Wings of Discovery 2008-2009

2008–09 EDGEWOOD HIGH SCHOOL STUDENT SCIENCE JOURNAL

Featuring the work of: Caleb Burr Lindsay Fulton Philip Gorman Peter Guerin Noah Kachelski

Christopher Kuecker Lydia Lovell Charlotte Martin Sam McLaughlin Cedric Meyers Matt Neumann

Jay Sekhon Arial Shogren Lianna Schmidt Jon Seaton Kerry Sweeney Bethany Walker

In Loving Memory of Joseph E. Zaiman, Jr.

Wings of Discovery is an annual, independent journal of original science research by Advanced Science students at Edgewood High School. All contributions constitute the students’ own work and reproduction in whole or in part of any article without permission is prohibited.

Wings of Discovery Š 2009 Edgewood High School of the Sacred Heart.

Edgewood High School, 2219 Monroe Street, Madison WI 53711.

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Foreword As President of Edgewood High School, I write today to tell you how proud I am to have our science research journal, Wings of Discovery, in your hands at this moment.

This journal is a credit to our Advanced Science students, who care so much about adding to the knowledge we have about our world, and are capable of doing so.

It is also a credit to our award-winning science teachers, who nurture in their students not only current scientific understanding, but the confidence to become part of creating it. Edgewood strives to form young scientists in the Dominican Catholic values on which we were founded — truth, compassion, justice, community and partnership — and Wings of Discovery is part of that effort.

Few high schools around the country publish a journal like the one you now hold.

For this achievement, I would like to thank our science teachers — Mrs. Jessica Splitter, Mr. Jonathan Hessler, Mr. Eric Pantano, Mr. Derek Ralph, Mr. Robert Shannon and Mrs. Mekel Wiederholt Meier — for their guidance and leadership to our students. I also want to remember that this annual journal is a tribute to our beloved teacher Mr. Joseph Zaimann, whose dream this was, a dream that has come true in his memory.

In addition, I want to thank all EHS science supporters, without whom this annual journal would not have come to fruition in its first year nor in this, its third edition.

Your continued support will allow this exceptional growth experience for our science students to continue into the future and become another great Edgewood tradition.

The discipline of science embodies the search for truth. But without the additional essential values we instill in our students — compassion, justice, community and partnership — new knowledge of scientific truths may add little to the quality of life for the inhabitants of God’s earth. We strive to form the whole person of our young scientists, endowing them with a complete and honorable value system that will serve to create good through science. Our prayer is that this journal has been, and will continue to be for years to come, a means to this end.

Sincerely,

Judd Schemmel President

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In Loving Memory of Joe E. Zaiman, Jr. 1953-2003 For 23 years, Joseph E. Zaiman, Jr. was a science teacher at Edgewood High School in Madison, Wisconsin. During his time there he not only taught life science, but by his example he taught about life itself. Through his love and enthusiasm for teaching, he earned the admiration and respect of his students, colleagues, friends and family. Joe’s motto to learn constantly, laugh a lot, and love others is the best way to describe the way he lived his life.

Joe was a man who savored constant learning. Whether he was reading a scientific journal, identifying a rare aquatic insect, digging for fossils, or learning the stars and constellations, Joe wanted to know everything about the world around him. But he most enjoyed learning from his students. Likewise, his infectious desire to learn inspired his students as well. Joe always thought that the best way for students to understand nature was to teach through hands-on experiences. His students will never forget their DNA models, hiking the rain forest in Belize, bog walking on Madeline Island, marveling at newly hardened lava in Hawaii or canoeing the St. Croix River.

All of these activities made science fun and inspired students to strive to learn more.

When one thinks of Joe, one has to think of laughter. It was his trademark. A day would not go by when any student or friend of Joe’s did not hear him laugh. Joe’s sense of humor, like his love of learning, was contagious. His unforgettable jokes, pranks, and wonderful “magic tricks” brightened even the gloomiest day.

Joe emulated the Gospel teachings through his love of others. He showed an unconditional love for everyone and everything. His love for his students could be seen and felt immediately upon entering his classroom. Joe reached out to all students.

However, it seemed that he was in his glory helping those who struggled or did not quite fit in. Being around Joe and his warmth made you want to be a better person.

Joe was also a man of many dreams. Some were fulfilled in his lifetime while others were not. The publication of a scientific journal specifically designated for high school students was one dream he planned to pursue. It is our hope that this journal will in some way be a tribute to Joe, a man, a teacher, and friend who lived life to learn, love, and laugh and inspired all around him to do the same.

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Index Synthesis of Substituted Tetraphenylethylenes — a component of a study on captodative stability) ................PAGE 6

by Jay Sekhon Plasma Chemistry: Deposition of Hydrophobic Layers Onto Silicon and Glass Substrates ..............................PAGE 9

by Matthew Neumann Beaver Survey of the West St. Croix State Park ..............................................................................................PAGE 12

by Caleb Burr and Christopher Kuecker Relation between ErbB Signaling Pathways in Head and Neck Cancer and Intrinsic Resistance to EGFR Inhibition ..................................................................................................PAGE 15

by Philip Gorman Etching and Release Techniques of Elastically Relaxed Free-standing Strained-Silicon Nanomembranes ........PAGE 19

by Cedric Meyers Orconectes rusticus Populations in the St. Croix River and Tributaries in Relation to Human Interaction (Fishing) ..................................................................................................PAGE 22

by Arial Shogren, Lianna Schmidt and Charlotte Martin Presence of Bacteria and Protein in Grade A and Grade B of Milk ................................................................PAGE 25

by Kerry Sweeney Purification of Thermosensitive Oligo(lysine)-b-Elastin-like Polypeptides ......................................................PAGE 27

by Jon Seaton The Cloning of SNR7 into Saccharomyces cerevisiae........................................................................................PAGE 30

by Lydia Lovell Mushroom Survey at Five Different Sites, Testing Relative Humidity, Soil Composition, Amount of Sunlight, and Number of Mushroom in St. Croix State Park ......................................................PAGE 32

by Peter Guerin and Noah Kachelski Survey of Mussel Middens Found Along the St. Croix and Kettle Rivers........................................................PAGE 34

by Bethany Walker and Lindsay Fulton The Effects of Electron Donating and Accepting Radicals on the Geometry of Tetraphenylethylene..............PAGE 37

by Sam McLaughlin Acknowledgements ........................................................................................................................................PAGE 41

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Synthesis of Substituted Tetraphenylethylenes (a component of a study on captodative stability)

by Jay Sekhon Jay Sekhon graduated from Edgewood High School in 2007, and is currently a student at the University of Wisconsin, Madison.

The issue of captodative stability, an enhanced form of stability that occurs from the presence of both an electron donating group and electron withdrawing group on a radical that increases the stability of the radical to a greater level than the sum of the individual substituent groups has been of interest to chemists for years. Research has been conducted towards this end through the synthesis of substituted tetraphenylethylenes, but previous methods of synthesis have been determined to be exceedingly difficult and dangerous. It became necessary to determine the viability of an alternative scheme using a Pd-catalyzed reaction of phenylboronic acid with diphenylacetylene. The reaction provided the target compound even under abysmal conditions, so a new synthetic scheme was developed, and initial forays into using the Pd-catalyzed synthesis scheme have proven to be highly successful. The products of this reaction will be used for the overarching study on the captodative stability of tetraphenylethylenes.

Background aptodative stability is an enhanced form of stability resulting from the effects of an electron-donating subsistuent group and an electron-withdrawing substituent group that affects the stability of the parent molecule in a greater way than the sum of the two individual substituents. Theoretically proposed by Dewar in 19521, the idea was explored and given substantial experimental evidence by researchers in the 1970s and 1980s, such as Viehe2.

Tetraphenylethylene specifically was studied by Arnold and Leigh in 1981 using only the presence of a substituent group in the para position3. The study concluded that the biradical transition state of the molecule experienced a greater stability with the donor/acceptor combination than with the sum of the individually substituted donor or acceptor. An alternative group of researchers examined the stability of a triphenylmethyl radical, concluding that in some, but not all, cases, an acceptor/acceptor combination created greater stability for the radical than donor/acceptor4.

This group suggested new criteria for defining captodative stabilization.

Additional studies have led to similar conclusions, in which the Viehe definition of captodative stability appeared to be absent in certain systems5. However, these studies provide segments to the understanding of the captodative effect, since a large range of substances have been examined. Since connections between studies are few, it is necessary to examine similar systems to better understand the idea of captodative stability.

Research into the captodative effect of substituted tetraphenylethylenes by Stracener has been done6.

Geometry optimization and bond angle calculations through the use of the Gaussian computer program, specifically through density functional theory, yielded that the ethylene double bond length was at its greatest with the donor/acceptor substitution.

Previous efforts into the synthesis of tetraphenylethylenes for this specific project have been done with

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partial success7, 8. A previously proposed synthesis scheme using substituted benzenes that were connected to form benzophenones via a Grignard reaction, then combined to form tetraphenylethylene via the McMurry Coupling has been completed. This method involved the use of the Rosenmund von-Braun reaction with cuprous cyanide, converting a bromobenzen into a benzonitrile, as well as tested the conversion of a benzaldehyde to a benzonitrile. The Sandmeyer reaction, which converted an amino group into a nitrile was indicated to have the greatest degree of success. However, this scheme of Grignard and McMurry coupling have proven to be almost prohibitively difficult and dangerous, since the McMurry coupling requires the use of a closed, inert environment (usually argon) since one of the reactants, TiCl3, is highly reactive with oxygen. This suggests the examination of alternate synthesis schemes.

Recent research by Larock and his colleagues has indicated that a far easier method of synthesizing a tetrasubstituted olefin exists through the reaction of an internal alkyne and an arylboronic acid, catalyzed by palladium9. Additional research also gives the possibility of performing a similar reaction (tetrasubstitued olefin) by reaction of an aryl iodide, internal alkyne, and arylboronic acid10. These alternate methods of synthesis employ reaction procedures that are significantly safer and easier to follow. As such, the Larock research suggests a possibility of being able to use a Pd-catalyzed reaction to synthesize this project’s tetraphenylethylenes.

Goal The overarching goal of this project is the investigation of a possible correlation of phenyl torsional angle with the magnitude of captodative stabilization of the biradical transition state of thermal cis-trans isomerization of substituted tetraphenylethylenes7.

The specific goal of this section of the project was to examine the viability of the Larock Pd-coupling, determine an alternative synthetic scheme to the desired compounds using the Larock reaction, and begin synthesis of the desired compounds using the proposed scheme.

SYNTHESIS OF SUBSTITUTED TETRAPHENYLETHYLENES

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Figure 1

The project calls for a total of twenty different substituted tetraphenylethylenes (Fig. 1).

Method Larock Pd-coupling would be applicable to the project. To this end, diphenylacetylene and p-methoxyphenylboronic acid were purchased, as well as palladium acetate. The reaction was prepared in a 25 mL roundbottom flask according to Fig. 2.

Figure 2 Molecular sieves (4Å) were also placed in the reaction vessel. The vessel was closed with a septum and had two syringe needles placed through it, with one needle attached to an O2 tank. The vessel was flushed with oxygen before a balloon was attached to the second syringe and inflated to keep the contents under O2 pressure. The vessel was then placed in a heating mantle and brought to 50° C. These reaction conditions were held for three hours, then the reaction was left sitting for an additional 21 hours at room temperature.

During the reaction, difficulties were encountered in maintaining a steady temperature, as the mantle fluctuated the temperature from 50-70° C throughout the three-hour period. The balloon had also deflated upon the conclusion of the reaction. Regardless, the reacted solution was quenched with a saturated solution of NaCl, and the organic layer was extracted with t-butyl methyl ether three times. The solution was dried over MgSO4 and filtered, then placed in a rotary evaporator.

The resulting compound was a brownish-yellow and had a sticky nature. A melting point test gave a reading of ~145° C, which was between the literature value of the desired product and the reactants, so the product was inspected via NMR. The output of the NMR had small peaks, so it was re-evaluated using the BAPR (Block Averaging with Peak Registration) method of NMR overnight. The resultant output indicated a partial success with peaks appearing to indicate methoxy groups, but also that the diphenylacetylene was still present. The compound was then dissolved in heated

water and ethanol in an attempt to recrystallize, but this proved unsuccessful.

The reaction was attempted a second time and held under better reaction conditions. The molecular sieves were baked beforehand in preparation and the heating mantle was avoided entirely, resulting in the reaction being carried out at room temperature. The new product was significantly more firm than the previous reaction, and a melting point test indicated a melting point of ~179° C. NMR was carried out using the BAPR method again, and a significant amount of product was detected, so the Larock method was considered a success for determining a new synthesis scheme.

From there, it became necessary to determine how to obtain the necessary diphenylacetylenes and phenylboronic acids. It was learned that the desired phenylboronic acids could all be purchased. The diphenylacetylenes posed a different challenge, however, and a scheme became necessary to determine how to make them, as only a few could be purchased. The Larock research indicated that electron donating groups gave poor yields when placed on the diphenylacetylene, so it was determined that the –CN group would be placed there. The general synthesis scheme was thus determined (Fig 3).

Figure 3 The scheme begins with a benzyl alcohol (optionally with a nitrile attached, depending on the desired final compound). From there, the alcohol is reduced via pyridinium chlorochromate to an aldehyde.

The starting alcohol is also reacted with PBr3 to produce a benzyl bromide. From there, the bromide is prepared for a Wittig reaction by making it into a phosphonium salt by reaction with triphenylphosphine. The phosphonium ylide then reacts with the aldehyde to produce the alkene. The alkene is then dehydrogenated to produce the diphenylacetylene (alkyne), which then reacts with the phenylboronic acid by the Larock synthesis. Foreseen problems with taking a nitrile

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REFERENCES 1. Dewar, M. J. S. “A Molecular Orbital Theory of Organic Chemistry. IV. Free Radicals.” J. Am. Chem. Soc., 1952, 74, 3353.

2. Viehe, H. G.; Janousek, Z.; Merényi, R. “The Captodative Effect.” Acc. Chem. Res., 1985, 18, 148.

3. Arnold, D. R.; Leigh, W. J. “Merostabilization in radical ions, triplets, and biradicals. 5. The thermal cis-trans isomerization of para-substituted tetraphenylethylenes.”

Can. J. Chem., 1981, 59, 609.

4. Neumann, W. P.; Uzick, W.; Zarkadis, A. K. “Sterically Hindered Free Radicals. 14. Substituent-Dependent Stabilization of Para-Substituted Triphenylmethyl Radicals.” J. Am. Chem. Soc., 1986, 108, 3762.

through this reaction from the benzyl alcohol have been solved by using an amino group and conducting a Sandmeyer on the diphenylacetylene or by using substituted bromine and performing the Rosenmund von-Braun reaction on the diphenylacetylene. In either case, the scheme lends itself to a high potential for success, using significantly safer and easier methods than the McMurry coupling previously.

At this point, with the scheme determined, carrying it out began. Only one reaction was tested at the time of this writing—the conversion of the benzyl alcohol to benzyl bromide (Fig. 4).

5. Bordwell, F. G.; Lynch, T. -Y. “Radical Stabilization Energies and Synergistic (Captodative) Effects.” J. Am. Chem. Soc., 1989, 111, 7558.

6. Stracener, L.; Handorf, A.; Eannelli, K. POSTER PRESENTATION “Substituted Tetraphenylethylenes: Computational Studies and Efforts Towards Synthesis for Investigation of Captodative Effect.” Am. Chem. Soc. 7. Stracener, L. “Synthesis and Study of Substituted Tetraphenylethylenes: Effect of Torsional Angle on Captodative Stabilization” 8. Kinzel, Kyle. “Synthesis of Benzonitriles and Their Use in the Captodative Stability of Tetraphenylethylene.” Wings of Discovery., 2005-2006, 2, 31-33.

9. Larock, Richard C. and Chengxiang Zhou. “Synthesis of Tetrasubstituted Olefins by Pd-Catalyzed Addition of Arylboronic Acids to Internal Alkynes.” Organic Letters.

