellite cells [12]. To prove this, Thomas Rando performed an experiment where he suchered an elderly and a young mouse together and their blood was allowed to mix [11].

If the elderly mouse were injured when suchered to the younger mouse, it regained the capacity to regenerate its muscle. He also observed that the Notch pathway was reactivated in the elderly mouse after suchering. This study allowed him to conclude that the younger mouse’s microenvironment stimulated the regeneration of

The Stony Brook Young Investigators Review, Spring 2010

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! Reviews ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! the Notch pathway in elderly mouse because the muscle cells of each respective mouse did not crossover [11, 12]. Current research is attempting to utilize ES cells to reactivate the Notch pathway in older muscle in order to promote regeneration [13].

69++$ Hematopoietic stem cells, which are found in the bone marrow, gives rise to blood cells, which are vital for oxygen transport throughout the body via hemoglobin (red blood cells) and play a role in the immune response (white blood cells). They also possess a high turnover rate and regenerative capacity with life spans of 10 days [11]. Studies have shown that a single blood stem cell can produce all of the blood in an animal [14]. This was shown in an experiment performed by Drize et al., where the blood supply in a mouse was first irradiated. Then when a single blood stem cell was injected, the mouse survived because all of the blood cells reformed from that one cell. Currently, adult stem cells are used in a clinical capacity, specifically in bone marrow transplants [3]. In February 2010, scientists at UC San Diego identified the specific vertebrate region where adult stem cells arise during embryonic development.

This provides insight into the differentiation pathway between ES cells and adult stem cells, and could lead to future therapies for patients suffering from blood disorders by producing patient-specific hematopoietic stem cells [15].

Recently, scientists in Weill Cornell Medical School discovered a new method of propagating blood stem cells, which en-

hanced the life of these cells by 21 days or more. Endothelial cells were used in this study because they create a microenvironment that triggers the regeneration of blood stem cells in the bone marrow [16]. For this study, the gene, E4ORF1, from an adenovirus was inserted into endothelial cells. These genetically-modified cells were then propagated in a petri-dish with blood stem cells extracted from mice without the addition of serum or growth factors.

This resulted in the production of a large number of hematopoietic stem cells. When these cells were inserted into mice with irradiated blood cells, these cells differentiated into all of the necessary blood cells within the mouse. There were no signs of tumor development within these animals after more than a year. This method was highly effective because by eliminating the growth factors, the stem cells were only influenced by the signals from the endothelial cells, which dictated differentiation into blood cells. As a result, this decreases the possibility of tumor formation or improper cellular differentiation. Scientists are hoping to use this knowledge to devise therapies of inhibiting tumor proliferation in cancer patients [16].

>%(10-0< Diabetes mellitus is an autoimmune disorder where the beta cells of the pancreas are destroyed and thus fail to produce insulin (a vital hormone that signals cells to uptake glucose), resulting in high glucose level build-up in the bloodstream. Since the pancreas possesses a low cellular turnover and high regenerative potential

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The Stony Brook Young Investigators Review, Spring 2010

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[11], it has a limited regenerative capacity.

In 2000, scientists in University of Wisconsin in Madison showed that ES cells can differentiate and express the insulin gene in human patients [17]. In 2001, scientists differentiated mouse embryonic cells into structures that resembled pancreatic islets that are able to secrete beta cells [18]. Current research is focused on the introduction of ES cells into the pancreas in hopes of promoting their differentiation into beta cells to regain pancreatic insulin secretion capability [19].

Recently, Professor Anthony Atala of Wakeforest University School of Medicine has discovered amniotic fluid cells, which are in a state intermediate of embryonic stem cells and adult stem cells and possess the potential to differentiate into any cell in the human body [20]. Although the therapeutic potential of these cells is currently being evaluated, studies have shown that these cells do not form teratomas [21]. Atala’s team is currently attempting to coax these cells to differentiate into beta cells of the pancreas [20].

A,-<7)8)>1$@$%->,0$*#>#0#-,.7)0 Although it was believed that the heart possesses a low regenerative capacity due to its low cellular turnover rate and regenerative potential [11], recent studies have indicated that the heart possesses a minute reservoir of progenitor cells that are capable of some regenerative capacity [22], when Antonio Beltrami first isolated c-kit+ cells from the myocardium of an adult rat [23]. These cells have the capacity to self-renew, are both multipotent and clonogenic, and give rise to three different cardiogenic cell phenotypes (cardiomyocytes, smooth muscle cells, and endothelial cells).

When these cells were injected into rat hearts after myocardial infarction, the myocardium was regenerated [24].

Heart formation results when pluripotent cells differentiate into mesodermal progenitors, where pluripotent capability decreases and differentiation capacity increases due to increased activity of lineage-specific activators, such as brachyury and MESP from the microenvironment. From this stage, cardiac cell fates are determined when they are separated into distinct populations of cardiac progenitor cells, distinguished by the presence of NKX2.5 alone (first heart field progenitors) or a combined expression of NKX2.5 and ISL1 (second heart field progenitors). The fates of these progenitors are determined by additional transcription factors. First heart field progenitors differentiate into cardiac conduction cells (HF-1b expression) and cardiac muscle cells (GATA4 expression), while second heart field progenitors differentiate into cardiac muscle cells (GATA4 expression), endothelial cells (HOXB5 expression), and postnatal cardiac progenitors [22].

When scientists attempted to increase the number of progenitor cells through the injection of ES cells, teratomas developed due to high expression of a multitude of growth factors from the cardiac microenvironment, causing the ES cells to assume a multitude of cardiac fates simultaneously [24]. To avoid such malignant results, scientists are attempting to coax ES cells into assuming a particular cardiac differentiative fate prior to injection by exploiting cellular pathways, such as the IGF-1/PI3 kinase/Akt signaling pathway, to control their proliferation [24]. In addition, the use of ES cells has been shown to provoke an immune response, hindering the effectiveness of the treatment [25]. When Cohen and Leor used a scaf-

Reviews

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fold to transfer ES cells into the scar tissue of a rat after myocardial infarction, they observed enhanced cardiac repair as compared to regular injection [26] because the scaffold was site-specific and it gradually transferred ES cells into the site of injury, reducing the risk of teratomas.

Last year, scientists at the Mayo Clinic were the first to use iPS cells to treat acute myocardial infarction in mice [27]. In this study, vectors with the four human transcription factors were packaged into a plasmid and transduced into mouse embryonic fibroblasts and proliferated in vitro. These cells were then transplanted into 8-12 week old athymic mice via four injections into the heart. The iPS cell therapy restored myocardial performance that was initially lost after the heart attack, stopped further heart damage, and regenerated damaged heart tissue at the site of injury [27]. The success of this study shows the potential of iPS cells in cardiac repair and will allow scientists to devise patient-specific iPS cells for therapy in the future.

Doris Taylor has approached the question of cardiac regeneration from a different perspective. Professor Taylor’s lab developed a technique called “whole organ decellularization” where all of the cells are stripped from an organ, leaving only the extracellular matrix [28]. In this technique, a heart was removed from an elderly rat, which was then drained for 12 hours with a solution to strip the heart of all of its cells. The heart scaffold was then reseeded with the cells of the organism that would receive the transplant.

Within a week, the heart was rejuvenated and began to pump because the stem cells had differentiated into their respective roles in the organ [28].

Professor Atala employed a similar technique for organ regeneration when he engineered a heart valve. A pig valve (since it is most similar in design to a human heart valve) was stripped of all of its cells, leaving only a scaffold behind. Next, this scaffold was placed underneath an inkjet printer that coated the valve with stem cells. The valve was then subject to cyclic stretching, a form of exercise that resembles valve contractions within the body. After six weeks, the valve was ready for implantation into a human patient. This technique was employed by Professor Atala’s lab in 2004, when his team developed the first lab-grown organ (a bladder) that was implanted into a human [29]. Thus far, five such bladders have been transplanted into patients and all five work just as effectively as their native human counterparts [30]. This technique is highly effective because it allows organs to be created that are specifically-suited for each patient, in terms of size and length without any fear of rejection.

