University of Wyoming McNair Scholars Research Journal

2011-2012 Volume 17

Table of Contents The McNair Scholars Program ...... 3 Ronald Ervin McNair: ...... 5 Melvin Authur ...... 7 Scott Cheney ...... 33 Cameron Finley ...... 71 Nathan Fletcher ...... 84 Joshua Heyer ...... 92 Kimberli Ogg ...... 110 Laurie Petric ...... 124 Kevin Schilling ...... 137

McNair Scholars Research Journal 2011-2012 Volume 17

McNair Program Staff Zackie S. Salmon: Project Director Susan J. Stoddard: Assistant Director Pilar Flores: Director, Student Educational Opportunity

This McNair Journal is the official journal of the Ronald E. McNair Post- Baccalaureate Achievement Program at the University of Wyoming. The University of Wyoming’s McNair Scholars Program is 100% federally funded by the U.S. Department of Education at $288,000 annually.

The UW McNair Journal is published annually. Manuscripts and abstracts are accepted from McNair Scholars participating in the program at the University of Wyoming.

Zackie S. Salmon Director, McNair Scholars, University of Wyoming Dept. 3808 1000 E. University Ave. Laramie, WY 82071 (307) 766-6189 (307) 766-3073 TTY (307) 7664010 Fax

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The McNair Scholars Program

Background The University of Wyoming McNair Scholars Program was established in September 1992 and is one of the only 150 programs in the nation. The University of Wyoming and the United States Department of Education jointly support the program named for an accomplished scholar of physics and an astronaut on the Challenger space shuttle, Ronald E. McNair. Prospective scholars represent a wide range of disciplines. These students share the common desire to pursue graduate studies, attain the doctorate, and join the ranks of the next generation of faculty members.

Program Components The McNair Scholars Program provides exciting opportunities for undergraduate students at the junior and senior levels to prepare for acceptance into quality graduate programs of their choice. Program participants are provided services in academic skills, individual counseling support, and funded summer research internships. During the academic year a series of seminars provides information on graduate school financial aid, research skills and technical writing, the graduate school admission process, graduate school entrance exams, portfolio preparation, and more. Student concerns in financial, personal, and academic realms are addressed through individual counseling support services. Finally, the program offers participants site and mentors, provide workshops on research skills, help with report preparation, and assist as otherwise needed on an individual basis. At completion of the internship experience, McNair Scholars make formal presentations of their research to faculty and peers at the McNair Scholars Conference and submit papers summarizing their work. Opportunities to attend national research and graduate recruitment conferences and visits to other graduate campuses are encouraged and provided.

Funded Internships Research internships are offered to those Scholars who have earned at least 60 credits by the beginning of the internship period. Stipends for internships are awarded for an eight week summer session. During the eight week internship, students work 40 hours per week under the supervision of a faculty mentor and a graduate student advisor. Students seeking involvement submit an internship application to the program.

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Eligibility and Selection First generation students (neither parent has received a bachelor’s degree) and income eligible Or Black (non-Hispanic), Hispanic, American Indian, Alaskan Native, Native Hawaiian and Native American Pacific Islander Have at least 50 credit hours Meet GPA requirements U.S. Citizen or permanent U.S. resident Committed to attaining their Ph.D.

Prospective participants are encouraged to contact the project staff for information and application materials at any time during the year. Participants are selected from undergraduate applications attending the University of Wyoming on the Laramie campus. Participation in the program is limited to 33 students.

Forward As the McNair staff has grown in experience over the years, so have the faculty, graduate students, and McNair interns. Each year I am struck by the increased level of sophistication I see in both the projects and the presentations, oral and written. This continuous improvement can be attributed to the hard work and dedication of the McNair Scholars, and the faculty members and graduate students without whose help their success would have been impossible. One of the greatest joys of being a McNair staff member is the opportunity to work with such terrific colleagues as the students, faculty members, and graduate students represented in this journal. The McNair Scholars featured in this journal can be rightfully proud of what they have achieved. We wish them well and look forward to the great things they will achieve.