2005, 7, 259-262.

10. Larock, Richard C. and Chengxiang Zhou. “Regio- and Stereoselective Route to Tetrasubstituted Olefins by the Palladium-Catalyzed Three-Component Coupling of Aryl Iodides, Internal Alkynes, and Arylboronic Acids.” J. Org. Chem. 2005, 70, 3765-3777.

Figure 4 The reaction was carried out in a 50 mL roundbottom flask using t-butyl methyl ether (TBME) as a solvent. The vessel was placed in an ice bath as the HBr was added to the dissolved PBr3 and benzyl alcohol. The reaction was stirred for three hours, quenched with additional HBr, then extracted twice using TBME. The resulting compound was placed in a rotary evaporator, then fractionally recrystallized using ethanol and water. The crystals were then prepared for NMR. Large quantities of the desired benzyl bromide were indicated, and a high yield (approximately 75%) was achieved.

Conclusion Despite initial difficulties with the reaction conditions using the Larock Pd-catalyzed coupling, the two reactions performed indicated a strong potential for being able to synthesize the desired products for the overall study. The synthesis scheme proposed offers a significantly better method for creating the desired products versus the previous method utilizing the Grignard reaction and McMurry coupling. Initial forays into the new synthesis scheme by converting the benzyl alcohol to the benzyl bromide have proven very successful, and if such success continues, the desired compounds can all be readily synthesized.

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Plasma Chemistry: Deposition of Hydrophobic Layers Onto Silicon and Glass Substrates

by Matthew Neumann Plasma chemistry is a fairly new form of chemistry. Through the use of plasma chemistry, polymerization of an infinite number of monomers can occur, although it is extremely difficult to predict how much of the monomer will polymerize. One of the most useful monomers to polymerize is tetraflouroethylene into polytetrafluoroethylene (PTFE) commonly known as Teflon. PTFE’s fairly inert nature, low coefficient of friction and hydrophobic properties make it a widely used polymer. PTFE is used as a non-stick coating for cookware, and in containers and piping for reactive or corrosive chemicals. It can also be used to coat glass slides for Raman spectroscopy, which gives a “fingerprint” of the molecule. It can also be used to coat thin strained silicon membranes, making them waterproof.

Goal

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he overall goal of this research project was to amplify results from Raman spectroscopy tests and to deposit a hydrophobic coating to a highly strained thin silicon membrane.

A hydrophobic coating would also be applied to glass slides, which was hypothesized as helping amplify results from Raman spectroscopy tests.

continue to bond and cross link creating a thin film on the material” (Gordiets). “The fourth step is the recirculation and new molecules produced in the volume or at the wall will mix with the injected monomers.

Recirculation takes place in volume III and the plasma new molecules are produced in volume II” (Schram).

“The fifth step is the clustering of monomers, radicals, and ions which form a dust” (Bouchoule). Before the Plasma reactor running Ar treatment

Method/Results In order to create the hydrophobic layers on the glass slides and membranes plasma chemistry was used.

The plasma process can be divided into five steps.

“The first step is the production of the primary plasma.

This plasma ionization can be atomic or molecular and occurs in production volume I” (Schram). Normally microwave radiation is used to give the energy needed for ionization.

“The second step is the dissociation of the monomer in the recombining plasma from the source. Active radicals are formed which are ‘sticky’” (Schram).

“The third step is plasma surface interactions of arriving radicals with a surface lead to the formation of a chemically bounded surface layer. These radicals will

Plasma Reactor Schematic (Schram)

Matthew Neumann graduated from Edgewood High School in 2007. He would like to thank Hongquan Jiang PhD and Dr. Max Lagally a rrofessor of materials science and physics at the University of Wisconsin-Madison.

Currently he is attending the University of Wisconsin-Madison and is working towards a bachelor’s degree in biomedical engineering.

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hydrophobic layer could be applied to the substrates, a cleaning process would have to take place. In order to do this Ar or O2 was used. These gasses were injected into the plasma reactor and were converted into plasma. This treatment lasted for five minutes at 150 mTorr. After this treatment was completed all organic substances that were on the substrates would be removed. Next gaseous tetraflouroethylene was injected into the plasma reactor and was used to treat the substrates. Tetraflouroethylene polymerizes into polytetrafluoroethylene (PTFE) commonly known as Teflon. PTFE is fairly inert, has an extremely low coefficient of friction, and is extremely hydrophobic. When tetraflouroethylene is converted into plasma the π bond is broken to create a σ bond. This leaves the two carbons with free electron, called a radical.

Carbon radicals are very unstable and will “attack” any weak bonds like another π so that carbon can reform its octet, by creating a σ bond to another carbon. This

treatment was performed at 150mTorr. One of the current problems with plasma chemistry is how difficult it is to design a result. For Raman spectroscopy and the membranes, varying thicknesses of the PTFE would be needed. Because of the needed for varying thicknesses the treatment of tetraflouroethylene was done for varying durations of time. It was thought that the longer the substrate was allowed to remain in the treatment, the thicker the layer of PTFE would become. The treatment times were thirty seconds, one minute, two minutes thirty seconds, four minutes and six minutes. At each time duration, fifteen samples were created and then film thickness was measured and averaged. It was

concluded that treatment time affected the thickness of the tetraflouroethylene that polymerized into PTFE. A best fit line was also approximated to help design results of later plasma treatments.

Now that the plasma deposition had been perfected, a hydrophobic coating could be applied to the glass slides for Raman spectroscopy tests. Raman spectroscopy is a spectroscopic technique used in chemistry and physics to study vibration, rotational and other low-frequency modes in a system. It relies on Raman scattering of monochromatic light, usually from a laser in the visible, near infrared, or near ultraviolet range. The laser light interacts with the atoms in the sample of the crystalline lattice. These atoms are constantly vibrating at a specific frequency. When a photon hits these atoms, energy is exchanged causing the energy of the photon to be shifted up or down. This gives vibration information that is very specific for the chemical bonds in the molecule which play a major role in many of the physical properties of the sample, including the material’s thermal and electrical conductivities. This technique is typically very weak due to the fact that there is a lot of empty space between atoms that many of the photons pass through, but by coating the substrate in PTFE, the sample being measured bends up and dries with a small diameter. This small diameter allows for more photon to phonon interactions giving a better reading from Raman spectroscopy. The thickness of PTFE film on the glass slides used was zero nanometers, 26 nanometers, 40 nanometers, 80 nanometers, 154 nanometers, and 202 nanometers. The thicker the film the more the sample would be bent up making a smaller diameter of the sample. Raman spectroscopy was performed on a 633 nanometer sample on slides with varying thickness of the PTFE. As the film thickness of PTFE on the slides increased so did the intensity of the photons that gained or lost energy.

Now that the Raman experiments had been completed a hydrophobic coating could be applied to the thin strained silicon membranes. To apply the hydrophobic coating the silicon membranes would have

PLASMA CHEMISTRY: DEPOSITION OF HYDROPHOBIC LAYERS ONTO SILICON AND GLASS SUBSTRATES

Silicon membrane before release to be released from the bulk silicon and silicon dioxide they were grown upon. The silicon membranes are only nanometers thick and can only be as large as one millimeter by one millimeter. To release the membrane the silicon dioxide it was grown upon must be etched away, using full strength hydrofluoric acid, 48% hydrofluoric acid by volume. Hydrofluoric acid dissolves Silicon dioxide via:SiO2(s) + 4HF(aq) → SiF4(g) + 2H2O(l) or SiO2(s) + 6HF(aq) → H2[SiF6](aq) + 2H2O(l). Hydrofluoric acid is a weak acid meaning that the hydrogen and fluorine do not fully dissociate when in water. Nonetheless it is a highly corrosive and dangerous acid, needing only to come in contact with two percent of one’s body to cause death. First special lab coats, face masks, and several layers of gloves were put on. The hydrofluoric acid was carefully poured into a plastic beaker that was coated in PTFE. The beaker was then gently stirred around, taking anywhere from five to ten minutes to etch away the glass releasing the membrane. After the membrane was released the hydrofluoric acid was diluted using de-ionized water.

Silicon membrane after release The membrane was then taken out of the diluted hydrofluoric acid via a ladle or tweezers that were coated in PTFE. The membranes then were applied to glass slides. There are many problems associated with the releasing of these silicon membranes. First the silicon membranes thin nature made them prone to tears which would render then useless. Attempting to remove the silicon membranes from hydrofluoric acid often times lead to the tearing of the membranes. Secondly many times after release from the bulk silicon the silicon membranes would bond back to the bulk silicon. The coating of these membranes is still currently being worked upon.

Applications Currently Intel uses strained silicon for all computer chips they manufacture. This is due to the fact that a highly strained silicon membrane conducts electricity faster than a regular silicon membrane. The data collected from Raman spectroscopy can tell a lot about the thermal and electric conductivities. This would allow for a production of a silicon membrane that conducts extremely well without giving off a lot of heat. The silicon membranes can then be thinly coated in PTFE, making them resistant to water.

The membranes are thin and flexible and Silicon membrane attached to a can be applied to any plastic substrate substrate from clothes to paper. With a hydrophobic coating the silicon membranes can be used as sensors on airplane wings or they can be used in computer to make them faster.

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REFERENCES Bouchoule A. (ED.). Dusty Plasmas, Physics and Technological impacts on Plasma Processing, Wiley, Chicester (1999).

Gordiets B.F., Ferreira C.M., Pinheiro M.J., Ricard A. “Plasma Sources.” Sci. Technol. 7, 363-388 (1998).

Schram D.C, van de Sanden M.C.M., Severns R.J, Kessels W.M.M. J. Phys. IV 8, 217-230 (1998).

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Beaver Survey of the West St. Croix State Park Area

by Caleb Burr and Christopher Kuecker

Caleb Burr

Christopher Kuecker Caleb Burr will graduate from Edgewood High School in 2009. He plans on attending a liberal arts college and is considering entering a medical field.

He has loved science since he was a little boy and will graduate from high school with 6 science credits.

Caleb participated in the St. Croix research field course for two years and loved every minute of it. In this project, he was assisted by many of the counselors at St. Croix, especially Mary-Bridget Nowicki.

Christopher Kuecker is originally from Irvine, California and moved to Madison, Wisconsin in 1998. He will graduate from Edgewood High School in 2009. He plans to study business at the University of WisconsinMadison. Chris has participated in the St. Croix research field course for two years and enjoyed the experience of the great outdoors.

There are a variety of fascinating creatures in the Saint Croix State Park area, including the beaver. Beavers are intriguing mammals which have an enormous impact on the world with their ability to alter and sometimes create new ecosystems. Beavers are capable of producing this change by creating dams of sticks that impede water flow. This damming causes the river flow to build up behind the wall of sticks, creating both a new lentic body of water and ecosystem. Since beavers have a major impact on the environment, the purpose of this project was to document beaver activity on the St. Croix River, Backwaters and the Kettle River, and then determine whether the St. Croix River and Backwaters had more beaver activity than the Kettle River. Sites along the river banks were identified and examined for beavers and their activity including browse marks, logged trees, cut-not logged trees, caches and lodges. Through the research findings, it was evident that the St. Croix River and Backwaters had more beaver activity than the Kettle River. This was supported by evidence that the St. Croix River and Backwaters had 176 sites, including 110 new, whereas the Kettle River only had 116, including 86 new.

Introduction n the current environment, all animals have an effect on the ecosystem. The beaver, or Castor canadensis, is one of the animals which has the ability to create a major impact on the environment. As the largest rodent in North America, beavers do this by constructing blockades of sticks obstructing water flow, or dams. When a dam begins to reduce river flow, water builds up behind it creating a lentic or “still� body of water. This body of water results in a new underwater ecosystem which becomes a habitat for a diverse population of flora and fauna. The ecosystem begins developing because the dam prevents dirt from moving down steam, allowing for vitamin-rich soil to settle onto the pond bed. After the soil settles, new underwater plants as well as insects begin to grow and thrive in this calm area. Sticks left behind by the beavers sink to the bottom of these ponds, forming new habitats for aquatic life to thrive in as well.

Beavers were an important reason why Europeans settled the upper Midwest; to serve the needs of European fashion, fur-traders and trappers pursued beaver for more than 200 years across North America, almost extinguishing the beaver population in many areas. Beavers not only assist in producing a different ecosystem in the water, they also update the ecosystem on the land. Beavers play a significant role in forest diversity because as they cut down preferred trees for dams, lodges and food, they leave other tree species standing. Thus a new tree population is generated.

Also, in areas where beavers cut down favored trees, new shrubs and other tree types begin to grow. New organisms crucial for forest regeneration also begin to flourish.

As evidenced by this information, a study on beavers is important because they have a large impact on the environment. Since beavers can affect the bionetwork so drastically, obtaining additional knowledge through this study is an important step in wildlife management considerations in order to maintain a naturally balanced ecosystem. Therefore, the goal of this study is to

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document beaver activity of West St. Croix State Park Area on the St. Croix River, Backwaters and Kettle River, and determine if the hypothesis that the St. Croix River and Backwaters has more beaver activity than the Kettle River is true.

Method To carry out the study, the St. Croix River, Backwaters and Kettle River were examined for beaver signs. There were many different signs observed indicating beaver presence, including browse marks, cut trees, caches, and lodges. Therefore, it is important to describe these beaver activities. Browse marks are damage from tree gnawing, either in an attempt to cut down the tree or to consume the inner bark. Cut, but not logged, trees have been cut down but not dragged to Beaver browse mark the river. Logged trees have been cut down and pulled into the water. Caches are piles of sticks that have had their bark stripped or are being reserved for later consumption. There are two types of lodges; bank lodges, which are built up onto a protective bank, and water lodges found entirely in the water. All of these signs were sought out and, if found, coordinates were taken, activity age was recorded, and species of tree affected, as applicable, was noted.

By examining the data in Chart 1, it is apparent that there is more beaver activity on the St. Croix River (176 sites) than on the Kettle River (116 sites). There are more logged trees, browse marks, and lodges, which correlate to the fact that more beavers are living on the St. Croix and Backwaters than on the Kettle River.

Also, while two beavers were observed in the St. Croix/ Backwaters area, none were observed in the Kettle River area, giving credence to the fact again, that more beavers

BEAVER SURVEY OF THE WEST ST. CROIX STATE PARK AREA

are living on the St. Croix. It is also believed that there is more beaver activity on the St. Croix because the St. Croix River and Backwaters offer a better habitat than the Kettle River does. This can be concluded by examining the data in Chart 2 where it is evident that the beavers preferred trees are slippery elm, speckled alder, and burr oak, which, by observation, grow more abundantly on the shores of the St. Croix River and in the Backwaters. These preferred trees are not as numerous on the shores of the Kettle and therefore, beavers reside instead in the area where these trees are located, namely the St. Croix and Backwaters. These particular species grow very close to the river’s edge.

Since there were not as many trees near the river’s edge, the beavers would have to climb onto land and go into the forest to search for food. Beavers are very vulnerable to predators on land so they avoid spending extended periods out of the water. Also, the trees that grew closer to the water on the St. Croix and Backwaters generally had smaller circumferences and greater varieties of softer wood. Thus, they required less effort to cut down and to chew through to the preferred inner bark. Moreover, it was found that the aspen, paper birch and ash species have significantly less beaver activity because these trees grow farther away from the water. Through field research, it was observed that the only time that aspen and ash were cut down by beavers was on the Kettle River side. This is because there were not as many bankside trees which caused the beavers to go out into the forest to forage for food likely fearful of predators. In addition, by studying Chart 3, one can see that newer cuts are more numerous on the St. Croix and Backwaters than on the Kettle River. This points to the fact that beavers are more actively searching for and eating food on the St. Croix and Backwaters than on the Kettle River. Furthermore, the fact that the St. Croix River has more beaver activity than the Kettle River could also be related to the varying water speed in the two rivers.