The Stony Brook Young Investigators Review, Spring 2010

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! Reviews ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! .(,7%#<+#\<'>%<0(<0'' Parkinson’s disease is the gradual loss of nigrostrial dopaminecontaining neurons resulting in motor skill impairment [31]. Due the brain’s limited regenerative capacity [11], damage to the Central Nervous System is very difficult to repair.

Earlier this year, scientists at Stanford University School of Medicine transformed mouse fibroblasts into functioning nerve cells in vitro through the use of three transcription factors (Brn2, Myt1l, and Ascl1) via lentivirus transfection [32]. The resulting neurons looked similar to natural neurons, were produced faster (within a week) and with an enhanced 20% efficiency (compared to the normal 1-2% of cells that assume pluripotency in the typical iPS cell protocol). They also skipped the pluripotent phase of development, thus preventing the formation of tumors. These neurons expressed identical function to natural neurons, including the generation of action potentials, the formation of synapses, and the expression of multiple neuron-specific proteins [32]. This study suggests that the pluripotent phase maybe just a stage in embryonic development that could be skipped using the correct transcription factor combination [33]. This study is a major step forward in understanding the progression of Parkinson’s disease in patients and for developing patient-specific neurons from their own cells.

)-0?'8099'A#<-%-"-0'''''''''''' Since 2007, Germany has possessed a stem cell institute which offers stem cell treatments to patients suffering from detrimental maladies, including diabetes, stroke, spinal injuries, arthritis, heart disease, multiple sclerosis, and many more [34]. In March of 2010, the UC Davis Center for Regenerative Cures opened in Sacramento, California. This is the first stem cell institute in California to offer stem cell treatments to patients. This facility will house 200 scientists and medical personnel and will offer treatments for heart damage, vision impairment, HIV, Huntington’s disease, and peripheral vascular disease [35]. An individualized stem cell line will be created for each patient from their own stem cells to be used for their individual therapy. This shows just how much stem cell research has evolved since its start in 1998. It is now a sub-sector of translational medicine, where scientific discoveries at the bench will be applied bedside to patients in the pursuit of offering hope and novel treatment against today’s maladies.

8+#=9"<%+# Since the late 1990’s, stem cells have arisen as one of the most powerful and prominent research areas for disease therapy. Although the ethical debate has not been fully put to rest, the discovery of iPS cells opens a new door for cellular reprogramming with the quick creation of patient-specific stem cells for therapies.

Despite the current limitations of these cells, scientists are working assiduously to advance understanding and procedures to better control stem cell proliferation and differentiation. The progress of this field in its short existence illustrates the clinical potential that these cells may have to offer patients long-awaited therapies from their detrimental maladies. With the opening of more translational

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medicine stem cell institutes and the allocation of greater funding to this field of research, the future of this field and its potential to enhance the lives of humanity looks very bright.

;=7#+D90$&0?0#-< I thank Bin Zeng for reviewing this article and for our fruitful conversations about stem cell research.

*#&#-#0(#+ 1.Smith A. A glossary for stem-cell biology. 2006. Nature. 441, 1060.

2.Barrilleaux B, Phinney DG, Prockop DJ, and O’Connor KC. Tissue Engineering. 2006. 12(11), 3007-3019.

3.Melton, Douglas. Understanding Embryonic Stem Cells.

2008. The Howard Hughes Medical Institute Lecture Series.<http://www.researchchannel.org/prog/displayevent. aspx?rID=16091&fID=345>.

4.Kochar PG. What Are Stem Cells? 2004. ProQuest Information and Learning. <http://www.csa.com/discoveryguides/stemcell/ overview.php>.

5.Bordignon C. Stem-cell therapies for blood diseases. 2006. Nature. 441, 1100-1102.

6.Mauritz C, Schwanke K, Reppel M, Neef S, Katsirntaki K, Maier LS, Nguemo F, Menke S, Haustein M, Hescheler J, Hasenfuss G, and Martin U. Generation of Functional Murine Cardiac Myocytes From Induced Pluripotent Stem Cells. 2008. Circulation.

118, 507-517.

7.Okita K, Ichisaka T, and Yamanaka S. Generation of germlinecompetent induced pluripotent stem cells. 2007. Nature. 448, 313317.

8.Okita K, Nakagawa M, Hyenjong H, Ichisaka T, and Yamanaka S. Generation of Mouse Induced Pluripotent Stem Cells Without Viral Vectors. 2008. Science. 322, 949-953.

9.Zhou H, Wu S, Joo JY, Zhu S, Han DW, Lin T, Trauger S, Bien G, Yao S, Zhu Y, Siuzdak G, Schöler HR, Duan L, and Ding S. Generation of Induced Pluripotent Stem Cells Using Recombinant Proteins. 2009. Cell Stem Cell. 4, 381-384.

10.Choi CQ. Cell-Off: Induced Pluripotent Stem Cells Fall Short of Potential Found in Embryonic Version. 2010. Scientific American.

11.Rando TA. Stem cells, ageing and the quest for immortality.

2006. Nature. 441, 1080-1086.

12.Rosenthal, Nadia. Stem Cells and the End of Aging. 2008. The Howard Hughes Medical Institute Lecture Series.<http://www.researchchannel.org/prog/displayevent. aspx?rID=16094&fID=345>.

13.Carlson ME, Suetta C, Conboy MJ, Aagaard P, Mackey A, Kjaer M, and Conboy I. Molecular aging and rejuvenation of human muscle stem cells. 2009. EMBO Molecular Medicine. 1, 381391.

14.Drize N, Chertkov J, Sadovnikova E, Tiessen S, and Zander A. Long-Term Maintenance of Hematopoiesis in Irradiated Mice

by Retrovirally Transduced Peripheral Blood Stem Cells. 1997.

Blood. 89, 1811-1817.

15.McDonald, Kim. Biologists Image Birth of Blood-Form-

The Stony Brook Young Investigators Review, Spring 2010

! ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! ing, Stem Cells in Embryo. 2010. UC San Diego News Center. <http://ucsdnews.ucsd.edu/newsrel/science/02-09StemCells.asp>.

16.Butler JM, Nolan DJ, Vertes EL, Varnum-Finney B, Kobayashi H, Hooper AT, Seandel M, Shido K, White IA, Kobayashi M, Witte L, May C, Shawber C, Kimura Y, Kitajewski J, Rosenwaks Z, Bernstein ID, and Rafii S. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. 2010. Cell Stem Cell. 6, 251-264.

17.Schuldiner M, Yanuka O, Itskovitz-Eldor J, Melton D, and Benvenisty N. Effects of eight growth factors on the differentiation of cells derived from human embryonic stem cells. 2000. Proc.

Natl. Acad. Sci. U.S.A. 97, 11307–11312.

18.Lumelsky N, Blondel O, Laeng P, Velasco I, Ravin R, and McKay R. Differentiation of Embryonic Stem Cells to InsulinSecreting Structures Similar to Pancreatic Islets. 2001. Science.

292, 1389–1394.

19.Brennard K and Melton D. Slow and steady is the key to -cell replication. 2009. J Cell Mol Med. 3, 472-487.

20.Anthony Atala Lab. Engineering Pancreatic Beta Cells. Wake Forest University Center of Regenerative Medicine. <http://www. wfubmc.edu/Research/WFIRM/Diabetes-Treatment.htm>.

21. Furth ME and Atala A. Stem cell sources to treat diabetes.

2009. J Cell Biochem. 106, 507-511.

22.Shrivastava D and Ivey KN. Potential of stem-cell-based therapies for heart disease. 2006. Nature. 441, 1097-1099.

23.Beltrami AP, Barlucchi L, Torella D, et al. Adult cardiac stem cells are multipotent and support myocardial regeneration. 2003.

Cell. 114, 763–776.

