November 4, 2015 Justice Erikson Box 1418 jle13@hampshire.edu Dear Justice, I am pleased to announce that you have been awarded $350 from the Lowry-Bruno-McNeal Fund to help defray the actual expenses associated with your Division II project, “Investigation of Antioxidant Effects on Borrelia burgdorferi Biofilms in vitro.” This grant will end on May 2, 2016. Unfortunately, we were not able to provide you with an award from the Dr. Lucy Fund for which you also applied. The funding for your project will be paid to you as you submit receipts to Joan Barrett in the Natural Science office. We are unable to release any funds in advance. You must attend an instructional meeting to learn about Fund reimbursement guidelines and the process for filing reimbursement requests. Sessions will be held Tuesday November 10 at 5pm and Wednesday November 11 at 5pm, both in CSC 316. You must attend one of these sessions. All reimbursement requests must be submitted by June 1, 2016. Expenses for items not covered by the Lowry-Bruno-McNeal Fund will not be reimbursed. All items purchased with this grant are the property of Hampshire College. If specific concerns arise, please discuss them with your committee chair and with Steve Roof, who oversees this fund. As an award recipient, you must write a brief (one-page) report at the end of the project summarizing your findings and describing how the funds were spent. Please submit to https://www.hampshire.edu/natural-science/ns-student-endowed-fund-awards. In addition, you are expected to submit an abstract that will be posted to the Hampshire College website and to inform the community of any presentations or publications that result from your work. Finally, ten or so years after graduation, you are encouraged to return to Hampshire to talk to our then-current students about your fabulous career! Congratulations on your fine work thus far. We look forward to hearing about the results of your project. Sincerely,

Dean of the School of Natural Science Professor of Earth and Environmental Science This letter must be signed and returned to Joan Barrett by November 11, 2015, before disbursements are made. Please save a copy for your records. I accept the conditions of this award____________________________Date__________________ Note: Although income taxes will not be withheld from the (your) grant, portions of the grant may be considered taxable income by the IRS, depending on how you use the money. You will not receive a W-2 or 1099 form from Hampshire College for this grant. Generally, a scholarship or fellowship is not taxable if (1) you are a degree candidate at an eligible educational institution, and (2) the amounts you receive are used for required tuition and fees or required course materials, and are not received as payment for your services. Portions of the grant used for other expenses, such as room and board, are considered taxable income. For more Information, see topics 355, 421, or Publication 970 on the IRS web site (www.irs.gov).

Sch oo l o f Na tu ra l Sc i en ce Amherst, Massachusetts 01002

413-559-5757 Fax 413-559-5448

HAMPSHIRE COLLEGE

November 4, 2015 Justice Erikson Box 1418 jle13@hampshire.edu Dear Justice, I am pleased to announce that you have been awarded $150 from the Tara Nelson Award to help defray the actual expenses associated with your Division II project, “Investigation of Antioxidant Effects on Borrelia burgdorferi Biofilms in vitro.” This grant will end on May 2, 2016. Unfortunately, we were not able to provide you with an award from the Dr. Lucy Fund for which you also applied. The funding for your project will be paid to you as you submit receipts to Joan Barrett in the Natural Science office. We are unable to release any funds in advance. You must attend an instructional meeting to learn about Fund reimbursement guidelines and the process for filing reimbursement requests. Sessions will be held Tuesday November 10 at 5pm and Wednesday November 11 at 5pm, both in CSC 316. You must attend one of these sessions. All reimbursement requests must be submitted by June 1, 2016. Expenses for items not covered by the Tara Nelson Award will not be reimbursed. All items purchased with this grant are the property of Hampshire College. If specific concerns arise, please discuss them with your committee chair and with Chris Jarvis, who oversees this fund. As an award recipient, you must write a brief (one-page) report at the end of the project summarizing your findings and describing how the funds were spent. Please submit to https://www.hampshire.edu/natural-science/ns-student-endowed-fund-awards. In addition, you are expected to submit an abstract that will be posted to the Hampshire College website and to inform the community of any presentations or publications that result from your work. Finally, ten or so years after graduation, you are encouraged to return to Hampshire to talk to our then-current students about your fabulous career! Congratulations on your fine work thus far. We look forward to hearing about the results of your project. Sincerely,