Zackie Salmon, Director UW McNair Scholars Program Spring 2011

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Ronald Ervin McNair: The Man with a Mission

Ronald E. McNair, the second African American to fly in space, was born on October 21, 1950 in Lake City, South Carolina. While in junior high school, Ronald McNair was inspired by a teacher who recognized his science potential and believed in him. He graduated as valedictorian from Carver High School in 1967. In 1971, he received his Bachelor’s Degree Magna Cum Laude in Physics from North Carolina A & T State University (Greensboro). In 1976, at the age of 26, McNair earned a Ph.D. from the Massachusetts Institute of Technology (MIT). While working with the Hughes Research Laboratory as a staff physicist, McNair soon became an acknowledged expert in laser physics. NASA selected him for the 1978 space shuttle program and in 1984, McNair became the mission specialist aboard the flight of the shuttle Challenger. In addition, he received three honorary doctorate degrees as well as numerous fellowships and commendations. Dr. McNair’s life ended tragically on January 28, 1986 when the Challenger space shuttle exploded and crashed into the ocean, taking the lives of six other astronauts. After his death, Congress approved funding for the Ronald E. McNair Post-baccalaureate Achievement Program, which is dedicated to the support and promotion of the high standard achievement exemplified by McNair. The University of Wyoming McNair Scholars Program is dedicated to preserving his legacy of scholarship and accomplishments.

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McNair Scholars Research Papers

2011-2012

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Melvin Authur Mentor: Dr. Caskey Russell Major: Social Work

Research Topic: Complacency Over Land Boundaries on the Wind River Indian Reservation: A View of Inter-Tribal Relations and Reparations through Community Lenses

Currently American Indian Reservations and the indigenous occupants thereof are subordinate nations as products of their governmental discourse. Their environments have been created by the unfathomable mindset of the political decision makers, both contemporary and historical (Oxford University Press, 2008). On the Wind River Indian

Reservation in the state of Wyoming there exists a complacency among tribal members that has the potential for a great loss of tribal property.

The Wind River Indian Reservation is home to more than 12,000 enrolled Northern

Arapaho and Eastern Shoshone tribal members. The Northern Arapaho and Eastern

Shoshone Tribes are federally recognized tribes and have active treaties with the United

States of America. The Shoshone Tribe was the first tribe to be granted a reservation along with the Bannocks in the Fort Bridger Treaty of 1864; Chief Washakie solicited for and was subsequently granted permission to choose the geographical location of the reservation that would ultimately become the Wind River Indian Reservation. At Fort Washakie, or

Camp Brown as it was known in 1876, gold miners and hostile Plains Indians vied for

7 | P a g e control of the picturesque mountains and desert plated paradise. In the government’s perfectly flawed conduct with indigenous leaders, decisions were made to undermine

America Indian property rights. This flawed conduct occurred simultaneously with a severe blizzard that pummeled the area during the year 1874-1875. At that time the United

States Government decided to place the Arapaho people on the Shoshone Reservation with a total disregard for the two tribes’ historical conflicts. The government made promises to the Shoshone and Arapaho people and gave the Indians their word that the Arapaho people would be given a reservation in a different location (Stamm, 1999).

Today Northern Arapaho and Eastern Shoshone tribal members still feel the effects of this act. The environment on the Wind River Indian Reservation lacks the social structure that is required for advancement of Northern Arapaho and Eastern Shoshone

Tribal members in the State of Wyoming. Community capital is based on how well entities within a community get along with one another. The macro level of social work stipulates, when a community is assessed, one of the criteria for change is the potential degree of community capital a population exhibits. The tribal officials on the Wind River Indian reservation have not established discourse over property rights of each tribe. Non-native officials have politically incorrect opinions of Northern Arapaho and Eastern Shoshone people. The area in and around the city of Riverton, Wyoming has become the subject of a lawsuit filed by the Northern Arapaho tribe. In a situation where both tribes should have unified to protect their property rights in Wyoming, the tribes faltered in any of their efforts. Century old animosities linger in the ethos of tribal opinion, and the communities’ complacency stymies true discourse for resolutions of the one hundred years of

8 | P a g e irreconcilable differences based on this historical animosity (Markley, 1997; Fremont

County Assessor Map, 2011).

Historical Perspective

Indigenous Americans could be categorized into two major groups. The first major group would carry the label “Original American People” who in their cultures and social structures created a significant civilization that encompassed the entire western hemisphere. The second major group would consist of the American Indians who have been directly affected by Colonialism and Manifest Destiny. The Northern Arapaho and

Eastern Shoshone peoples have evolved from culture and tradition that existed for thousands of years. We can place the Shoshone Tribe in the area that is now the Wind River

Indian Reservation approximately in the 1500’s. At that same time we can linguistically place the Arapaho Tribe in the area now bordering the Minnesota and Canadian Border.