24. Murry CE, Reinecke H, and Pabon LM. Regeneration gaps: observations on stem cells and cardiac repair. 2006. J Am Coll Cardiol. 47, 1777-1785.

25. Mathur A and Martin JF. Stem cells and repair of the heart.

2004. Lancet. 364, 183–192.

26. Cohen S and Leor J. Rebuilding Broken Hearts. 2004. Scientific American. 45-51.

27. Nelson TJ, Fernandez AM, Yamada S, Terzic CP, Ikeda Y, and Terzic A. Repair of Acute Myocardial Infarction by Human Stemness Factors Induced Pluripotent Stem Cells. 2009. Circulation.

120, 408-416.

28. Doris Taylor Lab. University of Minnessota Stem Cell Institute. <http://www.stemcell.umn.edu/stemcell/faculty/Taylor_D/ home.html>.

29. Lab-grown bladders ‘a milestone’. 2006. BBC News. <http:// news.bbc.co.uk/2/hi/health/4871540.stm>.

30. Anthony Atala Lab. Engineering a Blood Vessel. Wake Forest University Center of Regenerative Medicine. <http://www. wfubmc.edu/Research/WFIRM/Blood-Vessel2.htm>.

31. Lindvall O and Kokaia Z. Stem cells for the treatment of neurological disorders. 2006. Nature. 441, 1094-1096.

32. Vierbuchen T, Ostermeier A, Pang ZP, Kokubu Y, Südhof TC, and Wernig M. Direct conversion of fibroblasts to functional neurons by defined factors. 2010. Nature. 463, 1035-1041.

33. Cogner K. Dramatic transformation: Researchers directly turn mouse skin cells into neurons, skipping IPS stage. 2010. Wernig Lab. <http://med.stanford.edu/ism/2010/january/wernig.html>.

34. X-Cell Institute of Regenerative Medicine. <http://www.xcellcenter.com/>.

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35. Tong A and Kalb L. $62 million UC Davis center puts Sacramento at hub of stem cell research. 2010. The Sacramento Bee.<http://www.sacbee.com/2010/03/10/2595672/uc-davis-62millionl-center-puts.html>.

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! Reviews ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! ;))*34$%2$26)$@2->*+$ =)B)( F*+$/;"F,&*+*6,+,.*+*-A"V*+1"K%(:/((%"U/62"#'233("iJI

Imaging techniques have and continue to foster our scientific understanding. From the invention of the light microscope, which led to the discovery of cells, to the use of X-ray crystallography to elucidate the structure of proteins and nucleic acids, imaging has allowed for our insight into biology to increase. Better imaging techniques and instruments continue to be developed in order to overcome the constraints of the human eye. While the resolution of a light microscope is limited by the wavelength of illuminating light, an electron microscope gives us a much greater resolution depending on the voltage applied [1]. For its part, the Atomic Force Microscope (AFM), a scanning instrument, may allow us to visualize the individual atoms of a molecule, and obtain results similar to the ball and stick models seen in chemistry textbooks. In fact, recent work by IBM scientist Leo Gross and his colleagues demonstrates how modifying the atomic microscope’s tip apex yields an atomic resolution image of pentacene [2].

The Atomic Force Microscope was originally created in 1986 to overcome the drawbacks of the Scanning Tunneling Microscope (STM). Since the STM can only create images of the samples placed on a conducting or semi-conducting surface, the types of samples that can be viewed are severely limited. The AFM, however, has the ability to create images on any kind of surface including polymer, biological and ceramic substrates. Before the findings of Gross et al., atomic force microscopy was used to resolve the structure of separate atoms, but not that of atoms within an adsorbed molecule on a surface [2]. The primary reason why the latter could not be accomplished was that the tunneling current was “primarily sensitive to the local electron density of states close to the Fermi level” [2]. A tunneling current is created when the tip of the STM

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is close enough to the sample, and then an applied voltage between the tip and the sample causes the electrons to tunnel through the junction. This current is dependent (exponentially) on the width of the electron junction. This sensitivity can be used to create an image of atomic resolution [2]. When working at an atomic level, the term Fermi Level is constantly used in order to describe the amount of electron energy levels at absolute zero. Electrons, which are known as Fermions, cannot exist at the same energy states according to Pauli’s exclusion principle. At absolute zero these fermions try to all exist at the lowest energy level, which creates a sea of electrons. The Fermi level occurs at absolute zero where no electrons are able to reach a higher energy level [3].

The key to resolving the problem caused by the sensitivity of the tunneling current was to terminate the tip of the microscope’s probe with a well-known and suitable molecule and understand that Pauli repulsion was the force that tampered with the image

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The Stony Brook Young Investigators Review, Spring 2010

! ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! (the Van der Waals and electrostatic forces were found to contribute to background of the image rather than the actual image itself ). Pentacene, a well-studied polycyclic hydrocarbon, was the molecule the Gross group chose to investigate. Previous scanning tunneling microscopy tests on pentacene had found that resolving this molecule on metals such as Cu and thin film insulators such as NaCl was difficult to accomplish using this technique. This difficulty arose because STM imaging was “affected by the density of the states when at the Fermi level” (Energy of electrons within a semi-conductor) [2].

In order to create a high-resolution image of an atom within a molecule with AFM, Gross et al. had to operate within a short range of forces while simultaneously using, preferably, a stiff cantilever with “oscillation amplitudes of about 1 Å” [2]. In order to successfully operate at even lower amplitudes such as 0.2 Å, it was necessary for the tuning fork to express a resonance frequency of 23,165 Hz. In addition to this setup, a carbon monoxide terminated tip was placed at the apex of the probe and resulted in the increased resolution of the image. The idea for modifying the tip came from a similar procedure used on STMs that produced enhanced resolution. By exploring this idea, Leo Gross et al. were able to create a cleaner and crisper image with higher resolution.

The tips were also modified with Cl, pentacene, and metals such as Ag, Au, and Cu. Out of its (CO) five competitors, Cl gave an image with the next best resolution. Unlike the CO tip; however, the Cl tip produced an image that had a smaller set of benzene rings as well as less pronounced “hollow sites above the minima” [2].

Leo Gross, et al. concluded that Non-Contact Atomic Force Microscopy could indeed resolve the structure of atoms within molecules, if and only if the AFM entered the realm of “repulsive forces” [2]. At 1.2 Å, it was observed that the strength of repulsive forces reached maxima, which, therefore, provided the best contrast and lateral resolution. Overall, the use of a CO terminated tip with AFM resulted in images that were crisp, clean, and informative. Leo Gross et al. point out that the uses of such a discovery include, but are not limited to, analyzing catalysts, investigating single-electron transport chains and metal-molecule systems on the atomic scale [2].

Being able to visualize a molecule at this level may result in obtaining greater insight into the electronic and chemical properties of any molecule at the atomic scale [7]. In fact, the Gross team and other IBM scientists hope to be able to understand electron transport through molecular networks and use this information to build “smaller, faster and more energy-efficient computing components” [7]. Thus, not only is the work of Leo Gross et al. notable for being the first time the chemical backbone of a molecule was imaged, but also for the profound impact that their results may have in nanotechnology.

Reviews

Hyperphysics. Georgia State U, n.d. Web. 12 Nov. 2009. <http:// hyperphysics.phy-astr.gsu.edu/hbase/solids/fermi.html>.

4. Voice of Progress. “Atomic Force Microscope (AFM).” Voice of Progress. N.p., 31 Mar. 2009. Web. 8 Nov. 2010 <http://www. voiceofprogress.com/?p=1343>.

5. Nanoscience Education. “Atomic Force Microscopy.” nano Science Instruments. nano Science Instruments, 2008. Web. 2 Nov. 2009. <http://www.nanoscience.com/education/AFM,html>.

6. Galloway Group. Atomic Force Microscopy: A Guide to Understanding and Using the AFM . 2004. <http://uweb.txstate. edu/~ab35/manuals/AFMmanuals/AFMLabManual.pdf> 7. Nanotechnology. “Scientists Image ‘Anatomy’ of a Molecule using Noncontact Atomic Force Microscopy.” Nanontechnology.