Chart 3

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Chart 1

Chart 2

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REFERENCES Muller-Schwarze, Dietland.

The Beaver. Cornell University Press: New York.

2003.

Kurta, Allen. Mammals of the Great Lake Region. The University of Michigan Press: Ann Arbor. 1995.

Long, Kim. Beavers: A Wildlife Handbook.

Johnson Books: Boulder.

2000.

"Protected Sanctuaries— The American Beaver."

Wildlife Land Trust. The Humane Society of the United States. 24 Aug. 2008 http://www.wlt.org/wildlife _closeup_beaver.asp

This conclusion was reached by observation of the speed.

The St. Croix River has a much lower water speed than the Kettle River. This is due to the contrasting inclines of the rivers. The St. Croix River has a gradual incline, causing the river to flow much more slowly. The Kettle River has a steep incline causing the river to flow rapidly.

Through research, it was been documented by many sources including the Wildlife Land Trust that “…slowmoving rivers are the ideal sites for beavers.” This is because it is easier for beavers to move their logged trees against a slower current to their caches, dams and lodges.

All of these facts provide evidence that the St. Croix and Backwaters has a better habitat than the Kettle River and thus, has more beaver activity.

There were a few errors that occurred during the beaver survey field research. First, not all of the beaver sites that exist along the Kettle River were recorded. This is because the cliffs along the Kettle River prevented the shore from being examined. Next, by concentrating observations along the shore line, the forest by the trail could not be investigated due to time constraints. Also, while surveying for beavers and their activity, signs could have been missed, hidden by shrubbery, or may have been too decomposed to determine with certainty that the sign was created by a beaver. These errors allow opportunity to gather more information through further studies. Future studies could be designed to allow more thorough observation to overcome the study errors.

A possible objective would be to determine if the beaver is significantly decreasing the types of tree species growing along the riverbank. An in-depth study of the Backwaters would further enhance understanding of the beaver and its whereabouts because the majority of the beaver activity in the backwaters is recent.

Conclusion There was adequate evidence supporting the hypothesis that there was more beaver activity on the St. Croix River and Backwaters than on the Kettle River.

This was shown through the observation of 176 beaver activity sites, with 110 being new, on the St. Croix/ Backwaters compared to the 116 beaver activity sites, with 86 being new, on the Kettle River. The difference between the number of beaver activities is a direct result of such factors as water speed and the abundance of tree species on the riverbanks. All of the data collected supports the observation that beavers prefer the habitat of the St. Croix and Backwaters to the Kettle River. The data collected is very significant because beavers need to be managed to ensure that the ecosystem remains stable.

Every species needs the same opportunity to survive and reproduce and the beaver can significantly hinder this with their dams. However, beavers play an important role in the ecosystem and they cannot be removed without resulting in significant disruption.

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Relation between ErbB Signaling Pathways in Head and Neck Cancer and Intrinsic Resistance to EGFR Inhibition

by Philip Gorman With Crucial Guidance from Tim J. Kruser, Paul M. Harari, and the University of Wisconsin Paul P. Carbone Comprehensive Cancer Center Epidermal growth factor receptor (EGFR) is a transmembrane receptor tyrosine kinase of the ErbB family and has a role in the regulation of numerous vital cell functions. Overexpression or dysregulation of this protein has been linked to increased cell proliferation, invasion, metastasis, and angiogenesis in various epithelial cancers. The monoclonal antibody Cetuximab inhibits EGFR. Unfortunately 70-90% of patients show an intrinsic resistance to Cetuximab. Previous studies have indicated a correlation between acquired resistance to EGFR inhibition and the expression and activity of other ErbB family proteins. The purpose of this research was to demonstrate a correlation between the expression and activity of ErbB family proteins and intrinsic resistance to EGFR inhibition. This was accomplished through performing cell proliferation assays to determine Cetuximab sensitivity and a western immunoblot to determine protein expression. Results were unclear in demonstrating a correlation, however, further research is warranted.

Introduction pidermal Growth Factor Receptor (EGFR) is a membrane protein found in many types of epithelial and mesenchymal cells which responds to the presence of certain extracellular ligands through the initiation of downstream signaling pathways. EGFR is a receptor tyrosine kinase (RTK) in that it is a transmembrane protein which, upon binding to a ligand, enables the transfer of a phosphate from ATP to a tyrosine residue (Krause172).

There are 58 RTKs in human cells divided in 20 subfamilies. EGFR is a member of the ErbB subfamily which consists of four proteins: EGFR, ErbB2, ErbB3, and ErbB4 (Baselga 2445). In members of this family, binding by a ligand to the extracellular domain results in homo- or heterodimerization with an adjacent ErbB family monomer forming an oligomer and modifying the conformation of the intracellular, enzymatic domain via the transmembrane domain (Krause 172). The re-conformed intracellular domain encourages autophosphorylation of tyrosine residues which serve as docking sites for several specific proteins with Src homology domains (SH2) and phosphoptyrosine binding domains.

These proteins in turn initiate specific signaling pathways within the cell (Weinberg 166).

EGFR responds to seven specific ligands: Epidermal Growth Factor (EGF), transforming growth factor-α, heparin-binding EGF, amphiregulin, epiregulin, neuregulin, and beatcellulin (Harari 690). The response to EGFR is in the cell is pleiotropic and its activity through signaling pathways, including the phospholipase Cγ (PLCγ1), signal transducer and transcription activation (STAT), phophatidylinositol 3-kinase(PI3K), and survival mediator AKT pathways (Gshwind 363), is closely associated with mitogenesis, apoptosis, changes in cellular motility, protein secretion, differentiation, and dedifferentiation (Harari 691). Modification of these cellular behaviors result in an association of EGFR with hyperproliferative disorders including cancer and cardiac hypertrophy as well as Staphylococcus aureus and

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Heliobacter pylori infections (Gshwind 363). Inhibition of EGFR in laboratory animals xenografted with human tumors produced results more significant than with cell culture alone, suggesting there is a multicellular component to EGFR activity, most likely it promotes angiogenesis (Harari 693). Overexpression in humans has been specifically observed in cancers of the head.

Studies indicate that in as many as one-third to one-half of human epithelial tumors EGFR is either overexpressed or dysregulated in some fashion, neck, bladder, brain, neuroglia, breast, ovaries, cervix, uterus, colon, stomach, esophagus, pancreas, kidney, and lung (Harari 691). In head and neck cancers EGFR overexpression is particularly prominent in that 70% of these tumors are strongly EGFR-positive (Harari 692). Due to the demonstrable correlation and probable causation relating EGFR activity and tumorigenesis, it is medically relevant to the treatment of cancer that means by which to inhibit this activity be devised.

EGFR may currently be inhibited through the use of either monoclonal (mAb) antibodies or small molecule tyrosine kinase inhibitors (TKI). TKIs function through binding to the active site on the intraceullar domain of EGFR, thus preventing autophosphorylation and the resultant initiation of a downstream signaling pathway.

The two TKIs currently approved by the Food and Drug Administration (FDA) for cancer treatments are erlotinib and gefitinib. Monoclonal antibodies inhibit EGFR by binding to the extracellular active site which typically binds to the ligand, thus preventing ligand- binding and the initiation of tumorigenic signaling pathways (Baselga). There are two monoclonal antibodies approved by the FDA for treatment in human cancers, Panitumumab (ABX-EGF, Vectibix) and Cetuximab (IMC-C225, Erbitux). Cetuximab is a chimeric antibody developed by ImClone systems and is created through introduction of human EGFR into a mouse which produces specific antibodies for the protein. The resulting antibodies are harvested and their constant region replaced with that of human immunoglobulin.

Philip Gorman Philip Gorman is a 2007 graduate of Edgewood High School and will be attending Kalamazoo College in the fall where he will pursue a biology major with the hopes of attending medical school. Philip was mentored in his research by Tim Kruser in the lab of Dr. Paul Harari at the University of Wisconsin’s Paul P. Carbon Comprehensive Cancer Center.

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Cetuximab has so far been approved for the treatment of irinotecan-resistant metastatic colorectal cancer, pancreatic cancer, head and neck squamous cell carcinoma, and non-small cell lung carcinoma (Bonner 568). Unfortunately, Cetuximab treatment is only effective in 10-30% of patients, while the remainder experience intrinsic resistance. Furthermore, those tumors that do initially respond to Cetuximab eventually become resistant through a phenomenon known as acquired resistance.

Some research has indicated there may be a correlation between the expression and activity of certain other proteins in the ErbB family and acquired resistance to Cetuximab. The role of these ErbB proteins in intrinsic resistance is currently unknown. The purpose of this research was to determine whether the expression and activity of ErbB family members was related to intrinsic resistance to Cetuximab. In order to test this hypothesis, a battery of cell lines were tested for in vitro Cetuximab sensitivity and the expression and activity of ErbB family proteins and their associated downstream pathways was determined.

Methods A cell proliferation assay was utilized in order to ascertain the comparative resistance or susceptibility of eight heterogeneous cell lines of HNSCC to treatment with Cetuximab. The eight cell lines were thawed from samples in frozen storage and grown in a nutrient medium. Concentration was determined by hemocytometer, and cells were seeded at a density of 25,000 cells per well. After allowing 24 hours for cell adhesion, Cetuximab was added to the wells at concentrations of 0.05, 0.2, 0.5, 1, 5, and 25 nM. Following treatment, the medium was aspirated and the remaining cell monolayer washed with phosphate buffered saline (PBS).

Cells were then fixed/stained using 0.5% crystal violet solution. The plates were air dried overnight and 0.1 M sodium citrate (pH 4.2) and ethanol in a 1:1 solution was utilized to elute the cell-associated dye. The elution was transferred to a 96-well plate and the concentration of dye was determined at 540 nm light wavelength.

Information concerning the concentration of dye indicated the total number of cells and was used to construct growth curves which demonstrated the effect of Cetuximab on cell proliferation.

Following assessment of drug sensitivity in the designated HNSCC lines, a western immunoblot analysis was performed to determine the expression of certain proteins of interest. Secondary samples of the eight lines were thawed and grown in medium. Cells were grown to 70-80% confluence in 100 mm dishes.

Cells were lysed using a Tween-20 lysis buffer consisting of 50 mM HEPES buffer (pH 7.4), 150 mM NaCl, 0.1% Tween-20 detergent, 10% glycerol, 2.5 mM EGTA, 1 mM EDTA, 1 mM DTT, and 1 mM PMSF. The various components of Tween-20 lysis buffer simultaneously release the cells from the dish, dissolve the plasma membrane, encourage proteins to dissolve in a solution, and prevent the cells protease enzymes from digesting the desire protein. The cell lysate resulting

from interaction with the lysis buffer was then sonicated to encourage lysis and mix contents. Protein was quantitated using a Bradford absorbance assay which measures protein concentration through the use of a Coomassie blue dye which causes light to be absorbed at 595 nm wave length in the presence of protein binding.

Based upon the measured concentrations, an equal amount of protein from each cell line was distributed for fractionation by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In SDS-PAGE a SDS buffer induces denaturation of proteins and forces them to acquire a negative charge. When a positive charge is placed at the opposite end of a section of polyacrylamide gel the protein is forced through the gel via this electric gradient. Smaller proteins move through this gel more quickly and thus a protein gradient based upon molecular weight develops. The rough weight of proteins may be determined through comparing them with a ladder of known proteins dyed for visibility. The fractionated protein was then transferred to a polyvinylidene difluoride (PVDF) membrane via electrophoresis. The membrane was incubated with a primary antibody specific to nine target proteins (EGFR, pEGFR, ErbB2, pErbB2, ErbB3, pErbB3, pMAPK, pAkt, and tubulin). The membrane was then incubated again in a secondary antibody with a variable region specific to the consant region of the primary antibody.

The secondary antibody is conjugated with horseradish peroxidase (HRP), an enzyme that releases light when chemically treated through a process called chemiluminescence. By measuring the light released via photographic film, the location and relative amount of protein targeted by the primary antibody may be determined. Prior to incubation with either antibody the membrane was kept in treated milk so as to saturate the membrane and prevent any non-specific binding of antibodies to plastic.

All antibodies were purchased commercially from the indicated providers: EGFR, pEGFR(tyr1173), ErbB3, and HRP-conjugated gaot-anti-rabbit IgG, goat-antimouse IgG, and donkey-anti-goat IgG antibodies were from Santa Cruz Biotechnology Inc; pErbB2, pErbB3, pAkt, and pMapk antibodies were from Cell Signalling Technology (Berkely, Mass.); and ErbB2 antibody was from Neomarker (Freemont, California).

Results/Conclusions Analysis of the cell proliferation assay through the creation of growth curves revealed obvious differences in the sensitivity and resistance of the eight heterogenous cell lines of HNSCC to Cetuximab. Three of the lines (designated SCC-5PT, SCC-17B, SCC-22B) displayed a pronounced resistance to the drug (Fig. 1). Three of the lines (designated SCC-11A, SCC-14A, and SCC-37) displayed sensitivity to the drug with a rate of growth roughly 50-60% of that of the untreated cells (Fig. 2).

The two remaining lines (designated SCC-1483, and SCC-1) displayed a moderate sensitivity to the drug with a rate of growth roughly 80-85% of that of the untreated cells (Fig. 3). There appeared to be a point in each of the growth curves, typically between 0.2 nM and 0.5 nM of

ERBB SIGNALING PATHWAYS IN HEAD AND NECK CANCER AND INTRINSIC RESISTANCE TO EGFR INHIBITION

Figure 1: Cetuximab-resistant lines SSC-5PT, SSC-17B and SCC-22B

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Figure 2: Cetuximab-sensitive lines SSC-37A, SSC-14A and SCC-11A Figure 3: Moderate sensitivity to Cetuximab lines SSC-1 and 1483 (not shown)

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REFERENCES Baselga, José, Arteaga, Carlos L. “Critical Update and Emerging Trends in Epidermal Growth Factor Receptor Targeting in Cancer.” Journal of Clinical Oncology Vol. 23; 11 (April 10, 2005): 2445-2459.

Bonner J. A., Harari P. M., Giralt J., Azarnia N., Shin D. M., Cohen R. B., Jones C. U., Sur R., Raben D., Jassem J., Ove R., Kies M. S., Baselga J., Youssoufian H., Amellal N., Rowinsky E. K., Ang K. K. “Radiotherapy plus Cetuximab for SquamousCell Carcinoma of the Head and Neck.” New England Journal of Medicine Vol. 354 (February 9, 2006):567578.

Cetuxiab, at which the drug reached its maximum level of effectiveness and any higher concentration of the drug produced little result. That the cells were treated under identical conditions in vitro but demonstrated differing responses to Cetuximab leaves only their heterogenous character as a variable. Susceptibility and resistance must therefore be related in some way to the expression and activity of certain proteins within the HNSCC cells.

The film results from western immunoblot were scanned and computer-enhanced to ease analysis (Fig. 4). Expression and activity of the nine proteins was variable throughout the eight cell lines with the exception of tubulin which, as a common component of the cytoskeleton, should be equally expressed in all cells.

The equal distribution of tubulin indicates that equal concentration of protein was utilized in the experiment.

Those ErbB proteins preceded by a “p” represent the phosphorylated version of that protein which has been

made active and is capable of initiating tumorigenic signalling pathways. pAkt and PMAPK are proteins which serve as a downstream component of the signalling pathways trigged by EGFR, their phosphorylation would indicate active EGFR. In looking for a correlation between expression and activity and resistance it was neccesary to analyze the expression of certain proteins in sensitive cells (SCC-11A, SCC-14A, and SCC-37) versus resistant cells (SCC-5PT, SCC-17B, SCC-22B). Based upon the information present in this particular western immunoblot, such a correlation was not observed despite intensive analysis. Due, however, to the close association of the ErbB family proteins to EGFR, and the apparent relationship between ErbB family proteins and acquired Cetuximab resistance further research into the relationship between intrinsic resistance and ErbB expression/activity is warranted.

Gshwind, Andreas, Fischer, Oliver M., Ullrich, Axel.