The historical significance of the Arapaho and Shoshone Tribes was essential in pre-

Columbus civilizations, as both tribes have been represented cross culturally in oral traditions (Anderson 2011).

The Wind River Mountains and the area that is now Fremont County became very sought after by settlers and miners. Gold was discovered in the late 1860’s around the same time the Eastern Shoshone acquired their reservation. After gold was discovered in the area above Lander, Wyoming the Shoshone Reservation was subsequently reduced in size by the United States Government. The reconciliation was intended to accommodate the influx of miners and white settlers. From the signing of the Fort Bridger Treaty of 1868 to the placement of the Northern Arapaho people in 1878, the Eastern Shoshone Tribe did not

9 | P a g e have any control over the impositions placed on them by the federal government (Markley,

1997).

The Northern Arapaho placement in the Wind River Indian Reservation is the catalyst for all the contemporary conflict of today. The Eastern Shoshone and Northern

Arapaho Tribes had historical conflict before reservation life, which usually involved the settling of hunting and property rights, and did not involve government policy. The two warring tribes carried their historical animosity onto the reservation and they were forced to adapt to the situation.

The white community as well as the Shoshone people considered the Northern

Arapaho hostile after their participation in the Battle of Little Big Horn and the Indian stronghold of the Bozeman Trail. With the Northern Arapaho Tribe came a certain amount apprehension toward their behavior. The Arapaho traditionally had a reputation that preceded colonialism and they were typically feared by other tribes (Frosch, 2010). This is attributed to the perceived relationship Arapahos always had with the creator. The

Arapaho also brought with them an extensive war record with the U.S. Cavalries. The soldier station at Camp Brown was fortified because of the influx of settlers and miners and there would be enough soldiers to combat any Indian uprising.

The Eastern Shoshone Tribe prided themselves on their ability to assimilate into non-Native roles, and their population has thrived because of the adoption process they embrace. Chief Washakie was very dedicated in his interaction with the federal government and he secured the best location for the Eastern Shoshone Reservation. He also guided his people into assimilation because he wanted them to survive in the new world.

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The Wind River Indian Reservation became important because of its proximity to the gold rush, and Shoshone people were in good standing with the U.S. Soldiers stationed at Camp

Brown (Stamm, 1999).

This environment created the perception among the whites of two types of Indians on the Wind River Indian Reservation, the good Indian and the bad Indian. Arapahos were destined to be viewed as the antagonists while the Shoshones where viewed as more assimilated. The tribes lived in constant civil conflict as the Wind River area had not fully been settled in terms of civil obedience. The environment at this place and time was unforgiving, and Northern Arapaho and Eastern Shoshone people were still in the habit of going on traditional war parties (Fowler, 1978).

The stage was now set for the polarization of the two present tribes. There is no clear reasoning for the U.S. Government’s decision to place the Northern Arapaho on the

Shoshone Reservation. The preconceived notions of the Arapahos and the burdens placed on the Eastern Shoshone people have created negative perceptions of the Indian people in the Wind River community. We do know that the federal government was pursuing ownership of the remaining parcels of Indian land at the turn of the century (Eastern

Shoshone and Northern Arapaho, 2008). Placing the Northern Arapaho on an occupied reservation would negate the promise of locating another reservation for the Northern

Arapaho.

The Northern Arapaho and Eastern Shoshone Tribes have opposing thoughts on how to adapt to their situations on the Wind River Indian Reservation. The Eastern

Shoshone Tribe wanted to assimilate to the white settlers’ culture at the turn of the 20th

11 | P a g e century. The Shoshone Tribe modeled themselves after the non-Natives in the hopes of bettering their relationship with the federal government. This was a very complex time in the history of the Wind River as the Arapaho enlisted their warriors into the U.S. Military in the hopes of possibly getting their own reservation. The Shoshones were promised that the

Arapaho would only live on the Shoshone Reservation temporarily, and the Arapaho did not want to be on the reservation when there was a possibility of getting a reservation of their own. At the turn of the century the situation for the Indians on the Wind River Indian

Reservation was one of compromised livelihood of both tribes. By this time (1876) the two tribes were led to believe that all the land north and south of the Big Wind River belonged to the tribes (Markley, 1997).