AZoNetwork Sites, 28 Aug. 2009. Web. 1 Nov. 2009. <http:// www.azonano.com/news.asp?newsID=13363>

*#&#-#0(#+ 1. Karp, Gerald. “Techniques in Cell and Molecular Biology.” Cell and Molecular Biology: Fifth Edition. pp. 734-735. John Wiley & Sons, Inc., Hoboken, NJ. 2. Leo Gross et al. “The Chemical Structure of a Molecule Resolved

by Atomic Force Microscopy”. Science, Vol 325, 1110-1114, 2009.

3. Georgia State University (Physics Department). “Fermi Level.”

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Each year thousands of students enter college with the hope of pursuing medicine. However, a significant percentage of these students never become physicians. The latter is often the result of the immense number of premedical requirements that undergraduates must satisfy. In addition to this intense workload, many of the premedical students who do master the requirements are often not admitted to medical schools because of Medical College Examination Test (MCAT) score cutoffs and/ or the limited number of first-year positions available [1]. For example, there were 42,269 applicants for medical school this year alone with only 18,390 first-year slots available [2]. It is no secret that the undergraduate years serve as a “weeding out” process for medical schools. At times, even some of the most successful students do not gain admission into medical school. Thus, it is apparent that the road to becoming a doctor in the United States is one fraught with a variety of challenges.

!"#$%-7>70+$)&$B-#C#<7(,8$*#D;7-#C#0.+$ In the 19th century, the majority of medical institutions in the United States had very few formal premedical requirements.The common belief was that men aspiring to practice medicine simply needed to have a general understanding of the sciences and thus essentially anyone with the means to afford medical school could study medicine [3]. However, in the early 20th century the American Medical Association (AMA) created a council to standardize premedical requirements for entrance into medical schools and to restructure US medical education as a whole.The proposed premedical requirements were formalized and enumerated in Abraham Flexner’s 1910 report, Medical Education in the United States and Canada. As a result of Flexner’s report and work by the AMA council, premedical requirements necessitating competencies in biology, classical physics, and organic chemistry were finally set in the 1930s. With the exception of the addition of a calculus requirement and an additional semester of organic chemistry, these requirements remain virtually unchanged. Consequently, as one might expect, there has been much disagreement in the past century over the purported benefits of the premedical curriculum, since many feel that the premedical requirements fail to achieve the intended goals.

I5(-\<'I,+#&'I%-5'-50'8",,0#-'.,0?0$%=(9' *#D;7-#C#0.+E Though almost a century has passed since the Flexner report, it appears that we are not any closer to understanding what may or may not be wrong with premedical education [4]. The intended goals of the premedical curriculum are to give students a broad education so as to ultimately shape them as physicians who are able to reason morally and assess problems analytically and critically. However, many medical educators fervently argue that these goals are rarely achieved. Much of the criticism

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of the current premedical curriculum is situated around its rigid emphasis on the basic sciences, for many feel that this produces students who become narrowly focused and excessively concerned with grades. Some individuals favor the current curriculum because they see it as an effective way to thin out the medical school applicant pool. Nevertheless, many medical educators still feel that several of the courses that satisfy premedical requirements do not truly enhance undergraduate premedical training.

They argue that these courses are narrowly focused, for they often neglect to present any medical relevance [5]. Medical educators contend that premedical college courses mainly prepare students for material covered on standardized tests and thus cater to the MCAT. Consequently, there is now a call for undergraduate institutions to create premedical courses that will prepare students more adequately, by focusing on human biological principles and areas of science relevant to medicine [5].

In addition to this, many medical educators think that because of the narrow focus of premedical courses students often fail to grasp the crossdisciplinary nature of modern science. It is apparent that because we are complex organisms we are comprised of discrete systems, which are intricately linked within the body and modified profoundly by external influences [5]. Consequently, many individuals argue that premedical courses should be taught in a manner that truly reflects this interconnectivity.This can be accomplished by transcending the old-fashioned compartmentalization of undergraduate and medical school departments.

Most premedical curriculum reforms only examine the undergraduate level; however, many medical educators insist that revamping the curriculum alone is simply not enough.They argue that for one to truly assess and improve the issues with the premedical curriculum it is essential to reexamine the current structure of the medical system as a whole. Medical educators also stress that there needs to be more communication between undergraduate institutions and medical schools, for there is an evident disconnect between the sort of premedical preparation medical school faculty desire and the preparation that the current premedical curriculum actually provides. As a result, in the preclinical years medical school faculty often devote an extraordinary amount of time to elementary biochemistry and cell biology course material [5]. Moreover, medical school instructors argue that by neglecting to adequately address biologically relevant material, premedical science courses fail to prepare students for the rigor of preclinical course work. Medical educators also believe that many of the issues with the premedical curriculum stem from the current structure of the MCAT, for they argue that premedical science courses mainly prepare students for this exam. Consequently, they contend that by the end of undergraduate education most premedical students can only master the material covered on the MCAT. Currently, premedical students are required to take calculus, physics, and chemistry courses that mainly cover material presented on the MCAT, but rarely address biologically relevant material.Thus, many medical educators believe that while it is necessary for premedical students

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Perspectives to be familiar with material from such subject areas, it is essential that they learn this material in a manner that stresses their medical relevance.

In particular, numerous medical educators argue that instead of taking a second semester of organic chemistry that focuses on synthesis, premedical students should take a second semester organic chemistry course that introduces and focuses on relevant biochemical principles.They argue that dividing organic chemistry courses in such a manner will provide premedical students with a solid foundation in essential biochemical principles, which first-year medical school courses can then build upon.

In addition, many critics also argue that the curriculum focuses too heavily on satisfying specific requirements and they point out that very few students have time to reflect on the experience of being premedical students. While academic advisors are aware that the undergraduate years should both develop and reflect character, they often advise their students in a manner that is quite contradictory. On the one hand they encourage students to take stimulating courses that will challenge their intellect, while on the other hand they simultaneously remind students of the GPA driven, competitive nature of the medical school application process [6]. As a result, many students only take courses that are required by medical schools and directly relevant to material covered on the MCAT. Subsequently, very few students enroll in courses outside of the premedical requirements due to fear that outside courses may lower their GPA and thus their chance of getting into medical school, even though these courses may be beneficial in shaping them as physicians. Also, many students often limit themselves by only participating in extracurricular activities that they feel will portray them as the type of student medical schools are “looking” for.

While most universities advise premedical students to strategically choose their courses, very few discourage students from further challenging themselves by asking for courses outside the premedical requirements.

For example, the Stony Brook University pre-medical advising website says that while it is important for premedical students to tactically choose courses relevant to their course of study, “there is no substitute for good grades built upon a solid foundation of hard work in challenging courses which [students] have chosen prudently” [7]. However, most universities’ pre-professional advising services strongly encourage premedical students to focus on courses and extracurricular activities that will demonstrate to medical school boards that they are “good” candidates. For example, premedical advising services at the University of Virginia encourage premedical students to do research, for undergraduate research “[demonstrates] in-depth, sustained scholarly exploration, as well as the presence of lifelong learning skills that are essential in these professions” [8]. Here we see that premedical students are not told that research will help to develop these qualities, but that the advice is strategically oriented and focused on primarily engaging in activities that will demonstrate character. As a result of this, we see that many premedical students are only concerned with satisfying a checklist of courses and extracurricular activities, which will hopefully prove that they are good candidates for admission to medical school. Yet, because of this narrow mentality many premeds become automation-like and fail to see the crucial role their premedical years truly play in shaping their characters and values.

across the country to discuss ways to improve the current premedical requirements. A major goal of these meetings was to devise a set of skills, which premed students would need to satisfy, that accurately reflect the multidisciplinary nature of modern medicine. The report, “Scientific Foundations for Future Physicians”, published in June by the AAMC and Howard Hughes Medical Institute (HHMI) lists 16 competency skills, 8 of which articulate the science skills that medical students must gain before they leave medical school. The remaining 8 competencies are a checklist of skills that premedical students would need to satisfy in place of the traditional required premedical science and mathematics courses.