“The discovery of receptor tyrosine kinases: targets for cancer therapy.” Nature Reviews: Cancer Vol. 4;5 (May, 2005): 361-370.

Harari, Paul M. “Epidermal growth factor receptor inhibition strategies in oncology.”

Endocrine-Related Cancer Vol. 11 (December 1, 2004): 689-708.

Krause, Daniela S., Van Ettenm Richard A. “Tyrosine Kinases as Targets for Cancer Therapy.” The New England Journal of Medicine Vol. 353;2 (July 14, 2005): 172-187.

Weinberg, Robert A. The Biology of Cancer. New York: Garland Science, Taylor and Francis Group, LLC, 2007.

Figure 4: Western Immunoblot with relative concentration of the indicated proteins for the indicated cell lines.

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Etching and Release Techniques of Elastically Relaxed Free-standing Strained-Silicon Nanomembranes

by Cedric Meyers Although silicon theoretically can be highly strained by growing multiple layers of SiGe on its surface, in reality these membranes become brittle and break. Etching away the top Si/SiGe layers so that only the strained silicon layer is left bonded to the substrate can solve this problem. This, however, roughens the surface of the silicon, which inhibits electrons mobility through the crystal. One goal of this project was to determine which etchants roughen the membrane least. The roughness of the silicon was determined through AFM (atomic force microscopy) analysis. Only a full strength KOH etch of the silicon top layer was performed, and showed little increase in rms roughness when compared to the unreleased membrane. In addition two popular membrane release techniques were performed and the amount of tensile strain on the silicon was measured using XRD (x-ray diffraction). Method 1, release with photo resist (PR) removed, failed because the membrane re-attached to the membrane; but method 2, release with 13μm of PR coating the membrane, produced large (approx.

2mm x 2mm), intact membranes.

Introduction or years scientists have known about the effect of strain on various materials. Silicon is one such material that strain affects. Recently, Dr. Lagally's research group MRSEC IRG #1 on Chemical Vapor Deposition at the University of Wisconsin-Madison found that in silicon/silicon-germanium (Si/SiGe) semiconductor heterostructures, strain provides a mechanism for control of both carrier mobility and band offsets (Roberts). They found that the more tensile strain exerted on a silicon membrane, the greater the electron mobility becomes.

Therefore the silicon membrane becomes a better semiconductor.

To increase the semi-conductivity of the Si membrane, a film of silicon bonded to silicon dioxide (Buried Oxide or BOX) and then to silicon wafer (silicon on insulator or SOI) is placed in an ultra-high vacuum chamber (UHV) where the vacuum exceeds 10-9 Torr (Ritz). The sample is heated while silane and germane gases bombard the surface forming a crystal lattice, a process called chemical vapor deposition. The result is a SiGe alloy film on the surface of the SOI. Because germanium is a larger molecule than silicon, its alloy with silicon occupies 4% larger volume and has a physically larger crystal structure so that its lattice must compress to fit on the surface of the SOI (Ritz).

When released, the compressed SiGe layer mechanically dominates, stretching the surrounding silicon membranes. To prevent curvature of the released membrane, a second layer of silicon is grown on the SiGe layer. 49% hydrofluoric acid dissolves the BOX layer, releasing the membranes to be re-attached to another substrate with special adhesive at 500° C. In the past, additional higher Ge percentage SiGe alloy (lattice mismatch >4%) was grown to further strain the membranes. This, however, caused extreme brittleness due to the excessive thickness of the membrane along with a greater number of dislocations, or crystalline

F

imperfections, in the membrane. More recently, experiments have etched away the top Si and SiGe layers, leaving behind only the ultra-thin strained silicon layer. Higher Ge content SiGe could then be grown on the thin membrane to further strain the silicon. The purpose of this project is to test etching and release processes and analyze the results with AFM and XRD. Cleaning Methods In order to perform any experiments on silicon, all samples must be chemically cleaned and hydrogen terminated. The process started with a thorough washing of all samples, beakers, and utensils in highly filtered de-ionized water. Next the silicon samples were placed in a 10% v/v solution of hydrofluoric acid for 15 seconds to remove any silicon dioxide that had formed on the samples’ surfaces. Then the samples were rinsed and placed in a piranha solution of approximately equal volumes of sulphuric acid and hydrogen peroxide for 5 minutes. After another rinsing, the samples were placed in an AHP (ammonia and hydrogen peroxide) at 75° C for 15 minutes to hydrogen terminate the surfaces. The resulting samples were then ready to release.

Because work was done with many dangerous chemicals along with clean materials, there were several protocols to follow in the lab. First, when handling any chemicals or even touching their containers or storage cabinets—especially hydrofluoric acid (HF)—nitrile gloves, heavy rubber gloves, and PVC gloves must be worn, in that order. Also, heavy aprons and full-face masks must be worn at all times. Whenever handling clean materials, PVC gloves must be worn and materials handled only in clean vessels with clean utensils. The silicon was never to be touched with metal utensils, as this deposits metal ion on the surface, which affect its performance. When working with HF, only Teflon lab ware was used, as HF will etch glass.

Cedric Meyers graduated from Edgewood High School in 2007 and is currently studying chemistry and physics at Kalamazoo College in Michigan. Cedric performed his research at MRSEC IRG #1 on Chemical Vapor Deposition at UWMadison under his fourthyear graduate student mentor Clark Ritz and lab director Dr. Max Lagally.

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Goals of Releases While strained silicon membranes are produced in large segments, these large sections are virtually impossible to release from the handle wafer. Instead, these sheets are divided into many 2mm x 2mm sections. Despite their small size, these membranes are still difficult to release intact and tend to tear easily or reattach to the handle wafer. The goal of this part of the project is to determine a process that releases these 2mm x 2mm sections effectively and without significant damage to the membrane. This goal was met, and a membrane approximately 1.9mm x 1.8mm was released, with only one tear.

Methods of Release After cleaning, the membrane is released from its handle wafer. After lithographically etching many small access holes in the membrane, the membrane is released in a bath of full strength 49% hydrofluoric acid (HF).

Two release techniques were tested. The first technique was release with PR (photo resist) removed. PR is a polyhydroxystyrene-based polymer with photoacid generator used in photolithography of silicon. PR coatings allow patterns to be etched into a silicon surface using UV light. The first technique began by dissolving the PR coating in a beaker of isopropanol and acetone inside an ultrasonic vibrator. The PR-free membrane was placed in the HF bath for 1.5 hours and allowed to release. The second technique involved leaving approximately 13Îźm of PR coating the surface of the membrane. This sample was left in HF overnight because of an initial failure to release. Because of worry that the PR would hinder the complete elastic relaxation of the membrane, XRD was performed to analyze the amount each membrane relaxed.

Figure 1

Results of Release Techniques The first release technique yielded a virtually unusable membrane. As HF dissolved the buried oxide (BOX) layer, the membrane floated off of the substrate and then re-bonded due to van der Waals forces. The resulting sample resembled a very small, highly wrinkled piece of aluminum foil bonded to the substrate. The second technique yielded a large 1.9mm x 1.8mm membrane with minimal tearing. As the HF dissolved the BOX layer, the polymerized nature of the PR coating pulled the edges of the membrane away from the substrate, allowing for a direct path between the HF and BOX, promoting fast and even dissolution of the BOX. XRD analysis confirmed the full relaxation of the PRcoated membrane (see bold dotted line in Figure 1). The peak shift from unreleased to released membrane shows clearly that the membrane thickness decreased, meaning that the membranes stretched horizontally. Therefore, to consistently release large, intact membranes, a thick PR coating should remain on the membrane’s surface.

Goals of Etching After releasing the membrane, it is re-bonded to a new substrate and is ready for etching. Although wet etching, which is etching with liquid chemicals, can effectively thin the membrane, the chemicals used to etch back these layers do not etch evenly (Virginia).

They turn an atomically smooth surface into a rough, jagged, and pitted surface. A rough surface significantly slows the speed of electrons, negating any increases in electron mobility gained from straining the silicon membrane. Another goal of this project was to determine what combinations of etchants and their strengths would etch the layers without roughening the silicon surface.

ETCHING AND RELEASE TECHNIQUES OF ELASTICALLY RELAXED FREE-STANDING STRAINED-SILICON NANOMEMBRANES

Methods of Etching The etchant that was used to etch back the silicon layer was KOH. End caps were added to the sample to prevent the dissolution of the silicon substrate. Then the silicon layer was bathed in full strength KOH for several minutes in the clean room. The KOH attacks the diagonal lattice of silicon, etching quickly at a 45° angle.

There were several methods for analyzing surface structure. In this project, AFM was used to map the surface of the silicon membranes. In this process, a tiny “needle” that is about 10 picometers wide was dragged across the sample. A laser is bounced off of this tip and is reflected into a detector, which sends the information to a computer, where the information is assembled into a picture of the surface, represented graphically. Using this data, it could be determined whether or not the etching recipe roughened the surface substantially or not.

Results of Etching

Figure 2. Unreleased Membrane Because of insufficient time, only KOH was tested as a silicon etchant. Figure 2 shows the AFM surface map of the unreleased membrane. The rms roughness of .535nm is the average roughness of membranes grown in the CVD chamber. The white spots show bumps on the sample surface that are consistent with imperfections from CVD growth. Similarly, Figure 3 shows a released membrane mounted on a new substrate.

Figure 3. Released Membrane bonded to new substrate The large white splotches are caused by dust particles on the sample. The rms roughness inside the bolded box is .561nm, indicating virtually no change in surface roughness resulting from the release process. The .026nm increase in rms roughness can be attributed to imperfections in the adhesion between the membrane

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REFERENCES

Ritz, Clark. Lecture.

University of WisconsinMadison. 20 Dec 2006 and 12 Jan 2007.

Figure 4: Released Membrane, KOH etch and substrate. Figure 4 shows the released membrane after KOH etch of the silicon top layer. Although the surface roughness increased from .535nm to .542nm, this change is negligible. However, the surface appears more pitted, as seen by the dark circles throughout the image. These results are much better than expected.

Most etchants bore deep holes into the surfaces they are etching. However, because KOH etches at a 45° angle, it doesn’t bore into the surface. One drawback of KOH is that all silicon surfaces, except the one being etched, must be shielded so that they do not dissolve. Despite this minor setback, KOH etching of silicon has little effect on the overall surface roughness and would be a good choice to etch the silicon top layer.

Conclusion The goals of this project were to determine what combinations of etchants and their strengths would etch the layers without roughening the silicon surface and to determine a release process that releases large membrane sections effectively and without significant damage to the membrane. Measurements were performed using AFM for roughness and XRD for thickness and strain. This research is important because it will allow for the production of highly strained silicon membranes, while keeping them ultra-thin and flexible. Not only will this make future research in this area easier, but it will also make it possible to grow silicon membranes that are strained more than what is now possible. These membranes can be patterned into microprocessor chips and attached to virtually any surface allowing for “smart” prosthetics and clothing, ultra thin, flexible displays, or even “smart” cash or checks.

Roberts, Michelle M. et al. Elastically Relaxed Free-standing Strained-Si Nanomembranes.

MRSEC IRG #1, University of WisconsinMadison, Wisconsin.

Virginia Semiconductor, Inc. Wet-Chemical Etching and Cleaning of Silicon. Jan 2003. www.virginiasemi.com/ pdf/siliconetchingandcleaning.pdf. 15 Jan 2007.

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Orconectes rusticus Populations in the St. Croix River and Tributaries in Relation to Human Interaction (Fishing)

by Arial Shogren, Lianna Schmidt and Charlotte Martin Arial Shogren Since the late 1960s, Orconectes rusticus, more commonly known as rusty crayfish, have been plaguing the waterways of Wisconsin and Minnesota. Rusty crayfish can grow to nearly 4 inches in length; are omnivores, feeding on detritus, vegetation, and other aquatic life; and are extremely aggressive. They inhabit many different types of waterways, with different bottom types. However, rusty crayfish are more than just a nuisance, they are a threat to the biodiversity of an area, and, eventually, the ecosystem itself. Therefore, preventing the spread of this invasive pest is imperative. One of the ways that rusty crayfish are likely to have been spread is by fishermen releasing live bait. By comparing several different sites and classifying them as “fishing” (where there was physical evidence of fishing activity) or “non-fishing” (where there was little to no physical evidence of activity), the group was able to show the drastic difference in crayfish populations in areas with and without this sort of human recreation. The research suggests that areas that have high frequencies of fishing recreation should be monitored more closely for the spread of O. rusticus than areas not associated with wishing or other human activities.

Lianna Schmidt Arial Shogren was a third year student at the time this research was conducted, and she graduated in May of 2009.

In the future, she hopes to study environmental science.

Lianna Schmidt graduated from Edgewood High School in May of 2009.

She would love to become a forensic anthropologist once she finishes college.

She thanks the St. Croix Environmental Field Course instructors and moderators for their time and support.

Charlotte Martin graduated from Edgewood High School in May of 2009. She plans to study genetics should she enter into the scientific field after high school. She thanks Mekel, Toby, Jerry, and the entire St. Croix Environmental Field Course staff for all of their invaluable time and knowledge.

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asic ecology is the study of the interactions between organisms and their environment.

These interactions are essential to the environment, because these complex relationships make up the whole of the ecosystem, and are thus crucial to understanding what occurs when the system becomes imbalanced. Because these relationships are so delicate, the ecology of an ecosystem can be severely disrupted by any slight disturbance in the fragile systems that compose it. When something happens to a species in a community that causes its population to either increase or decline, most of the other organisms will somehow be affected. The effects of such disruption could be described as a ripple effect, which spans outward from the center, the center representing the initially affected species. One of the ways that the ecology of an environment can be affected in such a way is when a foreign species is introduced into the native area. These species are called invasive species, and are defined as species that take hold outside their normal geographic range. Usually, humans introduce invasive species into an ecosystem. Invasive species are so ultimately detrimental because they disrupt natural competition between organisms or populations, and have no natural predators to compete with. In Wisconsin and Minnesota, many ecosystems are being ravaged by invasive species. In the St. Croix specifically, the rusty crayfish has disturbed the natural balance of the waterways.

It is thought that rusty crayfish, or Orconectes rusticus, are native to the Ohio River Basin. It is believed that rusty crayfish found their way into the St. Croix River and tributaries through waters in Wisconsin and Iowa, after being introduced by nonresident fishermen who used rusty crayfish as bait, and then unknowingly threw unused bait into the water. Consequently, rusty crayfish

were not recorded in Wisconsin or Minnesota waters until 1960 and 1967 respectively. Numerous other theories exist concerning how the rusty crayfish got into the St. Croix, including biological supply companies failing to warn schools not to release the crayfish, as well as the harvest of the “rusty” for bait and food.

Nevertheless, it is important to take note of the rusty crayfish population close to and far from common fishing areas of the St. Croix, as to estimate the extent of the invasion. This is a main focus of this project.

Rusty crayfish are arthropods. They inhabit ponds, lakes, and streams, and live under rocks, logs, or other debris, in many different bottom types, including clay, sand, gravel, silt, or rock. Rusty crayfish reach maturity at a length of 1-3/8 inches, and can grow to nearly 4 inches long. This type of crayfish feeds on a variety of underwater life, including aquatic plants, macro-invertebrates, detritus, fish eggs, and small species of fish, among other things. It is are an aggressive species, often displacing native species of crayfish by increasing interspecific competition and fish predation. Instead of swimming away from a threat, like many native species, they will raise their claws and become defensive. This plays a role in why they are so harmful to native species; fish and other predators learn to avoid rusty crayfish, so they have no natural predators. Rusty crayfish have the most detrimental impact on the aquatic plant beds of the invaded body of water. Because these crayfish reduce plant abundance and therefore species diversity, it is especially damaging in northern areas, where aquatic plant beds are not as abundant. As a result, native species such as O. immunis, O. virilis, and O. propinquus have been displaced and their numbers are dwindling.