Dean of the School of Natural Science Professor of Earth and Environmental Science This letter must be signed and returned to Joan Barrett by November 11, 2015, before disbursements are made. Please save a copy for your records. I accept the conditions of this award____________________________Date__________________ Note: Although income taxes will not be withheld from the (your) grant, portions of the grant may be considered taxable income by the IRS, depending on how you use the money. You will not receive a W-2 or 1099 form from Hampshire College for this grant. Generally, a scholarship or fellowship is not taxable if (1) you are a degree candidate at an eligible educational institution, and (2) the amounts you receive are used for required tuition and fees or required course materials, and are not received as payment for your services. Portions of the grant used for other expenses, such as room and board, are considered taxable income. For more Information, see topics 355, 421, or Publication 970 on the IRS web site (www.irs.gov).

Sch oo l o f Na tu ra l Sc i en ce Amherst, Massachusetts 01002

413-559-5757 Fax 413-559-5448

HAMPSHIRE COLLEGE

November 4, 2015 Justice Erikson Box 1418 jle13@hampshire.edu Dear Justice, I am pleased to announce that you have been awarded $1393 from the Justine Salton Memorial Fund to help defray the actual expenses associated with your Division II project, “Investigation of Antioxidant Effects on Borrelia burgdorferi Biofilms in vitro.” This grant will end on May 2, 2016. Unfortunately, we were not able to provide you with an award from the Dr. Lucy Fund for which you also applied. The funding for your project will be paid to you as you submit receipts to Joan Barrett in the Natural Science office. We are unable to release any funds in advance. You must attend an instructional meeting to learn about Fund reimbursement guidelines and the process for filing reimbursement requests. Sessions will be held Tuesday November 10 at 5pm and Wednesday November 11 at 5pm, both in CSC 316. You must attend one of these sessions. All reimbursement requests must be submitted by June 1, 2016. Expenses for items not covered by the Justine Salton Memorial Fund will not be reimbursed. All items purchased with this grant are the property of Hampshire College. If specific concerns arise, please discuss them with your committee chair and with Ross, who oversees this fund. As an award recipient, you must write a brief (one-page) report at the end of the project summarizing your findings and describing how the funds were spent. Please submit to https://www.hampshire.edu/natural-science/ns-student-endowed-fund-awards. In addition, you are expected to submit an abstract that will be posted to the Hampshire College website and to inform the community of any presentations or publications that result from your work. Finally, ten or so years after graduation, you are encouraged to return to Hampshire to talk to our then-current students about your fabulous career! Congratulations on your fine work thus far. We look forward to hearing about the results of your project. Sincerely,

Dean of the School of Natural Science Professor of Earth and Environmental Science This letter must be signed and returned to Joan Barrett by November 11, 2015, before disbursements are made. Please save a copy for your records. I accept the conditions of this award____________________________Date__________________ Note: Although income taxes will not be withheld from the (your) grant, portions of the grant may be considered taxable income by the IRS, depending on how you use the money. You will not receive a W-2 or 1099 form from Hampshire College for this grant. Generally, a scholarship or fellowship is not taxable if (1) you are a degree candidate at an eligible educational institution, and (2) the amounts you receive are used for required tuition and fees or required course materials, and are not received as payment for your services. Portions of the grant used for other expenses, such as room and board, are considered taxable income. For more Information, see topics 355, 421, or Publication 970 on the IRS web site (www.irs.gov).

Sch oo l o f Na tu ra l Sc i en ce Amherst, Massachusetts 01002

413-559-5757 Fax 413-559-5448

HAMPSHIRE COLLEGE

Justice Lynn Erikson Division II, Completing Spring 2016

Chair: Christopher Jarvis jle13@hampshire.edu | Hampshire College Box #1418

31 Cottage Street, Belchertown, MA 01006

Investigation of Antioxidant Effects on Borrelia burgdorferi Biofilms in vitro.