The competencies are: 1. “Apply quantitative reasoning and appropriate mathematics to describe or explain phenomena in the natural world.”

2. “Demonstrate understanding of the process of scientific inquiry, and explain how scientific knowledge is discovered and validated.”

3. “Demonstrate knowledge of basics principles and their applications of living systems.”

4. “Demonstrate knowledge of basic principles of chemistry and some of their applications to the understanding of living systems.”

5. “Demonstrate knowledge of how biomolecules contribute to the structure and function of cells.”

6. “Apply understanding of how molecular and cell assemblies, organs, and organisms develops structure and carry out function.”

7. “Explain how organisms sense and control their environment and how they respond to external change.”

8. “Demonstrate an understanding of how the organizing principle of evolution by natural selection explains the diversity of life.” [9]

This checklist aims to restructure the current premedical curriculum by moving away from a rigid set of required courses, thus giving students the option to study a wider range of subjects. In the report, the committee attributes the need to reform the premedical curriculum to address “[concerns] that premedical course requirements have been static for decades and may not accurately reflect essential competencies every medical student must have mastered, today and in the future” [9]. Ideally, the proposed changes are meant to address the needs of modern medicine by widening the range of applicable courses, which many hope will implicitly encourage more interdisciplinary learning. For example, the proposed chemistry competencies continue to emphasize the need for organic chemistry in premedical studies; however, now they do so in a manner that emphasizes organic chemistry’s biomedical relevance. What sets the new competencies apart from the current chemistry requirements is that they explicitly emphasize using organic chemistry to understand and “explain biochemical reactions” [9].

As a result, many medical educators view the change to competencybased educational goals in a positive light. The latter is due to the report’s emphasis on incorporating more biologically relevant topics in premedical science courses. In particular, medical educators feel that this reform will allow undergraduate institutions to design curricula that truly reflect the integrated nature of modern science. Several institutions have already made changes to the way they teach introductory sciences courses *#=,C?70>$."#$B-#C#<7(,8$*#D;7-#C#0.+F$5",.$G)#+$ by developing interdisciplinary introductory courses. In 2006, Harvard launched a series of introductory courses that place an emphasis on the !"7+$*#,881$H#,0E$ interdisciplinary nature of the sciences. These courses, Life Sciences 1a In June 2009, the Association of American Medical Colleges and 1b, incorporate a wide range of topics from cellular and molecular (AAMC) met with physicians, scientists, and science educators from biology to general and organic chemistry. For example, in this course reacThe Stony Brook Young Investigators Review, Spring 2010

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! Perspectives ! ! ! ! ! ! ! ! ! ! !!!!!!!!!! tion thermodynamics and kinetics are taught in the context of the reaction catalyzed by HIV protease and inhibited by HIV protease inhibitors and pKa, pH, and the concept of equilibrium are taught using the phosphate group of DNA and the side chains of amino acids [10]. Princeton University has also designed a series of such courses, which incorporate unifying principles from biology, chemistry and physics into a two-semester course. University faculty have noticed that such interdisciplinary courses in design and conception are a step in the right direction, for premedical students are now able to begin to see medical relevance in the chemical and physical concepts they learn in their undergraduate years.

Many of these integrated courses start off with introductory material and then apply these topics to particular areas of research. Science educators believe that this integrative course approach can be extremely beneficial, as premedical students can be introduced to a diverse array of material in a manner that shows how the relevant biological, physical, or chemical topics play central roles in the problem solving process in medicine. In an interview with the American Chemical Society (ACS), Dr. Robert A. Lue, a professor of molecular and cellular biology at Harvard, said that courses like Life Sciences 1a and 1b are beneficial to many students interested in the sciences, for they avoid making a “distinction between science and non-science majors that may go on to medical school”; thus, they prepare both types of student in a way that is reflective of the multidisciplinary nature of modern science [11]. Many medical schools are also realizing the benefits of interdepartmental collaboration. Institutions such as Harvard Medical School and Weil Cornell Medical School have already added new departments of systems biology, which use multidisciplinary approaches to elucidate the complex interactions of body systems and to further understand diseases [5]. Some individuals contend that creating new interdisciplinary science courses may prove difficult due to the “varying availability of resources; depth of faculty; and political will of traditional departments” [5]. However, for modern medicine to keep pace with the ever-evolving nature of science interdepartmental collaborations, at both the premedical and medical school level, are essential.

The premedical years play a crucial role in preparing and shaping students for medical school and later years as physicians, both in the United States and abroad. While the AAMC’s competency-based educational reforms, to emphasize premedical courses that are relevant to the biomedical sciences, are a step in the right direction there is still much room for improvement. Though the AAMC report does note the value of a broad, liberal arts education, specifically by stating that medical school students should not be “limited to the study of science with little time available to pursue other academically challenging scholarly avenues …”, it only presents competencies that are relevant to the natural and physical science [9]. In addition, it is evident that if we are to see any major changes in the structure and content of premedical courses then parallel changes need to be made to the MCAT. While a separate AAMC committee is reviewing the MCAT requirements, a revised MCAT will be introduced no earlier than 2013 [9].

For the American medical system to truly be on par with the leading medical systems across the world we must make sure our students have the opportunity to take a diverse array of courses in their premedical education, which will ultimately prepare them for the various biological and ethical problems that they will encounter as physicians. Also, if we are to see substantial changes in American medical education, scientists and medical professionals must constantly question whether or not established pedagogic policies actually further the advancement of modern medical practices and needs. With the latter, medical educators and scientists will

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be able to eliminate policies and requirements that no longer meet the needs of modern medicine. For the American medical system to effectively train physicians who are in touch with the evolving nature of modern science and the diverse medical needs of their patients, medical educators must remember that the realms of premedical and medical education are inextricably linked. Furthermore, to truly facilitate change we must see an increase in interaction and dialogue between these two bodies.

*#&#-#0(#+F 1. Dalen J., Alpert J. Premed Requirements: The Time for Change Is Long Overdue! The American Journal of Medicine:122(2):104-106.

2. Association of American Medical Colleges. AAMC Data Warehouse: Applicant Matriculant File As of October 19 2009. http://www.aamc. org/newsroom/pressrel/2009/enrollmentdata2009.pdf .Accessed November 15, 2009.

3. Flexner A. Medical Education in the United States and Canada: A Report to the Carnegie Foundation for the Advancement of Teaching.

Bulletin No. 4. Boston, MA: Updyke; 1910.

4. Gross JP, Mommaerts CD, Earl D, De Vries RG. After a century of criticizing premedical education, are we missing the point? Acad Med. 2008:83(5):516-520.

5. Dienstag J. Relevance and Rigor in Premedical Education. New England Journal of Medicine. 2008:359(3):221-224.

6. De Vries R., Gross J. The Winnowing Fork of Premedical Education: Are We Really Separating the Wheat from the Chaff? American Medical Association Journal of Ethics, Virtual Mentor. 2009:11(11):859-863.

7. Stony Brook University. Pre-Health Advising.2009 http://www.stonybrook.edu/ healthed/basic.shtml. Accessed November 22, 2009.

8. University of Virginia. University career services. 2009 http://www. career.virginia.edu/students/preprof/prehealth/extra.php. Accessed November 15, 2009.

9. Association of American Medical Colleges. Scientific foundations for future physicians. 2009. https://services.aamc.org/publications/showfile. cfm?file=version132.pdf &prd_id=262&prv_id=321&pdf_id=132. Accessed November 15, 2009.

10. Arnaud C. Chemical & Engineering 2009: 87(44):35-38 http://pubs. acs.org/cen/education/87/8744education.html. Accessed November 15, 2009. 11. Arnaud C. Chemical & Engineering 2006: 84(29):43-45 http://pubs.acs.org/cen/education/84/8429education.html.