The goal of this project was to find out if rusty crayfish numbers increase in areas that are associated with human use—fishing, specifically—and to compare

ORCONECTES RUSTICUS POPULATIONS IN THE ST. CROIX RIVER AND TRIBUTARIES IN RELATION TO HUMAN INTERACTION (FISHING)

the rusty crayfish populations in the St. Croix River to those of its tributaries. Accordingly, the group hypothesized that in researching population sizes of rusty crayfish in the St. Croix River and its tributaries, the most abundant sources would be found in areas of high human traffic and activity. Such sites include boat launches and fishing areas along the banks. Although rusty crayfish may be found along parts of the St. Croix that are not regularly used by humans, the population densities would be significantly higher in areas that are.

To catch the crayfish, the group used two main methods. The first was “netting,” or taking a net and scraping it along the bottom for 30 minutes at each site to eliminate the factor of a differing amount of effort.

The second was using modified minnow traps and bait canisters filled with cat food, and leaving said traps overnight for two nights. Three traps were placed at each location.

Graph 1

This project was based on finding the correlation between human interaction with the environment— specifically fishing—and the rusty crayfish distribution in the St. Croix and its tributaries. By testing several sites in which evidence of fishing was present and comparing the data to sites where there was little to no evidence of fishing activity, one could see the impact that it has on an ecosystem, especially when monitoring the populations of invasive species. The sites that were tested and were categorized as having a high frequency of human traffic were the boat launch, the Kettle River Overlook area, Big Eddy, Kennedy Brook, and the Sand Creek Overlook. At all of these sites, there was physical evidence that fishing occurred, such as fishing line or lures. Likewise, the tested sites that exhibited few occurrences of fishing included Bear Creek, St. Croix backwaters and various sites along the St. Croix River.

As shown in Graph 1, there is an obvious correlation between human activity and a higher density of crayfish

in an area. In the sites where human activity was prevalent, there was typically a higher number of crayfish caught in either the dip net or the trap as compared to sites where fishing was not as apparent. At the boat launch, there were 28 crayfish caught. Similarly, in the Kettle River Overlook area, 25 crayfish were caught, and the Kennedy Brook data shows that 32 crayfish were found. The data for the Kennedy Brook area may be skewed because, of the three traps set, only one could be retrieved due to high water levels brought about during a severe thunderstorm. At another site where fishing was obvious (as the group witnessed a fisherman on the banks), 56 crayfish were caught. At the Sand Creek overlook location (the point at which the river meets Sand Creek), no crayfish were caught in either the net or the traps. It is probable that this is the result of the abnormally high levels of water that followed severe storms. Fishing was not as ubiquitous at Bear Creek, the St. Croix backwaters behind the swimming hole, or the St. Croix River, the sites that were not classified as having a high frequency of human interaction.

However, in Bear Creek, the data collection was incomplete because no crayfish were caught in nets, and two traps were unattainable due to aforementioned storms.

The St. Croix Riverway yielded no crayfish in the nets, and traps were not utilized due to time constraints and natural occurrences. In the backwaters, seven crayfish were caught either in the nets or in the traps, which

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Graph 2

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REFERENCES 1.

Campbell, Neil A., and Jane B. Reece.

Biology. 7th ed. San Francisco: Benjamin Cummings, 2005.

1136-1166.

2. “Harmful Aquatic Hitchhikers: Crustaceans: Rusty Crayfish.” Stop Aquatic Hitchhikers! US Coast Guard. 15 Feb. 2008 www.protectyourwaters.net.

3. “Invasive Species of Plants and Wild Animals in Minnesota.”

Minnesota Department of Natural Resources.

28 Feb. 2008 http://files.dnr.state. mn.us/.

4. “Rusty Crayfish: a Nasty Invader.” Minnesota Sea Grant. 8 Aug. 2007. Minnesota Department of Natural Resources. 13 Feb. 2008 www.seagrant .umn.edu/.

5. Voshell Jr., J. Reese.

A Guide to Common Freshwater Invertebrates of North America. The McDonald &Woodward Publishing Company: Blacksburg. 2002.

although the number is less than those found in fishing areas, was much higher than in either Bear Creek or the St. Croix. This suggests that rusty crayfish may have worked their way from the boat launch and distributed themselves throughout the St. Croix backwaters.

As the study was conducted to determine whether human activity affects crayfish populations, it is interesting to note that the majority of the crayfish collected were taken from areas within about 20 feet downstream of fishing sites and boat launches. This explains the denser population of crayfish in areas of high levels of human traffic. It’s common for fishermen to rid themselves of live bait after coming in from a fishing trip. This usually occurs at the points nearest a boat launch, which is where the group found most of the specimens, so it makes sense that crayfish would be downstream of a boat launch.

As mentioned before, there were two methodologies used to capture the crayfish to sample the populations.

To make sure that each method was successful, the numbers caught by either net or trap was recorded. The

methodology success rate tended towards the nets as being more successful at catching the crayfish, and the traps were successful but not at the rate the nets were.

After collecting and analyzing the data, the hypothesis was proven correct. Although Orconectes rusticus did not densely populate areas along the St. Croix, boat launches and fishing sites on its tributaries had higher populations as compared to non-fishing areas. Nevertheless, the majority of the sites with a large number of crayfish were at the mouths of the tributaries that fed into the St. Croix River. While not entirely what the group expected, this is good news for the river’s ecosystem; the absence of rusty crayfish—or perhaps the scarcity of them—in the riverway suggests that few have made their way into the St. Croix. Though rusty crayfish are an aggressive species, it is extremely important that the populations of these detrimental creatures are monitored to ensure that the waters of the St. Croix are protected.

The only way for prevention to happen is the continued research and testing of the waters of and around the St. Croix.

PRESENCE OF BACTERIA AND PROTEIN IN GRADE A AND GRADE B OF MILK

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Presence of Bacteria and Protein in Grade A and Grade B of Milk

by Kerry Sweeney The presence of proteins in Grade A and Grade B unpasteurized milk and the configurations of proteins is the major purpose of the experiments. The first process was separation of protein from the rest of the components of milk by ultracentrifugation. The proteins were then isolated by a polysaccharide gel electrophoresis. The identification of the proteins was accomplished by the mass spectrometer based on molecular weight and ion charge. Protein identification results were then researched and a conclusion based on the presence of certain proteins was drawn. As a separate experiment, the presence of certain bacteria in Grade A and Grade B unpasteurized milk was determined by plating the milk in MacConkey agar and testing for gram-negative bacteria. The results were then determined by comparing colonies’ size, texture and shape.

Introduction

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ilk has long been a prevalent part of a well-balanced diet and a culturally accepted beverage. The pasteurization of milk in the early 1900s began to make milk available to all consumers. The presence of both calcium and protein in milk became a point of study and questions for scientists. It has been assumed that the most prevalent proteins that exist in all types of milk are casein, beta lactaglobulin, and alpha lactaglobulin. The purpose of the experiment is to decipher the presence of these proteins and other proteins that exist in milk and to determine the safety based on the presence of the aforementioned proteins.

The experiment will focus on separating, isolating, and identifying the proteins that exist within Grade A and Grade B unpasteurized milk. The techniques used for separating will be ultracentrifugation. The process of isolating will be accomplished by a polysaccharide gel electrophoresis while the identification of proteins will be a MSDS. The question and focus will not only be on the proteins present, but also on the bacteria present in milk.

The process by which bacterial colony presence will be determined is agar plates and streaking.

Methods Mass Spectrometry In MSDS a small sample of a compound is initially ionized, this usually happens by a loss of an electron (CEM 1). The ions are sorted and separated according to their mass and charge (American Society 1). Once the separated ions are detected they are tagged and the results of their specific mass and charge are displayed through a separate database on a different computer

http://mass-spec.lsu.edu/mswiki/images/e/eb/MS_MS.png

(American Society 1). A MSDS usually is illustrated as a vertical bar graph, these bars represent the ion and its specific mass to charge ratio (American Society 1). The length and width of the bar indicates the abundance of the ion (American Society 1).

Gel Electrophoresis Gel electrophoresis is a method that separates macromolecules, usually proteins, based on their size and electrical charge (Bergen 3). Electrophoresis describes the migration of charged particles in an electric field (Bergen 3). The properties of a molecule determine the rate at which the electrical field moves a molecule through the gelatin medium (Bergen 3).

Ultracentrifuge The high speed and convection-free conditions of the ultracentrifuge are used to measure molecular weights of solutes (Answers 1). The machine provides its data based on molecular weight distribution (Answers 1). The ultracentrifuge separates and characterizes macromolecules on the basis of the density of the solutes (Answers 1).

Results In order to separate proteins from the milk compound, two spins of ultracentrifugation were used on two samples of unpasturized Grade A and Grade B milk. The two consecutive spins were run for 45 minutes each. The spins were conducted at 10ËšC. The first spin was set at 10,000 rpms and the second spin was conducted at 50,000 rpms. After centrifugation, only liquid (miscellaneous) and the protein pellet at the bottom of the epindorf tubes remained. The protein pellet was removed from both samples A and B. Small samples from the respective samples A and B were then removed and a protein assay was conducted to determine the concentration of proteins present in the samples. Once it was determined that there was a significant amount of protein present in the samples, the process of isolation of the proteins proceeded.

In order to identify the specific proteins that existed the process of polysaccharide gel electrophoresis was used. The gel was preprepared and there were 10 wells used for

Kerry Sweeney graduated from Edgewood High School in 2007 and is currently attending the University of Arizona in Tucson as a Biomedical Studies major. Her research was completed at the Biomedical Studies building in the Mass Spectrometry Department under the direction of Amy Harms, PhD.

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REFERENCES 1. Mass Spectrometry.

CEM education.

Pg 1-3. www.cem.msu.edu/ reasearch/Massspec.

2. American Society for Mass Spectrometry.

Pgs 1-5. 2007 www.asms.org 3. Gel Electrophoresis.

Bergen society. Pgs 1-2. www.bergen.org/gel 4. Ultracentrifugation.

Answers.com. Pg 2. www.Answers.com/ ultracentrifuation 5. Wikkipedia/“Mass Spec. Image 3.� 2006. http://mass-spec.lsu .edu/ mswiki/images/ e/eb/MS_MS.png

identification of the three types of protein samples and two control wells. Prior to the entry of the samples into their respective wells the Samples A and Samples B as well as a small-presumed sample of whey protein was diluted in water and other organic materials. The liquefied substance of the proteins, presumed Casein A and whey proteins were entered into their respective wells. The two control wells were on either end of the gel so that we could compare the presence and shading of the bands that existed. There were three wells of Casein A, three wells of Casein B as well as two wells of presumed combined whey proteins. The PAGE gel is then hooked up to an electric current and the electromagnetic charge is dispersed through the aqueous liquid.

After the liquid was entered into the wells, there was an hour-long incubation period. After the incubation period, the gel was removed from the aqueous solution and the entire gel was diluted again three times with water and left in water and in the refrigerator overnight.

After 24 hours of incubation, the process of identification began. The identification of the proteins was the purpose of the experiment and the most important in insuring the accurate identification of the proteins. The bands were excised from the gel plate after the gel was taken out of the aqueous solution. Four bands from the Casein A, Casein B, and whey proteins each were excised and diluted. They were first diluted with 500 microliters of distilled water two consecutive times. They were then diluted with 500 microliters of an organic substance twice. A solution that tagged the protein was then added to the epindorf tubes containing liquid protein. The liquid was then added via the 100 microliter pipette to a metal plate that was then put in a temperaturecontrolled chamber of a large computer. The mass spectrometer ionizes the solutions by causing them to lose an electron. The MSDS then separates the ions based on their charge and electricity and then, after approximately 10 minutes, the laser was fired at the center of each substance on the plate and the bar graphs based on the weight/charge which shown on the monitor after approximately sixty seconds of firing the laser.

Once the machine cooled and the bar graphs were shown based on the separation of the ions due to their size, the MSDS sent the information to a separate computer. The computer then analyzed the data of the nine wells of protein that were analyzed via the MSDS. Because all proteins are so similar in molecular weight the computer that calculated the results was precise to the nearest hundred thousandth of a decimal. The proteins found in the Casein A wells showed the highest concentration of proteins. These proteins included the presence of casein, alpha globulin, and beta globulin.

The most noteable proteins include keratin, a protein found in hair and skin (thus proving the presence of contamination), beta casein precursor, kappa casein precursor, pregnancy-associated glycoprotein and actingrelated protein Blactaglobulin precursor, as well as leukemia inhibitory factor precursor were found in the presumed

Casein A protein samples. The Casein B samples included proteins such as alpha S1 casein precursor, kappa casein precursor, neurofibromin, zinc finger protein, alpha lactaglobulin precursor, and Aden late kinase isoenzyme. The presumed whey protein samples yielded proteins such as hemoglobin beta chain alpha S1 casein precursor, thyroid hormone-induced hepaticprotein, and DNA replication licensing factor MCM 8.

A side experiment was conducted to question the presence of certain bacteria in Grade A and Grade B unpasteurized milk. The two samples were streaked in a fume hood on regular sugar-based agar. The milk was then incubated for 48 hours in a temperature-controlled area. The temperature was approximately 37Ëš C. After the incubation period, the colonies in Grade A and Grade B were compared with the assistance of a bacteriology specialist. Small size-specific colonies were found in large numbers in the Grade A sample. Fewer numbers of colonies but large size-specific colonies were found to exist in the Grade B samples. The most diverse amount of bacteria colonies (decided based on texture, sizea nd height) were used from both Grade A and Grade B. These specific colonies were re-streaked on a plate of MacConkey agar, which tests for gram-negative bacteria. They were re-streaked using a fume hood once again. After streaking, the new plates were incubated for another 48 hours. Only one colony tested positive for gram-negative bacteria. The gram-negative bacterium remains to be identified positively. Two other colonies, which did not test positive for gram-negative, were positively identified as Lactobacillus (Grade B) and Staphylococcus (Grade A).

Conclusion The proteins that existed within the specific grades of milk (both A and B) were relative to the assumptions we made involving the presence of casein, alpha globulin, and beta globulin. These proteins showed no alarming results or proteins that are dangerous or not expected to be present in milk. The results satisfied the question of whether the proteins that exist in milk are safe for consumption and exist in quantities safe for the consumer.

The specific bacteria identified were Lactobacillus and Staphylococcus. Staph is a bacterium that, as a resistant strain, has become overwhelmingly rampant in many hospitals and was shown to appear in grade unpasteurized milk. Lactobacillus also appeared in Grade A unpasteurized milk and is especially disconcerting because this is a bacterium that is directly linked to miscarriage in expectant mothers. The gram-negative unidentified bacterium was also disconcerting because a gram-negative strain of bacteria is more resistant to antibiotics.

The conclusions that were made based on the data and results was the presence of uncertain bacteria and proteins and the overall verifications of the safety of our dairy system and producers.

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Purification of Thermosensitive Oligo(lysine)-b-Elastin-like Polypeptides

by Jon Seaton The purification of K8-ELP(1-60) was noted to have much lower yields than other elastin-like polypeptides. The addition of lysine residues to ELP(1-60) resulted in an increase of the native ELP phase transition temperature (Tt) from 51˚ C to 63˚ C; moreover, yields of the recombinant block copolymer were significantly lower possibly due to the added cationic block and/or the resulting higher Tt. To elucidate this purification dilemma, modifications to the traditional inverse transition cycling (ITC) method were completed by analyzing the effect of differing salts, buffers, and polymer concentrations. It was hypothesized that the addition of salts following the Hofmeister series would lower the Tt and increase product yield. However, E. coli expression levels of K8-ELP(1-60) may also contribute to poor yields.

Introduction ancer is characterized by the uncontrollable growth of cells that threaten normal body function by damaging tissues and organs. It is estimated that more than 500,000 people in the United States die each year from cancer (ACS). There is currently no definitive cure for cancer, but there are many treatment options; however, treatment is often difficult because tumors are often slow to pronounce themselves and quick to grow out of control in the body. Another difficulty is that each cancerous tumor is specific to each person and will often react differently to various treatments. Scientists involved in cancer research have been trying to develop new treatment methods to make treatments of this disease more efficient.