Since the tribal land north of the Big Wind River was seeing economic growth and influx of white settlers between 1891 and 1893, the government tried to acquire entitlement to all the land north of the Big Wind River. In these instances the negotiations faltered and the government’s first offer of $600,000 and second offer $750.000 never became reality. The land north of the Big Wind River remains in possession of the two tribes. At this point in time the Northern Arapaho Tribe was making decisions concerning the reservation which they were temporarily placed on with the Eastern Shoshone people

(Eastern Shoshone and Northern Arapaho, 2008).

The land in and around the Wind River Indian Reservation was now valued because

Wyoming had just got it’s state hood in 1890 and there was an influx of non-Natives from the gold rush in South Pass. In 1896, the Indian Inspector James McLaughlin struck an

12 | P a g e agreement with the Eastern Shoshone and Northern Arapaho Tribes to acquire the near-by hot springs the and city of Thermopolis.

Since the inception of the reservation, the Indians of the Wind River have had a multitude of experiences that have shaped their perceptions of their new existence as subordinate peoples who had very few choices concerning their futures. The outside forces on the state and federal level are now interested in the land that has been given to the

Eastern Shoshone and Northern Arapaho Tribes. These forces made two previous attempts to acquire the land north of the Big Wind River; one was rejected by the tribes outright, and the other faltered in congress (Oxford University Press, 2008).

By 1896, the Wind River Indian Reservation’s geographic location was becoming its biggest downfall, as the white entities wanted to capitalize on the resources, the beauty, and the economy of the area. The indigenous cultures of the plains (Sioux, Cheyenne, Crow, and Arapaho) valued the area long before the idealism behind Manifest Destiny. The inevitability of encroachment made it impossible for Northern Arapaho and Eastern

Shoshone Indians to fully lay claims to their own reservation. The Indians of the Wind River clearly impeded on the capitalistic endeavors of the whites and the two tribes suffered significant land loss as a result. All land north of the Big Wind River would now be seen as desirable for settlement. Indians were pushed between the confines of the Big Wind River and the Popo Aggie River which lies south of the Big Wind River. The Indians would not only be viewed as a danger, but they also stood in the way of the white settler’s progress

(Fremont County, Map, 2011).

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In 1905, the government perused the land again and offered a new round of talks concerning the land north of the Big Wind River. Mr. McLaughlin now put the issue of settlement on the table and asked the Eastern Shoshone and Northern Arapaho Tribes to relinquish their right to the land north of the Big Wind River. The government was interested in separating the indigenous people from the rest of the land and they implemented the allotment process. The Indians of the Wind River Indian Reservation had clearly been through bad negotiations in the past and the prospect of money was a motivating factor among tribal members. At the turn of the century all the indigenous inhabitants of the United Sates experienced poverty on some level. The negotiations for the land north of the Big Wind River meant that tribal members would get a much needed economic boost (Stamm, 1999).

Some tribal members opposed the negotiations altogether and tribal tensions escalated resulting in a murder of a tribal member. Eastern Shoshone and Arapaho tribal members were on full alert and travel on the reservation became very dangerous. The negotiations produced an agreement and on March 3, 1905 the Act was an extension of the allotment process allowing for white settlement north of the Big Wind River, with the proceeds from the land sale to be put into a trust and paid to the tribes in a manner to be determined in the future.

The segregation of the Eastern Shoshone and Northern Arapaho Tribes was now polarized in the perceptions of many tribal members, and there is not any discourse between the two tribes in any political or business sense. The tribes were now very savvy concerning governmental promises, and both tribes rejected the auspices of the Indian

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Reorganization Act of 1939 (IRA). The tribes rejected the IRA out right, but they did adopt some of the policies such as creating a business council to oversee the affairs of the tribes.

The tribes now had the authority to operate their own governments and the government forced the Eastern Shoshone and Northern Arapaho Tribes to form a Joint Business Council

(JBC) because they both had an equal share of the reservation (Oxford University Press,

2008).

The property rights of the Eastern Shoshone and Northern Arapaho Tribes remained in dispute, and collectively there was a plethora of legal wrangling that attempted to establish the definition of Indian country in Wyoming. Tribal perceptions of property rights were evident by the economic development plan the tribes published in

1963, which states that they believed the reservation boundaries were:

Bounded roughly on north by the South fork of Owl Creek, with the Arapahoe

Ranch lying just north of the stream. The east boundary is a north-south line

running about 6 miles east of the Big Wind River. The south line runs just

north of Hudson west of the Continental Divide. The west boundary lies

approximately north and south from the East fork of the Wind River, or

generally the western boundary of Range 6 West. Contained within the

boundaries of the Reservation are the City of Riverton and the Riverton

reclamation Irrigation Project. The reservation is approximately 70 miles

east to west, and 55 miles from north to south… The Wind River Indian

Reservation contains 2,268,000acres (Eastern Shoshone Tribe and Northern

Arapaho Tribe of the Wind River Indian Reservation, p. 39).