Accessed November 15, 2009.

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You know what Google is doing each time you press the search button: it compiles a list of results and enumerates thousands of websites in record time. Rather the question is: what is Google doing to your brain and what long term effects could it have on your cognitive function?

A study by researchers at UCLA suggests that when elderly subjects between the ages of 55 and 76 perform internet searches, they show increased activation of neural circuitry. It seems that additional areas of the brain are stimulated when these subjects are carrying out an internet search than when reading a book [1].

That is, when an elderly person uses Google to find information, he utilizes regions of the brain that are not active when simply reading basic text.

To study neural activity, the UCLA researchers compared the activation patterns that are present when performing an online search task to those present when reading text. The study was conducted using internet savvy subjects with internet use ranging from one to several times a day, and internet naïve subjects with internet use of less than once a month. As the subjects carried out each activity, the intensity of blood flow during memory and cognitive tasks was monitored and the areas of brain activation were visualized using functional magnetic resonance imaging (fMRI) [1].

During the text reading task, it was found that the internet naïve and internet savvy subjects showed increased signaling in the frontal, temporal, and parietal regions of the left hemisphere.

There was also activation of the visual cortex, hippocampus and posterior cingulate.

When subjects then performed an internet search task, it was found that in the internet naïve, regions of brain activation were similar to the text reading task. Striking results were found, however, in internet savvy subjects. There was significant additional activation in the frontal pole, right anterior temporal cortex, the anterior and posterior cingulate, and the right and left hippocampus when they performed an internet search compared to when they completed the basic reading task. Overall, the fMRI study shows a greater level of brain activity in both internet naïve and internet savvy subjects when executing an internet search task than when reading basic text, but this increase is much more pronounced in internet savvy individuals [1].

This study entitled “Your Brain on Google”, published in the American Journal of Geriatric Psychiatry, suggests that internet savvy elderly subjects show more than a two-fold increase in spatial extent of activation when performing an internet search task than when reading a book [1]. The results of this study are of great importance. They suggest that computer based technology can be used to improve cognitive ability and brain function in older adults.

Moreover, the study yields promising results because even af-

ter continued repetitions of the internet search task, internet savvy subjects were shown to retain the increased brain activity associated with using a search engine [1]. The activity did not simply become routine; it continued to provide a cognitive challenge that elicited increased brain stimulation in order to complete the task.

Consequently, “Googling” could potentially be used as a cognitive therapy for the elderly. Naturally, this begs the question: what could this ubiquitous internet tool mean for the younger generations?

As we age, our brains undergo many transformations: amyloid plaques form, and there are even differences in glucose metabolism and size. Among the most significant changes are the decreases in cognitive ability, processing speed, and working memory.

Therefore, in light of this research and the growing pervasiveness of the Internet and other computer technology, one must wonder whether the tasks that apparently enrich the cognitive capabilities of the elderly can elicit the same response in younger brains.

Our daily use of the Internet and search engines such as Google to research and gather information could be providing us with a constant mental exercise. Most importantly, if the current trend in computer activity continues, the cognitive ability of an internet savvy individual in 60 years may even produce neural activation that surpasses that of a 60 year-old internet savvy person today.

Although it cannot yet be said that everyone experiences the same increased brain activity that the internet savvy elderly subjects show when performing an internet search task, the results of this study are hopeful nevertheless. With further investigation, we may find that our daily use of the internet and tools such as the Google search engine improves our brain function and provides continued exercise to help stave off the decline in cognitive ability that is typically associated with aging.

*#&#-#0(#+ 1. Small, Gary W. M.D.; Moody, Teena D. Ph.D.; Siddarth, Prabha Ph.D.; Bookheimer, Susan Y. Ph.D. “Your Brain on Google: Patterns of Cerebral Activation during Internet Searching.” American Journal of Geriatric Psychiatry 17.2 (2009): 116-26.

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Summer vacation: two to three glorious months that we, students, anxiously wait for after an arduous year of seemingly endless lectures, tedious assignments, and stressful exams. For a medical student, this highly anticipated period of relaxation really only happens between our first and second year of school. So what do we do if this may be the last summer vacation of our lives?

Some medical students choose to go into “bumming out” mode to avoid complete burn out. Some decide to work to try to minimize the heaps of debt that we will accumulate at the end of our four years. Others choose to volunteer, conduct a research project or travel to distant places. For me, I was able to combine the latter because I was fortunate enough to be awarded a grant from the Barry Coller Foundation at the Stony Brook School of Medicine to conduct international health research.

Through this program, I spent eight weeks in Barbados to work with the Barbados National Cancer Study, a collaborative effort between the Department of Preventive Medicine at Stony Brook University and the Chronic Disease Research Center, University of West Indies. My project investigated the knowledge, attitudes, and health practices of Barbadians in regards to prostate cancer and preventative screening. During my two-month stay, I interviewed prostate cancer survivors and their families, community leaders, and physicians about their perspectives on prostate health.

It was an extremely productive summer and probably the best decision of my life.

For those of you who don’t know, Barbados is one of the southern most Caribbean islands and its closest neighbors are St. Lucia, Martinique, St. Vincent and the Grenadines. It’s a very small country, spanning only 21 miles north to south and 14 miles east to west. To put this in perspective, traveling across this entire island is only one third of the distance from Stony

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Brook to New York City. Yet despite its small size, the culture of Barbados is quite rich and colorful, and experiencing it first hand was quite remarkable.

One of the first things that really struck me about Barbados was how friendly everyone was. Barbadians are the epitome of laid back and their welcoming nature is quite refreshing. Each morning when I climbed into a “Zed-R”, which was a little white public transportation van that I rode for my daily commute to the clinic, every person in the vehicle greeted me “hello” and they did the same for every new passenger that came aboard. Perhaps this is due to my New York upbringing, but this kind of behavior is almost unheard of. When was the last time you jumped onto the LIRR and everyone in your section said hi to you? Maybe on a good day you received a few nods and smiles, but for the most part I think we just keep to ourselves and go about our merry way. I don’t think it’s because we are rude but rather that we have a different pace of life. So with a simple hello, Barbados taught me to just take a step back, appreciate where I am, and not be so immersed in my personal agenda.

In the spirit of the stress-free Barbadian lifestyle, I was luckily able to take part in a good amount of recreational activities outside of work and met many interesting individuals.

I lived with several medical students from England and Germany who were completing elective rotations at the Queen Elizabeth Hospital, and together we spent many nights sharing war stories about how we were surviving medical school.

As we explored the local markets and beaches, we became good friends with several Barbadian natives who were generous enough to give us an exclusive tour of the island.

During our adventures, I surfed for the first time, watched sunsets from the edge of cliffs, hiked through the jungle to bathe in natural springs, ate some of the best home-cooked grilled flying fish, and participated in the annual Barbadian celebration known as “Crop Over.” Historically, “Crop Over” was a celebration of the end of the sugar cane harvest. Today, it is a two-month festival that ends with the Grand

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This event is similar to Trinidad’s and Brazil’s Carnival events. Now I could go on forever about what I saw and did on the island, but ultimately I don’t think I would have participated in any of these activities if I had not become friends with the locals. Through my new friends, I was ?5$%+"2/;/-6"$3"3-%"35"$2%"$*((%)$".3/-$)"/-" able to experience G*+4*13)8 the heart of Barbadian life and see a part of the country that most tourists rarely get to encounter.

When I did go to work, most of my days were spent at the Winston Scott Polyclinic in Bridgetown, Barbados, which is one of eight government-run health clinics on the island. Interestingly, the Barbadian health care system is a mixture of socialized and privatized medicine, where citizens are guaranteed access to

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the government clinics and hospitals, but can go to a private physician if he/she can afford it. With the U.S. government in heated debate over healthcare reform, I was curious to see how successful universal healthcare had been for Barbados.