Chemotherapy is the treatment of disease by use of anticancer or chemical substances capable of killing cancerous cells. In this treatment, a patient is administered a toxic chemical, which attacks and kills cells in the body, both healthy and cancerous. If the cancer has been detected relatively soon, then this method seems to bring promising results. However, chemotherapy is not an efficient treatment of cancer because it is not capable of targeting specific cancerous cells. Patients will often feel extremely ill when on chemotherapy because it is so harmful to the body.

Chemotherapy has even been known to cause death (Ray-Coquard). In order to make chemotherapy a more effective way of treating cancer, it must be engineered to deliver large amounts of toxin directly to the cancerous tumor and not to any other part of the body.

The ELP system has been designed to use thermosensitive biopolymers in aiding the delivery of chemotherapy drugs to cancerous tumors. ELPs or elastin-like polypeptides are polymers with the pentapeptide repeat VPGXG, X being any amino acid except proline (Gray). There are several advantages to using ELP for drug delivery. First, ELP purification is more cost effective than many other protein purification methods (Banki). The molecular weight of ELP can be

C

engineered to maximize the enhanced permeability and attention effect in tumors (Furgeson) and ELP has the ability to aggregate at temperatures above its Tt. The aggregation of ELP occurs because of the hydrophobic interactions that are driven by the positive entropies of dehydration in formation of the peptide bonds and the changes in the hydrogen bond dynamics in water and the polypeptide (Nicolini). If the Tt decreases, then the hydrophobicity of the ELP will increase; consequently, if the Tt increases, then the hydrophobicity will decrease (Urry). It can be thermodynamically quantified as the point in which Gibb’s free energy is zero because the available energy used for polymer folding is at a dynamic equilibrium. Therefore, the polymer will spontaneously fold at the point , where Tt is directly proportional to the change in enthalpy and inversely proportional to the change in entropy. Since the changes of enthalpy and entropy for ELP is temperature independent, the ratio can be adjusted to yield a transition temperature around human body temperature. In this way, through the application of hyperthermia, ELP and chemotherapy conjugates will become insoluble in the blood at temperatures >Tt and accumulate in the targeted tumor. An ELP-Dox conjugate has already been proven an effective means of drug delivery (Furgeson).

In this study, the purification of a novel ELP diblock copolymers, K8-ELP(1-60), was analyzed. K8-ELP(1-60) consists of 60 pentapeptide repeats (VPGXG), where X = V5:A2:G3, and eight lysine residues on the Nterminus. The addition of lysine to elastin-like polypeptides will increase its transition temperature (Urry). Under standard conditions the amino groups on lysine will be protonated, creating positive charges and preventing intermolecular attraction. Therefore, an effective method was needed to lower the transition temperature to improve the yield of K8-ELP(1-60).

Materials/Procedure Prior to this study, DNA coding for the expression of K8-ELP(1-60) was inserted into the pET-25b expression vector through Mini and Maxi preps, transcribed into

Jon Seaton Jon Seaton graduated from Edgewood High School in 2007 and is attending the University of Wisconsin-Madison where he is pursuing a degree in Applied Mathematics, Engineering, and Physics and Biomedical Engineering. After college he plans to go to medical school and earn a PhD/MD in Medical Physics and Radiology.

He hopes to complete residency at Johns Hopkins University Hospital.

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E. coli, and grown on agar plates supplemented with ampicillin. K8-ELP(1-60) was then purified through ITC and sequenced. The experiment in question involved the use of this prepared E. coli for expression of K8-ELP(1-60). E. coli was incubated and shaken at 37˚ C in 1L cultures of CircleGrowTM medium for 12 hours. Cultures were centrifuged at 3000 rpm for 20 minutes to collect E. coli. The supernatant was discarded and the E. coli resuspended in 30 mL 1X PBS buffer. In some samples, resuspending in 15 mL buffer solution and combining two cultures doubled the concentration of E. coli. 10X PBS was also used to determine the effect of its counter-ions on K8-ELP(1-60) yields. Experiments also involved resuspending E. coli in Tris buffer (50 mM Tris, 100 mM NaCl, and 20 mM calcium chloride or magnesium chloride adjusted to a pH of 7.0 with HCl). Samples were kept on ice and sonicated twice for cycles of 7 minutes, 10 seconds on and 20 seconds off. Samples were balanced, placed in high-speed centrifuge tubes, and centrifuged for 15 minutes at 16500 rpm and 4˚ C. The supernatant was transferred to a new centrifuge tube and the insoluble pellet discarded. Two milliliters of a 10% solution of a cationic, synthetic polymer (PEI – polyethylenimine) was added to the supernatant to remove DNA from the sample and centrifuged at 16500 rpm and 4˚ C for 15 minutes. The supernatant was transferred to another clean centrifuge tube and the pellet discarded. At this point, different salts of varying concentration were added to the sample to lower the Tt and encourage aggregation: NaCl, MgCl2, and CaCl2 were added to different samples in both 2M and 4M concentrations. The samples were then centrifuged at 37˚C at 16500 rpm for 15 minutes. The supernatant was discarded and the insoluble pellet resuspended in 3 mL 1X PBS. The samples were transferred to 1.5 mL eppendorf tubes and centrifuged at 4˚C and 13000 rpm for 7 minutes to remove any remaining cell debris. The supernatant was transferred to a clean eppendorf tube and the pellet discarded. 400μL of 10% PEI was added and the samples were centrifuged again for 7 minutes at 4˚ C and 13000 rpm. The supernatant was transferred to a new eppendorf tube and the pellet discarded. Salts were then added as before to the corresponding samples in the same concentrations to lower the Tt. The samples were then centrifuged at 37˚ C and 13000 rpm for 7 minutes to pellet the K8-ELP(1-60). The pellet was resuspended in 400 μL 1X PBS. Ultraviolet-visible spectroscopy was used to determine the concentration of K8-ELP(1-60) purified using Beer’s Law. Thermo-profiling was also preformed using ultraviolet-visible spectroscopy by slowly increasing the temperature of each sample and recording their absorbance.

Results/Discussion In this study, the effects of the concentration of E. coli, Tris and PBS buffers, NaCl, KCl, MgCl2, and CaCl2 on the yield of K8-ELP(1-60) were quantified.

Table 1 shows that the best method found for purifying

K8-ELP(1-60) was by 1X PBS and 4M NaCl. It was noticed that through this method, K8-ELP(1-60) would aggregate at room temperature. However, the data also illustrate a large variance in yields, which can be contributed to a number of problems that exist in purification of K8-ELP(1-60). First, the growth of E. coli in each culture was very different. The amount of E. coli in each culture was noticed to be within 5 grams. The inconsistency in the amounts of E. coli in different samples would therefore yield different amounts of product. Secondly, the efficiency and consistency of lysing E. coli by sonication was highly variable. A previous study demonstrated that the effectiveness of sonication is dependent on sample volume, acoustic power, cellular concentration, and ionic strength (Feliu).

In each sample, volume, cellular concentration, and ionic strength differed meaning the release of K8-ELP(160) from the E. coli was an uncontrolled variable. ELPs typically have yields of around 85 mg/L (Banki), but Table 1 suggests that the yields may be influenced by the ability of E. coli to express K8-ELP(1-60).

Method of Purification 1x PBS + 1x E.coli – 4M NaCl 1x PBS + 1x E.coli – 2M NaCl 1x PBS + 1x E.coli – 2M CaCl2 1x PBS + 1x E.coli – 2M MgCl2 1x PBS + 1x E.coli – 2M KCl 1x PBS + 1x E.coli – 4M NaCl 1x PBS + 2x E.coli – 4M NaCl 10x PBS + 1x E.coli – 4M NaCl 10x PBS + 2x E.coli – 4M NaCl Tris + CaCl2 – 4M NaCl Tris + MgCl2 – 4M NaCl Tris + CaCl2 – 4M KCl Tris + MgCl2 – 4M KCl

K8-ELP(1-60) 17.2 mg/L 9.32 mg/L 6.08 mg/L 0.72 mg/L 9.32 mg/L 17.9 mg/L 14.2 mg/L 6.43 mg/L 13.5 mg/L 3.58 mg/L 8.59 mg/L 11.3 mg/L 5.37 mg/L

Table 1. Yields of K8-ELP(1-60) per liter culture of E. coli calculated by ultraviolet-visible spectroscopy and Beer’s Law. Initially, it was thought that using salts with large ions such as calcium chloride and magnesium chloride would depress the Tt of K8-ELP(1-60) even more than monovalent salts because they would increase the change of entropy in the system, which would lower the transition temperature. It was hypothesized that by adding salts with larger entropies more disorder would exist in the water, increasing the hydrophobic effect, and encouraging protein folding. Thermal-profiling of different salts proved that the opposite was true. Figure 1 shows that salts following the Hofmeister series of increasing hydrophobic interaction decreased the Tt of K8-ELP(1-60). This verifies that the aggregation of ELP is both a thermo-mechanical and chemo-mechanical process (Urry). The yields in Table 1 follow a direct correlation to the thermal-profile. Therefore, the yield of ELP purified through ITC is dependent on the induced Tt by aqueous salts.

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REFERENCES American Cancer Society. Cancer Facts and Figures 20072008. Atlanta: American Cancer Society, 2007.

Banki, Mahmoud Reza, Wood, David W., “Inteins and affinity resin substitutes for protein purification and scale up.” Microbial Cell Factory (2005) 4:32.

Feliu, J.X., Cubarsi, R., Villaverde, A. “Optimized release of recombinant proteins by ultrasonication of E. coli cells.”

Biotechnological Bioengineering (1998) 58:536–540.

Furgeson, Darin Y., Dreher, Matthew R., Chilkoti, Ashutosh. “Structural optimization of a ‘smart’ doxorubicinpolypeptide conjugate for thermally targeted delivery to solid tumors.” Journal of Controlled Release (2006) 110:362-369.

Gray, W.R., Sandberg, L.B., Foster, J.A., “Molecular model for elastin structure and function.” Nature (1973) 246:461466.

Figure 1. Thermo-profiling of K8-ELP(1-60) using monovalent (NaCl) and divalent (MgCl2, CaCl2) salts.

Conclusion From this study, it was concluded that purification yields of K8-ELP(1-60) were highest for 1X PBS and 4M NaCl. Modifying the ITC purification method did not significantly increase K8-ELP(1-60) yields.

Therefore, further study must be conducted to determine why K8-ELP(1-60) cannot be purified with similar yields as other elastin-like polypeptides. The differing factor between K8-ELP(1-60) and other ELPs is an octalysine block, which suggests the presence of a repressor in E. coli that limits the transcription and translation of the cationic block copolymer. Further studies must be conducted in order to determine the presence of this repressor and methods to increase yields of K8-ELP(1-60).

Nicolini, C., et. al. “Characterization of the temperatureand pressure-induced inverse and reentrant transition of the minimum elastin-like polypeptide GVG(VPGVG) by DSC, PPC, CD, and FT-IR spectroscopy.” Biophysical Journal (2004) 83:1385-1392.

Ray-Coquard, I. et. al. “Identification of patients at risk for early death after conventional chemotherapy in solid tumours and lymphomas.” British Journal of Cancer (2001) 85:816-822.

Urry, Dan W. “Physical chemistry of biological free energy transduction as demonstrated by elastic protein-based polymers.” Journal of Physical Chemistry B (1997) 101:11007-11028.

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The Cloning of SNR7 into Saccharomyces cerevisiae

by Lydia Lovell

Lydia Lovell Lydia Lovell graduated from Edgewood High School in 2007. She is currently attending Hanover College and is double-majoring in biology and Spanish, with an emphasis on pre-medicine.

Her mentor, Jonathan Finkel, is a graduate student at the University of Wisconsin-Madison in the Culbertson Labs, and is currently working toward his PhD.

Saccharomyces cerevisiae, or baker’s yeast, has many unique features such as short doubling time, ability to form as a haploid or diploid, and simple life cycle, that have made it a valuable model organism. The goal of research was to use the methods and information about yeast learned to assist in transferring the SNR7 gene into yeast, through such techniques as polymerase chain reaction (PCR), gel electrophoresis, Miniprep extraction of E.coli DNA and transformation of DNA into E.coli and yeast. To accomplish this, the gene SNR7 was amplified via PCR and then inserted into a vector containing tracking markers URA3 and AMPr. The vector plus SNR7 was transformed into E.coli via electroporation to create multiple copies in a short time. A Miniprep was conducted to break open the cells and filter all components of the cell excluding the DNA. Gel electrophoresis was used to determine if the vector plus SNR7 had been isolated, and the DNA was sequenced to ensure that the vector and SNR7 had been properly ligated, both of which were confirmed. The cells were then transformed into yeast via a yeast chemical transformation. The clone is currently being studied for its effects on the yeast.

Background accharomyces cerevisiae, commonly referred to as baker’s yeast, is a single-cell eukaryotic organism. It has a simple life cycle, that can be in either the haploid or diploid phase. As well, it has a very short doubling time, about 2 hours. In yeast, cellular division occurs when the mother cell produces a bud, which after cytokinesis produces a daughter cell (Fig. 1). Due to these unique features, yeast have Figure 1. Mother and daughter cell become a valuable model organism. The structure and function of the yeast cells, as well as their genomic similarity to humans has important implications in studying human biology.

One advantage specific to using S. cerevisiae in research is in its ability to grow as a haploid or diploid: haploid meaning a cell containing one set of chromosomes, similar to bacteria, whereas diploid is a cell with two sets of chromosomes, as seen in humans. Thus, the spectrum in which S. cerevisiae can be used for research is doubled, as one can first identify functions of recessive mutations in the haploid cells. Another benefit to yeast’s ability to grow as either haploid or diploid is that two haploids with separate mutations can be mated, and through chromosomal recombination one set of chromosomes can obtain both mutations, which, once the cells are reduced back to haploid cells, provide for a cell with both mutations. This method for the creation of double mutants is easier, faster, and more efficient than most methods. Another important feature of S. cerevisiae is its rapid doubling time allows for larger populations of cells to be analyzed in less time than any other eukaryotic organism, (“as many as 108 yeast cells in 1.0 ml of culture” (Hartwell 639)).

S

Figure 2. Structure of yeast chromosome The structure of S. cerevisiae chromosomes consists of 16 linear chromosomes, with “long repeats” consistent of 30-40 kilobases, a centromere, multiple origins of replication, and subtelomeric repeats and telomeres at each end (Fig. 2). These are then packaged into nucleosomes. To view these chromosomes, one conducts pulsed-field gel electrophoresis, the process which separates intact yeast chromosomes. Through this, one can confirm the yeast chromosome and witness any changes in its structure. For S. cerevisiae the genome is sequenced and annotated (1996), with approximately 6000 open reading frames. The annotated genome has been converted into a website that provides a great tool to assist with yeast research. With this information as well as current research, the ability to mutate and observe the effects of mutations on S. cerevisiae has become much more efficient.

Goals The goal of research was to use the methods and information about yeast learned in a lab setting to assist in transferring the SNR7 gene into yeast. The specific goal was to learn both the background and methods of laboratory techniques such as polymerase chain reaction, gel electrophoresis, Miniprep extraction of E.coli DNA and transformation of DNA into E.coli and yeast. To accomplish this, the gene SNR7 was amplified, then inserted into a vector, and then the vector plus SNR7 was transformed into yeast. The clone is currently being studied for its effects on the yeast.

THE CLONING OF SNR7 INTO SACCHAROMYCES CEREVISIAE

Methods The cloning of the SNR7 gene and transformation into yeast involved multiple steps. The experiment began by performing a polymerase chain reaction (PCR). PCR is a process by which enzymes amplify the DNA, specifically the SNR7 gene. This PCR allowed for a large quantity of the SNR7 DNA to work with.