Fall 2015, Spring 2016

Supervised by John Castorino No IRB/IUCUC approval required

Submitted To: The Denice O’Neill Scholarship Fund

The Dr. Lucy Fund The Justine Salton Memorial Fund The Lowry­Bruno­McNeal Fund

The Tara Nelson Award

NS Student Funds Proposal Justice Erikson

Introduction

Lyme Disease (LD) is a major epidemic in the United States. The Center for Disease Control and Prevention (CDC) estimates over 329,000 reported cases of LD diagnoses every year,1 and in the past decade the two most affected age groups have been young children and seniors.2 The CDC, National Institute of Health (NIH), and Infectious Disease Society of America (IDSA) have narrow definitions of and limited treatment protocols for LD and “Post­Treatment Lyme Disease Syndrome” (PTLDS) which exclude many forms of “chronic” LD. These consist entirely of short­term antibiotic regimens, and include a list of treatments not recommended, including “vitamins or other nutritional managements.”3 More and more cases of PTLDS or chronic LD are coming into public awareness ­ and the stories of young lives ruined through rapid neurological degradation, immense chronic fatigue, etc., are piling up.4 I hope to provide a small piece of the puzzle that will be necessary to solve this critical public health issue.

A major impediment to treating chronic lyme is that we do not know how the causative agent­­ the spirochetal bacterium Borrelia burgdorferi (Bb)­­ evades the host immune system. One of several proposed mechanisms of Bb immune system evasion in chronic LD/PTLDS is that Bb may be forming biofilms.5 Biofilms, metabolically efficient organized aggregates, are a common morphology for many bacteria in nature. They have recently been linked to many chronic and difficult­to­treat diseases because of their resistance to antibiotics and the immune system.6 This resiliency is likely due to presence of extracellular polymeric substances (EPS) and extracellular DNA (eDNA).7 In vitro formation of Bb biofilms has been characterized previously, but as of yet no clinically relevant research has been done in vitro with biofilms. Additionally, while the biofilms were shown to occur, factors that may promote or inhibit Bb biofilm formation were not identified. My project seeks to provide a link between Bb biofilm research and potential treatments for chronic LD/PTLDS.

I would like to investigate whether a mechanism for the possible effectiveness of antioxidant therapy might be through direct action on Bb biofilms. Biofilms and antioxidants are topics of conversation among LD scholars, but I do not believe that they have been integrated before. Two major antioxidant therapies currently in clinical use are Thomas E. Levy's high­dose intravenous and oral vitamin C (ascorbic acid) regimen,8 and Stephen Harrod Buhner's application of an herbal formula containing Japanese Knotweed 9 (whose active component is likely resveratrol). Both treatments utilize high­concentration antioxidants, and while there is a lot of potential there for research investigating the intersections of oxidative stress, the innate immune system, LD, and antioxidant therapy, that work is being done, and was a focus of a Hampshire Division III by Peregrine Rothman Ostrow a few years ago. Methods

The first step of this experiment will be to establish viable Bb biofilms in vitro. I will be using methods described in Sapi et al. (2012) 7. My primary method of investigating the effects of antioxidants on Bb biofilms will be using the Minimum Biofilm Eradication Concentration(MBEC) assay. This assay first forms biofilms on pegs with coating specifically designed for optimum biofilm growth, and then includes a number of protocols for quantifying various challenge (antimicrobial) concentrations to biofilm growth. I will be using ascorbic acid and resveratrol as my primary challenges, and using pH­matched buffered media as controls for each of my challenge concentrations. The MBEC assay is designed to quantitatively determine at what concentrations the challenges inhibit growth, kill cells, and eradicate biofilms. I will be

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NS Student Funds Proposal Justice Erikson

running any promising results at least twice to confirm results. This data will provide a foundation of information for how these antioxidants directly affect Bb biofilms.