After speaking to many Barbadian residents and observing the clinic activities, I could see health disparities were still present and that resources were relatively limited. The patient lines to see a physician seemed infinite and locals told me to avoid going to the government hospitals because I might never see a doctor with the long wait. Facilities had limited air-conditioned rooms, were understaffed, and I remember a German medical student telling me that the hospital had run out disinfectant supplies due to the overflow of patients. I also learned that for a national population of 255,203 only two urologists were serving the region and, unfortunately, many men could not afford the treatments they offered.

After receiving this quick snapshot of the Barbadian health care system, I was really thankful for the facilities and resources that we have available to us in America and realized that although our system is not perfect, our situation could be much worse.

Without a doubt, working abroad was a life changing experience and something I wish that I had done more in the past. When I was a Stony Brook undergrad, I had heard all about the amazing programs in Tanzania, Italy, and Madagascar but sadly was under the impression that my pre-med plan did not allow time for a semester abroad. Clearly, this was just plain dumb on my part and I wish I could redo my undergraduate years to take part in one of these programs. No matter what major you are or what career path you aspire to, I highly encourage you to travel and really immerse yourself in the culture of the country that you are in. Don’t just do the tourist thing and see the landmarks, but rather meet the locals, find out what ‘hole in the wall’ hang out spots they go to, what ‘neverheard-of before’ holidays they celebrate, and really experience them for yourselves. You will undergo far more personal development in your travels than you will ever gain from a classroom. Further, not only will you have an amazing collection of memories, but you’ll gain a new perspective on life and have some exciting stories to talk about in your graduate school and job interviews.

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small proteins that have evolved and work as paralytics. Other blockers also have a natural source like curare and succinyl choline and they are used medically in surgical situations where muscle relaxation is required for incubation. It is used on people who are put on respirators to stop respiratory reflexes from interfering with respirator function. Nowadays, every hospital uses nicotinic acetylcholine receptor blockers and they are usually curare type blockers or succinyl choline.

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What is the role of the nicotinic acetylcholine receptor (nAChR) and what effect does it have in physiological processes?

Understanding the cellular and molecular mechanisms of a disease is important to developing new drug therapies which can specifically target affected cells. Dr. Joav Prives, a professor in the Department of Pharmacology, focuses his research on receptor –ligand interactions of the nicotinic acetylcholine receptor (nAChR). He investigates the intracellular interactions between the integral nACh receptor and organelles such as the mitochondria. Dr. Prives’ work seeks not only to better understand this ionotropic receptor’s interactions in its normal state, but also under aberrant conditions such as those found in Lou Gehrig’s disease and Myasthenia Gravis. Here, Young Investigators’ Esther Bilenkis, a freshman biology major, interviews Dr. Prives about his exciting research.

Briefly, can you please discuss which portion of the somatic nervous system transmits signals upon activation of the nicotinic acetylcholine receptor? What specifically makes this receptor an interesting drug target?

The nicotinic acetylcholine receptor is essential in the peripheral nervous system for allowing us to move our muscles. Without the receptor function as is compromised in myasthenia gravis, we become paralyzed. Nature has picked up on this vulnerability so that a variety of animals create toxins for the explicit purpose to paralyze their prey (snake neurotoxin). While the acetylcholine receptor is involved in voluntary muscle in the peripheral nervous system, it also plays a variety of roles in the central nervous system.

Like the name nicotinic acetylcholine receptor implies, in the case of people who are addicted to nicotine because of nicotine central nervous system functions, nicotine works as an activator of receptors that are very similar to muscle receptors and they are excitable receptors in the central nervous system. The downside is that these receptors are targeted by beta amyloid protein, a protein fragment that is a mediator for symptoms of Alzheimer’s disease.

Alzheimer’s disease also impacts the acetylcholine receptors in the central nervous system.

It is the somatic motor nerve ending that connects the central nervous system to the voluntary muscle. What is interesting is that it was the first receptor to be identified a long time ago, the first receptor to be characterized as a protein and the first receptor whose gene was clone. It is also a receptor that plays a part in many diseases, which makes it interesting.

What diseases does the nACh receptor play a part in?

One disease that it plays a role in is Myasthenia Gravis, which is an autoimmune disease where for some unknown reason antibodies are made. The immune system targets the nicotinic receptor of the same individual as non-self and produces destructive antibodies. There are a lot of autoimmune diseases but Myasthenia Gravis was one of the first autoimmune diseases to be characterized.

Alzheimer’s is another disease in which the acetylcholine receptor acts.

In modulating the effects of the autonomic nervous system, ganglionic blockers or antagonists of these receptors have been used in the management of various pathological states: can you please talk about the effectiveness of these antagonists?

The acetylcholine antagonists are used as research tools. Antagonists such as snake neurotoxins, which come from cobra, are

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The Stony Brook Young Investigators Review, Spring 2010

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Interviews Why did you choose to study chick muscle cells specifically?

Muscle can differentiate in a tissue culture plate even though it is an extensive differentiation that muscle goes through. The differentiation can happen outside of the body on a tissue culture plate.

Chick muscle easily goes through a full differentiation process including forming synapses with neuronal cells from the central nervous system of chick. It is also a matter of convenience. A lot of systems have an ideal animal that helps you solve a problem and chick is the ideal animal in this case.

What is your take on chronic nicotine administration and what effects do you think it has regarding diseases like Alzheimer’s?

There are currently clinical trials of nicotinic acetylcholine receptor activators as a possible attenuator (lessens the effects) of early Alzheimer’s. The irony is going to be that after nicotine has received a bad reputation for the last twenty to thirty years and it has reached its peak now, there will be the other side of the coin where people discover useful actions for nicotine, specifically in Alzheimer’s disease and also in schizophrenia, another central nervous system condition.

fied as relevant to susceptibility to various drugs.

What new understanding of key features common to the regulated expression of neurotransmitter receptors in muscle cells and neurons have you learned from your experiments?

Recently we have looked at acetylcholine receptors and the way that they are organized on the surface of the cell so that they are very constraint under the nerve ending, and the way that the nerve ending tells the postsynaptic cell to put its receptors right under the nerve ending. Lately, we have been looking at the way in which the mitochondria move with acetylcholine receptors to congregate under the nerve ending in the postsynaptic cell. This is exciting because the muscle mitochondria seem to be a possible source of one of the first events in the development of ALS, Lou Gehrig’s disease, a paralytic disease, and so we can study that in cell culture and some aspects of how the mitochondria respond to signals in the nerve. We have learned a lot about the way in which spatial topological aspects of nerve talking to muscle in terms of where to place the postsynaptic membrane and how to build a synapse. This is only the latest feature from the research.

What types of pharmacological and functional properties does the acetylcholine receptor exhibit?

It is relatively easy to make a variety of drugs that will block, activate, or partially activate receptors. Because of the fact that it is very well characterized in terms of what its’ active site looks for in terms of a ligand in terms of acetylcholine-like molecules which are normal activators. They have quaternary ammonium which is a charged nitrogen group that is pretty unique in that it is always charged in any pH (unusual to find). There are a variety of quaternary amines that will work. There is a big collection of potential target compounds that can be used, which is very unique about acetylcholine receptors.

What kind of therapeutic strategies do you think will come from a better understanding of how nicotinic acetylcholine receptors impart diverse effects on the system?

The nicotinic acetylcholine receptors have always led the way for other receptors because it was always ahead of other receptors.

What was discovered to be true with this receptor was later found to be true in other receptors. A very high proportion of medications, about sixty percent, target different kinds of receptors (not just acetylcholine receptors but also adrenergic receptors of various kinds, g protein coupled receptors). One direction in which pharmacology or pharmaceutical research is going is in pharmacological genomics. Different people have variations of receptor structure which renders them more or less sensitive to particular drugs and so having advanced knowledge of what specific profile a patient has in terms of sensitivity of its various receptors, the drug you wish to prescribe is going to be key so that the whole one size fits all idea of administering drugs will change drastically as we understand the human genome better. In the case of the acetylcholine receptor, it will lead the way so that people will be able to define individual changes in the acetylcholine receptor that can be identiThe Stony Brook Young Investigators Review, Spring 2010

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What would you have pursued if you were not in the field of Chemistry?