With the SNR7 gene amplified, a vessel was needed to transport it, first into E.coli and then into yeast. In cloning, this vessel is called a vector. The vector chosen in this experiment had two markers, which are genes that allow one to identify the presence of a vector in a cell. The markers used were AMPr, the gene that confers resistance to ampicillin, and URA3, the genes that allows for yeast to grow in media lacking uracil (Fig. 3). To place the SNR7 gene into the vector, both the vector and SNR7 needed to be digested.

Digestion involves using Figure 3. Vector with enzymes to remove a small URA3 and AMPr tags piece from each side of SNR7 and to cut the vector. In this experiment, SacII was used in the 5’ end, and BamHI in the 3’ end of the SNR7 gene; these enzymes were also used on corresponding spots in the vector. These are very specific enzymes which make “sticky ends;” this creates specificity allowing for the SNR7 gene to be inserted in the correct orientation when the vector and SNR7 are put together. With both the vector and SNR7 digested, the two pieces were ligated. Ligation is the process by which one uses a “ligase” to ligate or bond these “sticky ends” of the SNR7 gene to the “sticky ends” of the desired vector (Fig. 4).

Figure 4: Ligation of SNR7 into vector With the vector and the SNR7 gene ligated, it then had to be transformed into E.coli. This bacterium is used because it allows for the creation of multiple copies of the vector plus SNR7 to be created in a short time. To transform the vector and SNR7 into the E.coli, we conducted electroporation. Electroporation is the process that shocks the cells and thus opens the cell’s channels, allowing for intake of the vector plus SNR7. After the transformation, since the vector had an AMPr marker, we were able to screen for the specific E.coli that contained our vector plus SNR7, since only those cells that have our vector plus SNR7 will grow in its presence of ampicillin. Three colonies were picked and then

31

placed and grown in media containing ampicillin.

The three colonies were grown to saturation. Then the samples were “miniprepped.” A Miniprep breaks open the cells and filters out all components of the cell except for the DNA. This allowed for isolation of a large quantity of the desired vector plus SNR7.

To ensure that we had isolated our vector plus SNR7, gel electrophoresis was performed. Gel electrophoresis is the process where an agarose gel is created and used to separate and track the DNA, with a negative and positive side. DNA is placed in wells on the positive side; since DNA is negatively charged, Figure 5: Results from gel when a current is run, electrophoresis the DNA will be attracted to the negative end and will stream that way. As can be seen in Figure 5, we were able to isolate three samples of DNA. Then to ensure that the vector and SNR7 was properly ligated, the DNA was sequenced. When the DNA was sequenced, it confirmed both the presence of the vector and the SNR7 gene in the correct orientation.

With the vector and SNR7 confirmed, the vector plus SNR7 needed to get into the yeast. This was done via a yeast chemical transformation. A transformation stresses the yeast cells through heat and chemicals, allowing the yeast to uptake the vector plus SNR7. The cells were then screened on media lacking uracil, with only those cells which contain our vector plus SNR7 able to grow.

The new yeast strain is currently being analyzed in the lab.

REFERENCES Hartwell, Leland, et al.

Genetics: From Genes to Genomes. Saccharomyces Cerevisiae: A Genetic Portrait of Yeast. The McGraw-Hill Companies, Inc. 2000.

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Mushroom Survey at Five Different Sites, Testing Relative Humidity, Soil Composition, Amount of Sunlight, and Number of Mushroom in St. Croix State Park

by Peter Guerin and Noah Kachelski Peter Guerin

Noah Kachelski

Peter and Noah are currently seniors at Edgewood High School.

The purpose was to do a survey of five different habitats and record at each site the amount of sunlight, the relative humidity, the amount of plant litter, temperature, and the soil composition and then analyze the data and see what seemed to be the best habitat for the mushrooms surveyed. It was found that there is a direct correlation between the amount of sunlight and number of mushrooms; i.e. the less sunlight, the more mushrooms. However, this was not followed on the riverbank, where there were a great deal of mushrooms and sunlight together. We believe this to be so because of the great amount of water on the river bank that makes this area a good place for mushroom growth.. Based on the number of different species and the number of different sites in each area, the coniferous forest has a more diverse population compared to the deciduous forest. Ultimately all the different mushrooms have too many individual factors and have individual preferences so we couldn’t determine one place that would be the best habitat for optimal growth.

M

ushrooms are part of the Fungi Kingdom, which means in some ways they are like plants and in other ways they are completely different. They grow and reproduce similar to plants, but lack stems, leaves, chlorophyll and other parts. Mushrooms obtain food through mycelia, filaments coming off the bottom of the mushroom, which soak up nutrients from the soil, decaying wood, or other plant material. The top of the mushroom, the stalk and cap, are only the reproductive part of a much larger organism that lives mostly underground and can extend many feet in all directions. The reproductive part of the mushroom sends off spores to reproduce, which allow the mushroom to move from place to place and survive.

Mushrooms generally have relationships with trees, whether dead or alive, so they are usually found in forests or near trees of some sort. But due to the great variety of species and the habitats and environments they prefer, there are species that shun trees and grow in grasslands and open areas and fields and there are mushrooms that grow best in swamps and bogs. “An estimated 6,000 common higher fungi in North America” alone means that basically there are so many different mushrooms that they will grow anywhere and everywhere (Miller & Miller).

Our goal was to do a survey of mushrooms in St. Croix National Park and test the relative humidity, amount of sunlight, plant litter, soil composition, and temperature.

There didn’t seem to be any correlation between relative humidity and anything else, the results seemed to be completely random depending on the weather.

The only noticeable correlation was that the relative humidity was higher when we were nearer to water, 83% in the border to the wetlands site, which was around 10% higher than the next highest site. The temperature of the areas within each site only depended

Key to chart and graphs A: Mesic Hardwood/Deciduous Forest B: Border to the Wetland C: Pine Chapel/Forest D: River Bank E: Clear Cut/Open Area Mini Site Within the Site (Averages)

A

B

C

D

E

Wet Bulb (˚F)

52.6

55.1

51.4

64.9

64.1

Dry Bulb (˚F)

58.3

58.1

57.0

71.8

70.4

Relative Humidity

0.68

0.83

0.67

0.72

0.72

Amount of sunlight (%)

30.3

36.0

15.7

28.5

68.5

Temperature (˚F)

58.3

58.1

57.2

71.8

70.5

Number of Mushrooms

62.8

28.2

27.1

84.4

19.2

Total Time

185

150

180

240

180

on the time of day that we were collecting data; the temperature of each site didn’t seem to be related to the number of mushrooms or species present. We believe this is because there would need to be a long-term

MUSHROOM SURVEY AT FIVE DIFFERENT SITES IN ST. CROIX STATE PARK

33

In the coniferous forest there are only 11 sites of mushrooms, while at the same time there are eight different species. On the other hand, in the border to the wetland there were 20 sites and only eight different species. Based on this, the coniferous forest is a more diverse area for its population than the border to the wetland.

The coniferous forest is followed closely by the river bank compared to the least diverse, the border to the wetland.

temperature streak to have any impact on mushroom population. In order to study temperature’s effect, we would need a longer observation period. There was no factual link between soil composition and better or worse growth. Many of the mushrooms found were either on decaying logs or living trees. This, however, could be because it is far easier to spot mushrooms on trees rather than the ground. Based on the data there is a direct correlation between the amount of sunlight and the number of mushrooms. Generally the more sunlight, the smaller number of mushrooms and reversely, the less sunlight, the more mushrooms, mushrooms prefer cool areas over warm areas. This pattern is not followed on the riverbank, there is a great amount of sunlight, on average 72% of the sky was open and yet there were still about 85 mushrooms per site in the area. So there were many mushrooms even though there was plenty of sunlight. We believed that this was due to the fact that it is on a riverbank, a source of water, so this water might counter the amount of sunlight, making it possible for the mushrooms to grow. Our earlier investigation showed that the relative humidity is higher there, so that combined with the moist ground from the river would make it a preferable spot for mushrooms to grow and reproduce.

Conclusion Each species of mushroom has an individual preference for its best growing conditions; finding out a direct answer to our question became a complex maze that we couldn’t completely untangle. But we did determine that sunlight directly affects mushroom growth. Generally, the trend was the more sunlight, the fewer mushrooms. On the river bank, however, there are plentiful mushrooms and sunshine. There are many mushrooms in the river bank even though there is a great deal of sunlight. The coniferous forest has a small population—only 11 sites were found—but out of these sites, eight of them were different species. This means that the coniferous forest has good diversity. On the other hand, at the border to the wetlands we found many sites, but relatively few different species. Temperature did not seem to make a difference in population. Relative humidity, along with temperature, did not seem to have an affect on mushroom growth.

REFERENCES 1. McKnight, Kent H. A Field Guide to Mushrooms of North America. 1987, Houghton Mifflin Company, 215 Park Avenue South, New York, New York.

2. National Audubon Society. A Field Guide to Mushrooms. 1981, Chanticleer Press, Inc. New York.

3. Dr. Miller, Orson K., and Hope H. Miller.

North American Mushrooms: A Field Guide to Edible and Inedible Fungi. 2006, Morris Book Publishing, LLC. 4. Mollen, Cora and Larry Weber. Fascinating Fungi of the North Woods. 2007, Service Printers, Duluth, Minnesota.

5. Arora, David.

Mushrooms Demystified.

1986, Ten Speed Press, Berkeley California.

6. Phillips, Roger.

Mushrooms and Other Fungi of North America.

2005, Firefly Books Ltd. Ellcott Station Buffalo, New York

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Survey of Mussel Middens Found Along the St. Croix and Kettle Rivers

by Bethany Walker and Lindsay Fulton

Bethany Walker

Lindsay Fulton

Bethany Walker graduated Edgewood High School in 2009. In college, she plans on majoring in Environmental science.

Bethany would like to thank Mekel WieierholtMeier and Toben Lafrancois for all of their help and support. They made this research experience positive and very worthwhile. She would also like to extend her thanks to Cindy Lee who was a great help while out in the field.

Lindsay Fulton is currently a senior at Edgewood High School.

Mussel middens were surveyed along the banks of the St. Croix and Kettle River in St. Croix State Park in spring, 2008. More middens were found along the banks of the St. Croix River than the Kettle River indicating that the St. Croix is a better habitat for muskrats. The most common species of mussels found at both sites were the Mucket (Actinonaias ligamentina) and Fat Mucket (Lampsilis siliquoidea), at 29.95% and 23.05% on the St. Croix and 8.42% and 21.98% on the Kettle River, respectively. Also common at both sites was the Spike (Elliptio dilatata) species, at 21.88% on the St. Croix and 5.13% on the Kettle River. Mussels found on both rivers in small numbers were the Round Pigtoe (Pleurobema coccineum) and Pink Heelsplitter (Potamilus alatus), at 2.73% and 0.91% on the St. Croix and 0.73% and 0.35% on the Kettle River. Mussels found only on the St. Croix River were the Wabash Pigtoe (Fusconaia flava), Pimpleback (Quadrula pustulosa pustulosa), Giant Floater (Pyganodon grandis), and Hickorynut (Obovaria olivaria). Middens along the St. Croix indicate a more diverse mussel population than the Kettle River.

F

or the past 2000 years, mussels have been a part of streams all over the United States. From previous mussel midden studies, archeologists have determined that mussels played a key role in the survival of indigenous tribes. Today, 213 of 297 mussel species are endangered, threatened, or of special concern (Freshwater Mussel Fact Sheet). This is a result of construction around riverways, the building of dams, and the common use of mussel shells to make buttons (The Conservation and Management of Freshwater Mussels II: Initiatives for the Future). Mussel shells found in middens along the banks of the St. Croix and Kettle Rivers were identified and counted. Middens are piles of mussel shells found along the banks of rivers.

Muskrats that prey on freshwater mussels will gather into middens when they find a mussel, break it open and leave the shells on the bank. Muskrats are known to live close to the banks of rivers and often build their homes into the banks. They do not like to travel far from their home, normally no more than 50 feet, to hunt and therefore middens are found near muskrat habitats (Kurta). By using the middens instead of taking the mussels out of their river homes, the mussels are not endangered and the information gathered can be used by park services on both a local and national level. Once a midden has been found, the types and amount of shells found can be identified and recorded. By taking a survey of mussel middens in the St. Croix River and Kettle River, the species and density of mussels present in these rivers can be observed. Scientists and researchers can use these middens to estimate the mussel population in the river they are found by. Midden studies are important because they give park services an understanding of what kinds of mussels can be found as well as approximate locations of these mussels. A proportion of the mussels found can also be prepared and used to show which species are denser in specific locations. The St. Croix Riverway is home to approximately 40 different species of mussels. Of these 40, 21 are classified as endangered, threatened, or of special concern. Using the results from

this survey of middens, the park service can be more aware of which mussel populations are growing and which are diminishing. It was hypothesized that if middens are observed on the Kettle and St. Croix River, the St. Croix will have a greater variety of species in a greater density than the Kettle River.

The St. Croix is home to more mussel types in a greater density than the Kettle River. Three sites in total were found along the Kettle River banks. Two of the sites found along the Kettle were old middens. All of the shells had been bleached and many of them had been crushed. Because of this, the shells could not be identified and could not be counted accurately. The halves of the shells that were still together were counted and recorded. The midden found at Kettle River Site 2 contained approximately 118 halves. The midden found at Kettle River Site 3 contained approximately 55 halves.

The last midden, Kettle River Site 1, was classified as a depositional area. It contained 100 halves that were five different species. The five species were Spike (Elliptio dilatata), Mucket (Actinonaias ligamentina), Round Pigote (Pleurobema coccineum), Pink Heelsplitter (Potamilus alatus), and Fat Mucket (Lampsilis siliquoidea). The most abundant species was Fat Mucket, 60%. The least abundant species was Pink Heelsplitter, 10%.

There were 19 middens found along the banks of the St. Croix. When the data from all nineteen sites along the St. Croix River is combined, the three most common are Spike (Elliptio dilatata), 21.73%, Mucket (Actinonaias ligamentina), 29.75%, and Fat Mucket (Lampsilis siliquoidea), 22.85%. When looking at the same combined data, the three least common are Pink Heelsplitter (Potamilus alatus), 0.91%, Hickorynut (Obovaria olivaria), 0.13%, and Giant Floater (Pyganodon grandis), 0.13%.

When data from the Kettle River is compared to data from the St. Croix, the two most common species of mussels found were the Mucket (Actinonaias ligamentina) and Fat Mucket (Lampsilis siliquoidea), at

THE EFFECTS OF DEPRESSION AND ANXIETY ON CARDIOVASCULAR DISEASES BACKGROUND RESEARCH

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Table 1. Kettle River: Summary of Mussel Half-Shells Found Site

Mucket Fat Mucket

Spike

Round Pigtoe

Pink Heelsplitter

Unidentified

1

23

60

14

2

1

0

2

0

0

0

0

0

118

3

0

0

0

0

0

55

Table 2. St. Croix River: Summary of Mussel Half-Shells Found Mucket

Fat Mucket

Spike

230

177

168

Round Pink Pigtoe Heelsplitter 21

7

Wabash Pigtoe

Three Ridge

87

34

29.95% and 23.05% on the St. Croix and 8.42% and 21.98% on the Kettle River, respectively. Also common at both sites was the Spike (Elliptio dilatata) species, at 21.88% on the St. Croix and 5.13% on the Kettle River.

Mussels found on both rivers in small numbers were the Round Pigtoe (Pleurobema coccineum) and Pink Heelsplitter (Potamilus alatus), at 2.73% and 0.91% on the St. Croix and 0.73% and 0.35% on the Kettle River.

All of the species found on both the Kettle River and St. Croix River are native to this area, explaining why the species were found in the middens. While none of the species found have a status of endangered, two have a status of threatened and one has a status of being a species of concern. Two of the three most common species found fall into this category. The Mucket (Actinonaias ligamentina) is one of the species that has a threatened status and Spike (Elliptio dilatata) has a status of species of concern. Even though these two species have a threatened or species of concern status, the fact that so many of these species were found is a positive sign. This shows that the population is staying steady, if not increasing. When comparing the middens found in the Kettle River to those found in the St. Croix River, five of the same species were found. This is a very positive sign for the Kettle River. Unlike the St. Croix River, the Kettle River is not protected on a federal level.