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The tribes have operated as perceived sovereign nations under the JBC and the respective business councils for each tribe, and there is a complete consensus pertaining to tribal boundaries on the reservation. Most tribal members felt that the above description is a clear representation of tribal property rights, and this perception has held to present day.

The JBC has not had any exclusive role in managing tribal affairs that require unison and a certain amount of symbiosis. In the mid nineties the tribes began to separate some aspects of the JBC and the first order of business was to establish separate housing authorities for each tribe. This was in response to the ratio difference which allowed for the

Eastern Shoshone Tribe more resources for their housing program. The historical conflict between the two tribes was never resolved and their animosity for one another remained intact.

In recent years, the tribes have had issues, one specifically concerning Class III gambling on the reservation. In 1996, the Northern Arapaho Tribe sued the state of

Wyoming over Class III Gambling rights on the Wind River Indian Reservation. The Eastern

Shoshone tribe decided to take a back seat on the issue and did not enter into litigation with the Northern Arapaho Tribe. The end result was that the Northern Arapaho Tribe won their right to Class III Gamming on the reservation. Subsequently Governor Dave

Frudenthal allowed the Eastern Shoshone Tribe the right to operate Class III Gaming on the reservation (Northern Arapaho Tribe v. State of Wyoming and Governor Jim Geringer,

2004) .

At the turn of the 21st century, the Northern Arapaho Tribe sued the state of

Wyoming to establish the definition of tribal property rights in the city of Riverton. In this

16 | P a g e lawsuit the judge decided that he could not proceed with the case unless the Eastern

Shoshone Tribe and the Federal Government were named in the litigation. This lawsuit filed by the Northern Arapaho Tribe followed a recent court decision which involved the murder of an enrolled member of the Northern Arapaho Tribe and the conviction of her enrolled father. Andrew Yellowbear was convicted of murder in the state of Wyoming for killing his daughter in the city of Riverton. In the Yellowbear case, the court decided that

Riverton does not lie within Indian Country (Yellowbear v. State of Wyoming, 2008). Such rulings show how the perceptions of property rights within the city of Riverton differ between the native and non-native groups. All of which exemplifies that complacency exists between the two tribes concerning tribal property rights.

As of today, the Eastern Shoshone and Northern Arapaho Business Councils have not addressed the issue of tribal property rights as a collective. The tribes have squabbled over trivial issues such as placement of casinos on joint tribal property and they do not actively pursue discourse with one another. The historical aspects of the Wind River Indian

Reservation reveal the situational adversity created by placing the Arapaho and Shoshone people in irreconcilable positions. The United States Government clearly made decisions that were counterproductive for both tribes, and in the process affected the Indians of the

Wind River perpetually. Today the non-native population north of the Big Wind River rivals that of the native population on the Wind River Indian Reservation (United States Census

Bureau2010).

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DATA ANALYSIS

Data was collected over from May 24 through July 30, 2011, on the Wind River

Indian Reservation, and 20 participants were interviewed and audio recorded for data analysis. Interviews included a series of 10 open ended questions which guided the discussion. All participants were at least 18 years of age at the time of their interview. All participants were living on the reservation at the time of their interview. The participants were also selected based on formal and informal leadership roles, long term personal relationships, and on the positions of authority hey hold by on the reservation.

Audio recording the interviews (30-45 min.) allowed participants to answer spontaneously, and responses lacked the political correctness. The recording device also provided opportunity for dialog concerning issues on the Wind River Indian Reservation.

The reservation encompasses 2,268,000 acres of land and rural participant’s homes were accessed by vehicle, because of the distances transportation became a very big factor on the Wind River Indian Reservation. Most of the population resides mainly in the most rural locations of the reservation. Many of these locations are only accessible through gravel and farm roads, making off-road vehicles more preferable.