The second aspect of this research is to evaluate microscopically the healthy and challenged biofilms. I will establish larger format culturing conditions and challenge with optimum concentrations as indicated by MBEC results. I will look to confirm the establishment of biofilms by staining for biofilm characteristics such as calcium complexes (using Alizarin Red) and extracellular DNA (using the red fluorophore DDAO). I will use immunofluorescence to visualize the biofilms at Hampshire and definitively confirm Borrelia burgdorferi aggregates. I will then use confocal laser scanning microscopy at Smith College (where I am currently undergoing training) to obtain more detailed three dimensional information about the biofilm structures. Timeline

Fall 2015 (November and December) goals will be to establish successful biofilms, determine growth curves, and optimize the MBEC assay. Challenge concentrations might be started as soon as December. The bulk of the work will be done within an independent study in the Spring of 2016. Goals will be to establish and confirm useful challenge concentrations, which is anticipated to take up to two months due to bacterial growth time and labor intensity. Over the last month or so of the semester I will develop larger format biofilms, prepare samples using targeted stains, and collect microscopic data. Outcomes and Implications

I believe this research will be an excellent addition to the field, as it addresses questions that have not been addressed by the current literature. If I find interesting data and am able to successfully visualize my samples, I can see it becoming an important bridge between fields and a stepping stone towards an effective treatment for the often overlooked but devastating condition of chronic lyme. I will be holding myself and this research to rigorous standards, so that my contributions may be of real use to the scientific community. However, this project alone will likely not be the extent of this pursuit; it is very possible that this will grow into my Division III project in the Fall of 2016, wherein I will be able to expand upon this research greatly and develop substantial experimental results in concert with a rigorous examination of the field. Academic Preparation

This work is an indirect continuation of my project last semester in Methods in Molecular Biology, where I examined antioxidant activity on macrophages. This is a key part of the immune system response to Bb infection. I was working with similar cell viability assays and testing resveratrol concentrations, which will be extremely similar to working with the MBEC assay. It gave me a solid set of lab skills and knowledge of this topic, which I am also developing via my research paper in the seminar Natural History of Infectious Diseases this semester. I am also using my study in organic chemistry and biochemistry this semester to study biochemical mechanisms of antioxidants in the body. Much of my past coursework is relevant as well. I have taken two semesters each of general chemistry and organic chemistry at Hampshire, all with labs. I am very comfortable in the lab, and have a good sense of good laboratory practices and problem­solving skills. I have also taken many courses in the biological sciences, where I have considered topics such as experimental design, data analysis, and underlying chemical and biological mechanisms of human health and disease.

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NS Student Funds Proposal Justice Erikson

Bibliography 1. Nelson, Christina A., Shubhayu Saha, Kiersten J. Kugeler, Mark J. Delorey, Manjunath B. Shankar, Alison F. Hinckley & Paul S. Mead 2015, Incidence of Clinician­Diagnosed Lyme Disease, United States, 2005–2010, Emerg Infect Dis. 21 pp. 1625­1631 2. CDC, 2015a “Confirmed Lyme disease cases by age and sex ­­ United States, 2001­2010” http://www.cdc.gov/lyme/stats/chartstables/incidencebyagesex.html 3. Wormser, G.P., Dattwyler, R.J., Shapiro, E.D., Halperin, J.J., Steere, A.C., Klempner, M.S., Krause, P.J., Bakken, J.S., Strle, F., Stanek, G., Bockenstedt, L., Fish, D., Dumler, S.J. & Nadelman, R.B. 2006, "The Clinical Assessment, Treatment, and Prevention of Lyme Disease, Human Granulocytic Anaplasmosis, and Babesiosis: Clinical Practice Guidelines by the Infectious Diseases Society of America". 4. Under Our Skin. Dir. Andy A. Wilson. Perf. Mandy Hughes and Ben Petrick. 2008. Open Eye Pictures, Film. 5. Berndtson, K. 2013, "Review of evidence for immune evasion and persistent infection in Lyme disease", International journal of general medicine, vol. 6, pp. 291­306 6. Bacteria and Chronic Infections, University of Copenhagen. https://www.coursera.org/course/bacteria 7. Sapi, E., Bastian, S.L., Mpoy, C.M., Scott, S., Rattelle, A., Pabbati, N., Poruri, A., Burugu, D., Theophilus, P.A.S., Pham, T.V., Datar, A., Dhaliwal, N.K., MacDonald, A., Rossi, M.J., Sinha, S.K. & Luecke, D.F. 2012, "Characterization of biofilm formation by Borrelia burgdorferi in vitro", PloS one, vol. 7, no. 10, pp. e48277 8. Levy, Thomas E. Primal Panacea 2011. Henderson, NV: MedFox Pub. Print. 9. Buhner, Stephen Harrod. 2005. Healing Lyme: Natural Prevention and Treatment of Lyme Borreliosis and Its Coinfections . Randolph, VT: Raven, Print.