This shows that the mussel community is beginning to have to ability to survive in areas that are not federally protected.

While many of the same species were found on the St. Croix River than on the Kettle River, the St. Croix contained a greater variety of species in a greater density.

This is because of a few factors. One of these factors is muskrat habitats. Muskrats like to live close to slowmoving water and many times like to make bank dens.

(Kurta) The Kettle River is faster moving than the St. Croix. The Kettle also has many rapids throughout.

This habitat would not be appealing to a muskrat. Thus explaining why there were very few middens along the Kettle River. The St. Croix River is slower moving and

Pimple- Hickory- Giant Unidentified back nut Floater 19

1

1

28

does not have rapids like the Kettle. This would be an appealing habitat for muskrats. More muskrats living by the river would provide more middens in the area.

“Muskrats also don’t like to travel far from their homes to find food. The normal distance of travel is no more than 50 feet” (Kurta). On the Kettle River, the banks are often raised and are not close to the water. This situation would require the muskrats to travel greater distances to obtain mussels. Since muskrats prefer to stay close to home, they would most likely not choose the Kettle River as their home. This explains why there were few middens along the banks of the Kettle River. The banks of the St. Croix are very different from the Kettle River.

They are not raised and in many spots there is only grass along the banks. This makes the river very accessible to muskrats. The grassy area also makes an easy spot for muskrats to create their home, either out of sticks and mud or burrowing into the ground. These conditions are ideal for muskrats. Therefore, middens would be more common along the St. Croix.

Another factor would be the distribution of mussels.

When mussels are ready to produce, the females produce mantle lures that are filled with glochidia. When a fish strikes at the mantle lure, the glochidia are released and attach to the fish. The glochidia stay attached to the fish until they have obtained the nutrients they need to survive (Freshwater Mussel Fact Sheet). Because of this, mussels are most commonly found in areas where the host fish travel. In the Kettle River, it is harder for fish to travel great distances on the river due to fast moving water and the various rapids. In the St. Croix River, the host fish would have an easier time traveling throughout the river due to the slower water speeds and fewer rapids.

This gives more mussels the opportunity to settle in a larger portion of the river and provide food for the muskrats, ultimately creating more middens.

Weather created the most error while performing this survey. Due to severe weather, surveying taking place along the banks of the St. Croix River accessible by the backwaters behind the Head of Rapids Group Camp

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REFERENCES Cummings, Kevin S. and Christine A. Mayer. Field Guide to Freshwater Mussels of the Midwest. Illinois National History Survey.

Champaign, Illinois.

December 1992.

Freshwater Mussel Fact Sheet. National Park Service: U.S. Department of the Interior, 2007.

“Freshwater Mussels of the Midwest-Introduction." 10 Jan. 2002. Illinois Natural History Survey. 24 Feb. 2008. www.inhs.uiuc.edu/ cbd/musselmanual/ introduction.htm.

Kurta, Allen. “Muskrat.”

Mammals of the Great Lakes Region. University of Michigan. Michigan. 1995.

“Mussel Study." Water Tenders. 24 Feb. 2008. www.watertenders.org/ musselstudy.html.

Sietman, Bernard E. Field Guide to the Freshwater Mussels of Minnesota.

Department of Natural Resources. St. Paul, Minnesota. 2003.

“The Conservation and Management of Freshwater Mussels II: Initiatives for the Future.” Oct. 1995.

Upper Mississippi River Conservation Committee.

24 Feb. 2008 http://ellipse.inhs.uiuc.edu/ FMCS/Meetings/ Symp1995Abs.html #SAMPLE_ABS.

was not able to be completed. It was clear that middens could be found there, seeing that one of the sites was found in that area. When the St. Croix River was accessed by the backwaters at a later date, it was discovered that the middens had been flooded out. This was the case for other locations on both the Kettle River and the St. Croix River. This made it extremely hard to find middens. The banks of both rivers had been flooded and were covered with about two feet of water. Shells that had been washed away were extremely hard to find due to the deep water. The higher banks that hadn’t been flooded were also very hard to get to, making it almost impossible to find more middens and survey the areas.

Many of today’s mussel species are endangered or threatened. This makes it crucial for the populations to continue to be monitored and make sure that no species become extinct. The data collected in this survey can help monitor the population of mussel species. The park services, on both a national and local level, can use the results to gain a better understanding of specific mussel communities. The information gathered, specifically the species and the amount of shells, can be used to estimate

the mussel population in a specific area. This information can be used even more specifically to monitor individual species and their population. By monitoring the mussel population, species that are in danger of becoming extinct can hopefully be salvaged.

The hypothesis was supported by the results achieved in this survey. The St. Croix is contains a wider variety of species than the Kettle River and has a greater density of the species present. Seeing that the St. Croix is a federally protected riverway, the abundance of mussels that can be found there shows that the mussel population is staying steady. The Kettle River, which is not protected on any level, also has a great quantity of mussels. This shows that mussels are still able to produce and survive in a natural, uncontrolled environment. In general, both rivers are showing healthy mussel populations. This research topic was both educational for the students and beneficial to park services. Students were able to learn more about mussels and their lifestyle while providing important information that can help monitor population levels of freshwater mussels.

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The Effects of Electron Donating and Accepting Radicals on the Geometry of Tetraphenylethylene

by Samuel McLaughlin The optimized geometries and infrared vibrational frequencies of the tetraphenylethylene (TPE) molecule with –OCH3 and –CN cis substituents in the para positions on the phenyl rings as well as –CH3 groups at the meta position on all phenyl rings were calculated.

Results suggest that the presence of the –CN and –OCH3 substituents may confer additional stability to the TPE molecule in a transition state between its cis and trans isomers. Additionally, central C-C bond length in the TPE molecule was greatest with both substituents present and was greater than the additive increase in bond length conferred by the lone substituents.

Samuel McLaughlin Samuel McLaughlin graduated from Edgewood High School in 2009.

be detected in fluorenyl radicals (Bordwell and Lynch).

Introduction he captodative effect is the theoretical Additional work by Bordwell, Zhang, and Alnajjar found phenomenon where the addition of both an antagonistic, rather than synergistic, effect on electron donating and electron withdrawing homolytic C-H bond dissociation energies and by substituents to radicals increases the extension radical stabilization when working with stabilization of the radical to a greater extent carbon-centered radicals (Bordwell). It is clear, then, that than the additive stabilization conferred by the two the captodative effect is fickle at best, and its presence substituents in isolation. Whether or not the captodative varies greatly depending on the radical in question; effect actually exists or if it is merely the result of other however, work done by Leigh and Arnold highlighted factors has been difficult to pin down. Dewar first the tetraphenylethylene molecule as a promising suggested that, theoretically, the addition of electron candidate for future research on the captodative effect. donating and withdrawing substituents to radicals would Leigh and Arnold found decreased barriers to cis-trans increase overall radical stabilization (Dewar). The isomerization of the tetraphenylethylene molecule in intervening research has been divided as to whether the benzene solution with both –OCH3 and –CN para captodative effect actually exists. Viehe expanded substituents versus single –CH3, –OCH3, and –CN para Dewar’s theoretical work on the captodative effect by substitutents, indicating greater stability of the radical explaining that single substitutents stabilize radicals by transition state with the double substitution. increasing available resonance structures and that donor Additionally, they calculated the rotational barrier and acceptor substituents work synergistically because between cis and trans conformation for the TPE they are attracted to different regions of the radical and molecule to be 35.5 kcal mol-1 (Leigh). The work of do not interfere with each other as two identical Leigh and Arnold proved to be the genesis of a research substituents would. Additionally, working with benzylic project (of which this paper is an extension) that utilized carbon radicals with –CN and –OCH3 substituents, Gaussian to calculate the effects of the addition of Viehe found increases in β-H hyperfine splittings that various substituents to the TPE molecule, summarized indicated increased spin delocalization and therefore in Table 1 (Handorf ). increased stability that was Table 1 greater than the additive Energy Average Phenyl C-C bond stabilization of the (kcal/mol) Torsion Angle (degrees) length (Å) individual substituents (Viehe). Work done by 1a (o-Me, no para) -727649.9484 58.0035 1.36505 Bordwell and Lynch on 2a cis (o-Me, para OCH3) -871338.0776 56.702 1.36702 fluorenyl radicals found a 3a cis (o-Me, para CN) -843419.9586 57.27325 1.3658 slight increase in radical 4a cis (o-Me, both OCH3 and CN) -987108.6389 56.1615 1.36798 stabilization energies with 1c (m-Me, no para) -727660.278 50.1295 1.36619 the presence of both 2c cis (m-Me, para OCH3) -871348.7345 49.2365 1.36775 donating and withdrawing substituents, but this 3c cis (m-Me, para CN) -843431.3418 49.82025 1.36708 increased stabilization was 4c cis (m-Me, both OCH3 and CN) -987120.4909 48.80325 1.36928 within the range of experi1e (no Me, no para) -629038.3194 49.92825 1.36617 mental error and Bordwell 2e cis (no Me, para OCH3) -772726.6198 48.389 1.36842 and Lynch concluded 3e cis (no Me, para CN) -744807.911 49.70625 1.36696 quite emphatically that 4e cis (no Me, both OCH3 and CN) -888496.8723 48.153 1.37004 the captodative could not

T

EDGEWOOD HIGH SCHOOL STUDENT SCIENCE JOURNAL 2008-09

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Figure 1

Objectives This project examined the effects of electron donating and accepting radicals on the geometry of the TPE molecule as well as the additional effects of methyl groups in various substituted positions on the TPE molecule in an expansion of work done by previous researchers; research to date has focused on the d category of substituent combinations with work ongoing with the b category – Figure 1 (Handorf ) outlines the classifying scheme used for this research project. Finally, the ongoing aim of this project is to identify a stable transitional state of the TPE molecule between cis and trans isomers.

Research Methods Research focused on the computational analysis of the TPE molecule in the presence of various substituent groups. All calculations were performed with Gaussian03 and viewed with GaussView; the density functional theory used was Becke’s three-parameter exchange functional with the correlation functional of Lee, Yang, and Parr (B3LYP) with the 6-31G(d) basis set. Geometry optimizations of cis isomers of species 1-4d species have been completed as well as the 2b and 3b species; Table 2

1d (m-diMe) 2d cis (m-diMe and p-OCH3) 3d cis (m-diMe and p-CN) 4d cis (m-diMe, p-OCH3, and p-CN) Table 3

1d (m-diMe) 2d cis (m-diMe and p-OCH3) 3d cis (m-diMe and p-CN) 4d cis (m-diMe, p-OCH3, and p-CN)

resulting geometries were verified as minima via harmonic frequency calculations; resulting dihedral angle measures, phenyl ring twist measures, double C-C bond length measures, and molecular energies are recorded in Tables 2-5. Finally, the labeling scheme for TPE molecules with various radicals as outlined in Figure 1 was used with an additional cis or trans tag, denoting position of X and Y substituents relative to one another.

Results and Analysis Completed calculations include the entire d category and the 2b and 3b species. Radical energies and C-C dihedral angles are recorded in Table 2; no major trends can be observed in the dihedral angles.

Phenyl ring torsional angles are recorded in Table 3; the trend in average phenyl torsion angles is toward increased angles with the addition of substituent groups and the presence of both donor and acceptor groups increases phenyl twist angles the most. This slight increase in torsional angles in the presence of both substituents along with the slight increase in C-C bond length discussed below may be attributable to greater stability of any particular single-electron density at the carbons; this lengthening and twisting of the phenyl

Energy (with scaled ZPE correction, hartrees/particle) -1316.763088 -1545.733741 -1501.257736 -1730.228671

Energy (corrected w/scaled ZPE, kcal/mol) -826282.0055 -969963.3797 -94205.2422 -1085735.794

C=C Angle #1 (degrees) 11.438 11.553 11.512 11.473

C=C Angle #2 (degrees) 11.436 11.434 11.535 11.651

PT #1 (degrees)

PT #2 (degrees)

PT #3 (degrees)

PT #4 (degrees)

Avg. PT (degrees)

51.012 51.462 51.633 50.330

51.025 51.465 51.632 50.335

51.011 49.830 49.773 50.280

51.019 49.828 49.772 50.287

51.019 50.646 50.703 50.308

THE EFFECTS OF ELECTRON DONATING AND ACCEPTING RADICALS ON THE GEOMETRY OF TETRAPHENYLETHYLENE

REFERENCES

rings allows the rings to come closer to planarity, which may in turn allow greater substituent influence, creating a sort of amplifying effect.

C-C bond lengths are recorded in Table 4. Results indicate increasing double C-C bond length with the addition of either –OCH3 or –CN radicals and even greater bond length with both radicals present than the additive bond lengthening of the two radicals as outlined in Table 5.

C-C bond length (Å) 1d (m-diMe)

1.36523

2d cis (m-diMe and p-OCH3)

1.36562

3d cis (m-diMe and p-CN)

1.36601

4d cis (m-diMe, p-OCH3, and p-CN)

1.36655

Bordwell, Frederick G., and Tsuei-Yun Lynch. “Radical Stabilization Energies and Synergistic (Captodative) Effects.” Journal of the American Chemical Society 111 (1989): 7558-562.

Bordwell, Frederick G., Xian-Man Zhang, and Mikhail S. Alnajjar. “Effects of Adjacent Acceptors and Donors on the Stabilities of Carbon-Centered Radicals.” Journal of the American Chemical Society 114 (1992): 7623-629.

Table 4

Table 5 0.00039

Bond length difference from donor

0.00078

Bond length difference from acceptor

0.00132

Bond length difference from donor and acceptor

0.00117

Sum of effects from donor alone and acceptor alone

0.00015

Difference between additive and “captodative” effect

The increase in double C-C bond length from the additive bond lengthening effects of both –OCH3 and –CN radicals in the cis position was calculated to be 0.00117, which was noticeable smaller than the actual increase in bond length of 0.00132. This extra lengthening of the double C-C bond suggests that the captodative effect may exist for the TPE molecule.

Extensions Various calculations still need to be completed for the cis substitution groups as well as most of the trans categories, at which point work can be redirected toward identifying possible transitional states between cis and trans conformations of the TPE molecule. Additionally, there is the possibility of actually synthesizing and analyzing the molecules being studied, though previous efforts have shown the process to be highly difficult.

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Dewar, M. J. S. “A Molecular Orbital Theory of Organic Chemistry. IV. Free Radicals.” Journal of the American Chemical Society 74 (1952): 3353-354.

Gaussian 03, Revision C.02, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, Jr., J. A.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; and Pople, J. A.; Gaussian, Inc., Wallingford CT, 2004.

GaussView, Version 3.09, Dennington II, Roy; Keith, Todd; Millam, John; Eppinnett, Ken; Hovell, W. Lee; and Gilliland, Ray; Semichem, Inc., Shawnee Mission, KS, 2003.

Handorf, Andrew, Kate Eanelli, and Louise Stracener.

“Substituted tetraphenylethylenes: Computational studies and efforts toward synthesis for the investigation of the captodative effect.” 232nd American Chemical Society National Meeting. San Francisco. Sept. 2006.

Katritzky, Alan R., Michael C. Zerner, and Mati M. Karelson. “A Quantitative Assessment of the Merostabilization Energy of Carbon-Centered Radicals.”

Journal of the American Chemical Society 108 (1986): 7213-214.

Leigh, William J., and Donald R. Arnold. “Merostabilization in radical ions, triplets, and biradicals. 5. The thermal cis-trans isomerization of para-substituted tetraphenylethylene.” Canadian Journal of Chemistry 59 (1981): 609-20.

Viehe, Heinz G., Zdenek Janousek, and Robert Merenyi.

“The Captodative Effect.” Accounts of Chemical Research (1985): 148-54.

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