Interviews were both scheduled and convenient in order to produce a participant pool representative of the reservation. The scheduled interviews were conducted in the participants’ homes and at their places of work. For participation convenience interviews were conducted when and wherever possible, such as in vehicles at gas stations and at convenient locations around the reservation. American Indian reservations in the plains

18 | P a g e states are generally geographically locations with social environments unique to one another. These environments require culturally competent analysis of the contemporary social structures of American Indian Reservations. The primary researcher’s affiliation with the Wind River Indian Reservation has helped assure their participants genuine openness.

Interviews were transcribed and coded to identify common themes. Questions consisted of ten simply stated inquiries pertaining to the perceived relationship between the Northern Arapaho and Eastern Shoshone people. The questions were also designed to provoke thought and knowledge of tribal members’ interpretation of tribal boundaries of the Wind River Indian Reservation and the property rights of the Northern Arapaho and

Eastern Shoshone Tribes. Responses were then coded based on common themes; listening for word, repetition, phrase repetition, and repetition of local terms.

Interview Questions:

1. How would you describe the current relationship between the Northern Arapaho and Eastern Shoshone People?

2. What do you feel are the benefits of having two American Indian tribes on the Wind River Indian Reservation?

3. What are the major issues that hinder good relationships between the two tribes?

4. Why do you think the Northern Arapaho and Eastern Shoshone people will not enter into litigation to settle disputed land issues in and around the reservation?

5. Do you think the lack of communication between the two tribes has been due to historical conflict or conflicts that have arisen from the ramifications of sharing a reservation?

6. Do you think the Arapaho/Shoshone business councils perpetuate negative attitudes and opinions?

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7. In your opinion; what steps could each tribe take to improve the relationship

between the Eastern Shoshone and Northern Arapaho people?

RESULTS

Twenty participant interviews were analyzed; the participant pool consisted of five former and current business council members, among the ten from each tribe. All members were asked to participate and accepted participation and audio recording as no interviews were declined. Participation rate indicates a desire for dialog and many tribal members expressed enthusiasm for future tribal endeavors.

Common themes:

Participants responded to question number one and two common responses were

“good” or “strained”. References to good relationships pertained to those based on inter- personal communication. Tribal members felt that Northern Arapaho and Eastern

Shoshone personal relationships are good and that it is possible to get along on this level.

In contrast, references to strained relationships pertained to the political and economic stressors of the environment on the reservation. In general, the tribal members on the

Wind River Indian Reservation believe that the historical animosity is culturally embedded in the political of the reservation. Participants specifically mentioned money as a perpetuating factor for continuing conflict.

Participants in the interview perceive tribal per-capita payments as a premise for animosity. A common theme among the Eastern Shoshone Tribal participants was that they feel that Arapaho Tribal members may resent Shoshone Tribal members because Shoshone

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Tribal members have traditionally received higher per-capita payments. In contrast, a common theme among Arapaho Tribal participants is that they feel Shoshone Tribal members perceive resentment from Arapahos because Arapahos may still think the reservation is unjustly divided. The Wind River Indian Reservation is owned jointly by the Northern Arapaho and Eastern Shoshone tribes. The Eastern Shoshone Tribe has consistently had an enrollment half that of the enrollment population for the Northern

Arapaho Tribe. Since the reservation is divided equally by ownership between the two tribes, Shoshone Tribal members receive higher per-capita payments from the royalties produced on the reservation. Because of this division of payments, the Shoshone participants feel Arapahos still hold animosity toward them.

Generational knowledge was a common theme shared by the participants of both tribes. The younger generations had not ever heard of the term “Diminished Reservation”, or how this term relates to the Wind River Indian Reservation. Many of these participants had never seen or realized that county maps have zoned the reservation as a Diminished

Reservation. The older adult participants could knowledge of “Diminished Reservation”, and the majority of the older generation sample used the term openly.

Intermarriages were also considered a common theme between the Northern

Arapaho and Eastern Shoshone participants. The majority of participants specifically mentioned intermarriage as a factor effecting interpersonal relationships between the two tribes. Participants felt this created a positive interaction between tribal members. Some participants specifically said that intermarriage is something that has occurred in their family and is an issue that they now have to accept.

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Northern Arapaho and Eastern Shoshone relationships have been strained throughout the history of the Wind River Indian Reservation and the overpowering effects of their lack of discourse are evident. The boundaries of the reservation have not been disputed formally by tribal officials, and tribal members perpetuate complacency by accepting the unjust situation. Participants express dissatisfaction over their discourse with white government and the effects of being dealt with in an unproductive manner.