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NS Student Funds Proposal Justice Erikson

Budget: Consideration: Most of this I will likely continue to use into my Division III, and/or will be useful stock for the CSC laboratory. The shipping charges are estimations. Fund sources listed are suggestions. Items listed in order of priority.

Item Supplier Item # Price Shipping Justification & Source

Borrelia burgdorferi strain B31

ATCC 35210 $185 $85 To establish cultures. Tara Nelson Award

BSK­H Medium, Complete (500 mL)

Sigma­Aldrich P8291 $188.50 $50 Necessary medium for Borrelia burgdorferi

cultures. Tara Nelson Award

MBEC™ Biofilm Inoculator with 96 well base x 25

Innovotech 19111 $562.50 $55 For quantification of biofilm development and

antioxidant effects. Denice O’Neill or Justine Salton

LIVE/DEAD Biofilm Viability Kit (100

rxns)

ThermoFisher L10316 $366.00

Free For biofilm staining, shows damaged bacteria.

Denice O’Neill or Lowry­Bruno­McNeal

Borrelia burgdorferi Antibody, FITC conjugate (1 mL)

ThermoFisher PA1 ­73005

$272.00 $50 For Immunofluorescent staining.

Denice O’Neill

DDAO, Reference Standard (10 mg)

ThermoFisher H6482 $107.00 $50 For eDNA staining. Denice O’Neill

Cole­Parmer 12­Channel

Adjustable­Volume Pipette, 40­300 uL

Cole­Parmer WU­21600­23

$415.00 $25 Will greatly improve efficiency of work, and

will be a useful addition to CSC labs.

Dr. Lucy Fund

Cole­Parmer 12­Channel

Adjustable­Volume Pipette, 2­20 uL

Cole­Parmer EW­21600­15

$415.00 $25 Same as above. Dr. Lucy Fund

TOTAL $2511 $340 With Shipping: $2,851

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NS Student Funds Proposal Justice Erikson

Dissemination During the time of research, I will be posting regular updates on Hampshire’s Life

Behind Open Walls public research blog. As an Admissions Intern for Hampshire, this will likely end up being shared with many prospective students and families as my research develops. Provided I obtain decent results, I will seek to to present this research at conferences in 2016. Speaking about these issues and this research excites me greatly, and I will take any opportunity available to give talks and presentations throughout the campus community as well. It is also possible that this research will culminate into a publishable piece of work, in which case I will pursue publication in an open access journal. I will also be in conversation with representatives at Bastyr University and other colleges, as I begin pursuing admission at Naturopathic medical schools.

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Division II Filed Contract

The state of this document is filed with Central Records

Title The Chemistry and Biology of Human Health and Medicinal Herbalism

File Date 03/24/2015

Revision Date

Anticipated Completion Date 04/15/2016

Pass Date

Study Areas Biochemistry, Human Biology, Botany

Hampshire College Members

Christopher Jarvis - Current Committee Chair

Dulasiri Amarasiriwardena - Current Committee Member

Committee Signatures/Approvals

Christopher Jarvis signed this document on 03/24/2015 at 11:30AM.

Dulasiri Amarasiriwardena signed this document on 03/10/2015 at 04:12PM.

Provide an overview of your concentration, including several fundamental questions or issues that will help frame

your studies. Describe the relevant areas of study.

I intend for my concentration to be a wide survey of the functioning of human bodies at a multitude of

scientific levels from the atomic to population levels. I intend to pursue deep questioning in how human health

is affected by factors such as chronic and acute illness, and how the fields of biomedicine and natural medicine

approach treating illness and maintaining health. Medicinal herbalism is a strong focus for me, and a significant

portion of my divisional work will be in exploring this topic from both biomedical and traditional angles, largely

through indipendent work. I will be approaching my coursework with an interest in challenging the status quo of

the biomedical and naturopathic fields, investigating research methods, and considering alternative medicine

practices. The primary goal of my concentration is to provide a strong basis for future pursuit of a doctorate in

Naturopathic Medicine.