You tend to move toward your strengths in life and my strengths were figuring out stuff. Chemistry is just a natural for me. I could have done other things, like be an automobile mechanic, which also requires figuring things out. The odd thing is there are problems to solve there too, and I love solving problems. I think that is what excites most people; problem solving constantly keeps you going. When I first came to Stony Brook, teaching I had to do, but I was mostly interested in research. But, as I went through it and I did my research, all of the sudden I got very excited about teaching. I really enjoy going into the classroom; it is a lot of fun.

Who is your favorite Nobel Prize winner?

I would have to say Linus Pauling. He is a hero. He was actually the first one to win two [unshared] Nobel Prizes, first in chemistry and then the Peace prize. He was one of the very early anti-war activists and just a brilliant man.

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Organic chemistry is the study of carbon based substances found only in living organisms [1]. It is the field in chemistry with the most Nobel Prizes; also known as one of the most complex puzzles to university students all around the world. Dr. Fowler met with YIR to tell us about his experience in the field.

Professor Frank W. Fowler is a Distinguished Stony Brook Professor of Organic Chemistry who reaches out to a lecture hall of some 1200 students daily. Professor Fowler received his B.A. from the University of South Florida and his Ph.D. from the University of Colorado. He was also a Leverhulme Visiting Fellow at the University of East Anglia in England [2]. Dr. Fowler is a synthetic chemist. The massive production of many goods such as plastics, drugs, and clothing is due to the understanding of how unusually long molecules are created. Think of polymer chemistry as a chain of paper clips assembled together to form a molecule. His current research involves working on topochemically controlled polymerization; that is, developing strategies for the preparation of designed materials. The thorough knowledge of molecular synthesis for the preparation of a molecule that will then assemble into the designed supramolecular structure is of utmost necessity [2]. As Professor Fowler puts it, “The problem of converting the materials we have into the materials we want is the domain of synthetic chemistry.”

Topochemical polymerization works in a zipper-like mechanism.

Stacks of difunctional monomers may react in a regularly ordered crystal lattice by rotation on their lattice positions. The rotation of one molecule brings its reactive ends into contact with two others and so on; they “zip” together to form a polymer. Take polydicetylene as an example. If polymerization is carried out in a liquid phase, these molecules will polymerize, but they will undergo a combination of a 1,4 and a 1,2 addition just because these molecules bump into each other at random and wherever they hit, they react. However, in a solid state, they can be brought together and are “frozen” in place and can only react by using the crystal lattice to organize the molecules. By bringing molecules together in this restricted space and by rotating, they form new bonds and make conjugated polymers. Because this is done at a molecular level, there are not many tools or machines to bring these molecules together. Therefore, the molecules own intermolecular forces are used to assemble them in a host-guest manner. According to Professor Fowler, “the extension of synthetic chemistry from the molecule to large ensembles of molecules, or supramolecular structures, is among the greatest challenges facing contemporary chemistry.”

If someone had never heard of organic chemistry before, how would you describe it to him metaphorically? Personally, I describe synthesis as a chess game where one has to think ahead in order to create a molecule.

Certainly there is a chess component to it. More generally, I think I would not divide chemistry into organic chemistry and other stuff. If I were to explain chemistry to my grandmother, I would say everything is molecular, your cell phones, your bodies, everything is operating at a molecular level. If you are going to understand how

deals well with understanding phenomena; it speaks well to material science. Biology understands living things; it speaks well in a technological sense to medicine and pharmacology. But chemistry speaks to both of them! That is why I like to think of this as the Department of Molecular Science rather than Chemistry.

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The Stony Brook Young Investigators Review, Spring 2010

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Interviews It takes a lot of passion and you have to be very observant, but the really great things are done accidentally or by recognizing the unexpected, by good scientists obviously. It takes a great person to recognize something when it passes through the lab. It is not to say we do not have a plan; we have to write research grants and you have to outline your plans and it looks pretty neat. You hopefully have an idea that someone else has not thought of and you pursue it. If your idea is good you will have made progress; you will have increased knowledge.

Why do research at Stony Brook?

There are not a whole lot of places in the country you can do research. Stony Brook and Brookhaven Labs are unique. I needed to come here because this is where the facilities are, where the students are.

What is the chemistry work you are most proud of?

I think it is these [topochemically controlled] polymers. I got involved in supramolecular chemistry, which is the assembly of the molecules produced through molecular synthesis. These supramolecular interactions are very weak and difficult to understand. At the time we went into this field, the understanding was very primitive and it has gotten a lot better. It is the assembled molecules that give you function like solar energy conversion, and all of biochemistry is involved in it. These weak interactions are non-covalent; they hold molecules together to give a molecule its shape or give you a certain function. This topochemical polymerization is still very important; however, it has not reached its impact yet. What we were excited about is to be able to do [this polymerization] in a designed way. There are examples of accidental polymerization where you make a molecule, it crystallizes and indeed it polymerizes, but there was no plan as to the assembly of them into the solid state. The reason I like this field is because we took some very simple ideas and did something people have not been able to do before.

What is the best teaching strategy for reaching out to a lecture hall of 1200 students?

handle these classes differently, but how I do not know.

Can you name one compound/reaction or topic discussed over the course of CHE 321-322/326 that we encounter in everyday use or is a big contribution to the way we live?

My challenge is to know what you guys do and then I can try and bring in the chemistry that is relevant to your lives. I know little about what students today do. I know you eat, so I talk about food. It sounds terrible if I talk about the chemicals in food, but those are the ones you appreciate, like aromas and flavors so they are not necessarily bad. I think the chemistry of the brain is fascinating. Can students relate to that? We all have brains, we all have the same chemicals in there, but we do not all have the same mix of chemicals and thus we are all different in a sense. I find understanding this on a molecular level particularly exciting. Understanding pleasure, why something is beautiful on a molecular level is something we could eventually do; we are certainly getting closer. …Things are changing so rapidly and that is the challenge of teaching. What can I communicate to you that is going to be useful twenty years from now? That is difficult because I do not know what will be important then say in the field of medicine or biology. That is why the main thing I like to focus on is problem solving, because that is not going to go away.

*#&#-#0(#+ 1.“Nobel Prizes in Organic Chemistry.” Nobelprize.org. Camille and Henry Dreyfus Special Grant Program in the Chemical Sciences, 2 Apr. 2009. Web. 23 Mar. 2010. <http://nobelprize.org/nobel_prizes/ chemistry/organic_chemistry.html>.

2. “Chemistry at the Brook.” Stony Brook Chemistry. Department of Chemistry, SUNY Stony Brook, 25 Nov. 2006. Web. 23 Mar. 2010. <http://www.chem.sunysb.edu:81/faculty/ffowler.htm>.

3. Solomons, T.W. Graham, and Craig B. Fryhle. Organic Chemistry. 9th ed. Hoboken: John Wiley and Sons, 2008. Print.

The times have changed a lot, and I was very pleased to read recently that the students have changed. When I first came to Stony Brook, it was 1968 and universities were going through a lot of turmoil. What has really helped reaching out to a large lecture course is technology. It allows us to be mobile, that is to walk around the classroom. It allows [the professor] to treat a large class like a small one; you can talk to individual students and with a little bit of luck, if you are carrying the microphone, you can have them ask questions. The clickers give everyone in the room a say, and they can see how difficult the concept is for everyone else. The main thing we now try to take advantage of in class is the social component. Students who come in to university at this age are very natural at socializing. They will come and talk about something in that lecture, and what we try and do is have them talk about chemistry. I really do believe that the challenge right now with big universities is to come in and do what we need to do with fewer resources. The one thing we are losing with technology is evaluating the students writing which is impossible to do with this many students. It is difficult in the sense that in five years we will The Stony Brook Young Investigators Review, Spring 2010

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