Consensus among the participants was the desire to have the boundaries of the reservation contested in litigation.

Social Justice

Northern Arapaho and Eastern Shoshone people have struggled with the historical and contemporary implications of sharing the Wind River Indian Reservation. Today the tribes require a resolution for the oppression that has resulted from the Northern Arapaho unjust placement on the Shoshone Reservation. Reparations should be made to address the complacency that has resulted because of white encroachment.

Ethically the Northern Arapaho Tribe should have been given a reservation of their own as promised. The Eastern Shoshone people had the opportunity to develop and maintain a reservation of their own, and the government elected to leave the Arapahos on the Shoshone Reservation. Today both tribes are in complete discord because of the decisions made by government officials one hundred years ago. In 1905, the agreement made between the Federal Government and the Northern Arapaho and Eastern Shoshone

Tribes specifically sates that all lands that were not sold under the Riverton Reclamation

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Act, were to be given directly back to the tribes (Eastern Shoshone and Northern Arapaho,

2008).

The city of Riverton now extends many miles north of the Big Wind River and white encroachment is extremely evident. The white community has prospered, and Riverton is considered a hot spot for economic development. Lander was established in the 19th century and Riverton in the 20th, and now Riverton is twice the size of Lander. The reservation has provided economic sustenance for the entire county, yet its contributions are never recognized by county officials. The county uses the population of the reservation to accommodate their budget reports, but the county does not provide services to tribal members. The Conflict Theory states that individuals who own the means of production generally make up the rules and the rules they impose are generally made to protect their resources. The Northern Arapaho and Eastern Shoshone Tribes have endured years of oppression and continue to struggle in their environment (Wilmont, 2010. Van Wormer,

2007).

The Northern Arapaho and Eastern Shoshone are in conflicting positions because they have been put in an awkward situation. The ramifications of the Northern Arapaho placement are far reaching and obstruct the advancement of tribal property rights today.

My study has shown that tribal members feel inter-personal relationships are positive, but they also feel the tribes cannot work together because tribal officials clash. The biggest ramification of sharing a reservation has been that Northern Arapaho and Eastern

Shoshone Tribal members cannot bury the proverbial hatchet. The U.S. Government

23 | P a g e disregarded the legal and human rights of each tribe and put them both in the worst possible situation.

The white community has capitalized on the complacency that tribal members have exhibited concerning tribal property rights. Tribal members feel their property rights are secure because all the maps and documents indicate that the reservation boundaries are legal. Today the white community does not include reservation officials in any aspect of land use or zoning that affects the reservation. When the city of Riverton was known as

Wadswoth and white settlers established their homesteads, Indians were not allowed to enter the city. In the years to come they were forbidden to enter the establishments that the white settlers built and would only serve them thorough the back door. These attitudes toward Indian people exist today, and many of the white people in the community view

Northern Arapaho and Eastern Shoshone people as a burden (Fremont County Planning

Commission, 2004).

The oppressive nature of stereotypical thinking is evident at the state level as former Governor Dave Frudenthal was asked about the property rights of the Northern

Arapaho and Eastern Shoshone Tribes and the area north of the Big Wind River. His response was; “What are the Indian going to do with all that land any way!” The rights of

American Indians in the state of Wyoming do not reflect the sovereign nature expressed in other Indian communities with Federal Reservation status. The city of Riverton has indicated that they will legally thwart any tribal law suit that attempts to define tribal property rights.

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The farms and homesteads are embedded in to suburbs like Hidden Valley, Pavilion,

Midvale, and Paradise Valley. The white settlers used the money that was put into the trust for the Shoshone and Arapaho people and built the irrigation district that serves the white community in these areas. The white people north of the Big Wind River have occupied these lands for generations and have never offered any explanation of their presence on tribal property. All indications are that white people have bought tribal property from county entities and have disregarded tribal property rights. The perception that the white people have is that they have sole ownership of the land they have purchased (Fremont

County, Map, 2011).

The perception of tribal property rights that are held by Northern Arapaho and

Eastern Shoshone people are blurred by their political and economic differences. Historical animosity creates indifference among the tribes and tribal property rights that do not get discussed. The environment on the reservation has been created by mistrust in the government and mistrust among tribal members and officials. Each tribe has traditionally viewed the other as obstacles that impede the interest of their tribal affairs. The older generations have memories of the atrocities that have occurred on the reservation and in the surrounding community. The reservation has suffered the effects of being one of the first Indian Reservations that incorporated a boarding school. The Indians of the Wind

River Indian Reservation have endured much of the hardships that shape American Indian culture today. Their stories are unique and require an understanding of each tribe’s interaction with the Federal Government. The Indian people of Wyoming feel that their land is all they have left and their property rights define who they are in America today.