Some additional questions include: "What medicine is effective? Why? How do we determine this?" and "How

can I prepare to be an effective and conscientious alternative healthcare provider?"

Write your goals. What do you want to know, understand, and be able to do by the end of Division II?

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I would like to use my Division II to study a wide range of pre-medical sciences, including biology, chemistry,

botany, psychology, and physics. My goal is to obtain a scientific foundation of understanding the human body,

brain, and mind as they relate to the sciences, with an additional focus of natural medicine and plant sciences.

Psychology and neuroscience will be present in addition to general human biology, molecular biology, etc. in

order to better understand mind-body connections. Emphasis will be on preparation for medical school,

specifically Naturopathic medical school requirements.

How will you achieve your goals and answer your questions? Describe the sequence of courses learning activities

and possibly internships, independent work, and field study that will shape your Division II. (Some advisors

recommend writing a semester-by-semester plan).

Course plan: https://docs.google.com/spreadsheets/d/1l-QdQG55Y-C94kN47_JUTDtP-

8EWpJ8ggtnailvdgTk/edit?usp=sharing

I want to know as much as possible about the human body and all forms of medical science. I intend to take all

classes that feel relevant to this goal, and am particulartly interested in completing courses such as: Organic

chemistry, virology/immunology, neuroendocrinology, epigenetics, and psychology. Courses in plant sciences and

herbalism will also play a major role. I also have an intense desire to complete some original research during my

Div II, whether it ends up directly feeding into my Div III or not. To that end, I want to be prepared to

conduct research in chemistry and molecular biology fields.

How do you plan to engage with multiple cultural perspectives in Div II? View a complete description of this

requirement.

I intend to continue my studies of Chinese medicine and other global medicine systems, analyzing how

traditional medicine systems play into modern medical systems, and analyzing the roles of power in colonization.

Additionally, I will be looking for an opportunity to do volunteer medical work in Latin America, where I will

think about race, empirialism, and systems of opression. It is vitally important to me to respect the cultures in

which the medicine systems I study have originated.

Below details what you plan to do to satisfy the Community Engagement and Learning requirement for Division

II.

I am hoping to go on a Global Medical Brigade to do volunteer medical work in Latin America.

I regularly engage in independent projects in herbalism, etc. with other students, this may present an opportunity

for CEL-2.

I may also use TAing for CEL-2 hours.

The list of previous and current work below may be included in Division II with the approval of your Division

II committee. Courses used to satisfy Division I requirements are not displayed. Students with advanced standing

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have items below marked as 'Other' may be used for up to 3 electives in Division I.

Include Term Course

2015F NS-0314 Organic Chemistry II Rayane Moreira

2015F NS-0353 Natural Hist Infectious Dis Lynn Miller

2015F NS-0262 Biochemistry John Castorino

2015F NS-0388 Adv Methods in Mol Biology John Castorino

2015S NS-0220 Human Physiology Cynthia Gill

2015S NS-0235 Methods in Molecular Biology John Castorino

2015S NS-0299 Learning Activity Projects Lynn Miller

2015S NS-0212 Organic Chemistry I Rayane Moreira

2015J JTW-0201 Intensive Neuroanatomy Jane Couperus

2014F NS-0244 Neuroendocrinology Cynthia Gill

2014F NS-0249 Epi: Genetics John Castorino

2014F LA-ACT Learning Activity - Teaching Assistant Training

2014F LS-0102 Elementary Spanish II Esther Lopez

2014F TA-NS Teaching Assistant - NS-101T: How Things Work Herbert Bernstein

2014S 5C-CRS St-Clinical Herbalism II

2014J JTW-0137 Plants of the Gods Nathaniel Putnam

2013F NS-0121 Human Biology Christopher Jarvis

2013F OPRA-0108 Intermediate/Advanced Yoga Meredyth Klotz

Other NS0154 Plants and Human Health

The following is your course of study and other learning activities which will satisfy your Division II

Contract requirements.