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Reparations for Northern Arapaho Placement

The unique situation that the Northern Arapaho and Eastern Shoshone Tribes have found themselves in offers many avenues for reparations for Arapaho placement on the

Shoshone Reservation. The government promised Chief Washakie that the Arapaho people would be moved to a reservation of their own. The Eastern Shoshone people made an effort to assimilate into the white culture as much as possible. They believed this would be the best way to adapt to a new way of life. Their people achieved recognition among the soldiers who occupied the camps in the area and began to farm the land (Markley, 1997).

Chief Washakie and Sacajawea represented Shoshone culture, and the white people in the area accepted their presence. Under these circumstances the Shoshone Tribe would have definitely thrived as farmers and God fearing Indians. Today the tribe would be the sole heirs to the Wind River Indian Reservation and all the resources produced on it.

The fraud-the placement of the Arapaho people on Shoshone Reservation- inflicted on the Eastern Shoshone Tribe should be addressed as an issue that has not been resolved.

The actions of the Federal Government need to be addressed and an assessment of the

Shoshone Tribe’s economic loss should be calculated. Reparations could be made in the form of recognition of property rights and compensation for encroachment. The Shoshone

Tribe should also be recognized for the Federal Government’s fraudulent behavior and the suffering they endured from this fraud.

The Northern Arapaho Tribe has had a very strained relationship with the Federal

Government (Northern Arapaho v. State of Wyoming, 2004). They adapted to a nomadic lifestyle and found a new existence on the plains, and in the course of their plight they

26 | P a g e battled the U.S. Military on many fronts (Stamm, 1999). The Arapaho people were first disbanded when the migration of the buffalo was disrupted by the Colorado Gold Rush

(Sherow, 2010). The southern band of Arapaho was attacked along with the Cheyenne people at the Sand Creek Colorado, Massacre in 1864. Arapahos were present at the Battle of Little Big Horn in 1868 and fought with the military until their surrender at Fort Laramie of that year. The Treaty of Fort Laramie 1868 produced an ample reservation for the

Arapaho and included land that extended from Colorado into Kansas and Nebraska.

Unfortunately the government systematically altered agreements with the tribe and the

Northern Arapaho people were shuffled into different locations in South Dakota and

Wyoming.

The Northern Arapaho Tribe did not receive a reservation as promised by the

Federal Government. At one point the Federal Government considered placing the Arapaho on Red Cloud’s Reservation in South Dakota (Markley, 1999). In this instance the Arapaho elders made it clear that they did not want to be assimilated with the Sioux. Instead the government capitalized on the Arapahos’ destitute condition and ordered them on the

Shoshone Reservation, which did not accept their presence. The Northern Arapaho people never had the opportunity to possess land that was rightfully theirs and they were left in a state of perpetual conflict on a land that was promised to another tribe.

By the end of the 20th century, the state of Colorado recognized the plight of the

Arapaho people and offered to give them land in their state. As a form of cultural reparation this land was offered in the mountains near Steamboat Springs for Arapaho preservation, but once again the Federal Government intervened and snatched up the land for Bureau of

27 | P a g e

Land Management. Unlike Colorado’s attitudes regarding tribal sovereignty, the state of

Wyoming has consistently opposed tribal sovereignty and the officials on this level have attitudes that are counterproductive to tribal promotion. It is safe to say that the state of

Wyoming would not recognize that Arapahos are entitled to any such reparations that include tribal sovereignty.

The historical aspects of the Wind River Indian Reservation and Fremont County have been overlooked at the expense of Indian people. Fremont County’s interest in the land north of the Big Wind River creates complacency. Tribal members are not fully educated on the intentions of white people concerning tribal property in this area, and they believe tribal property rights are secure because of government maps indicate ownership.

The white are actively encroaching on tribal property and they consider the area north of the Big Wind River theirs.

The situation on the Wind River Indian Reservation has occurred because all parties-Natives and whites-involved are badly informed. Northern Arapaho and Eastern

Shoshone Tribal members need to direct their attention to issues of encroachment north of the Big Wind River. Tribal members should be reeducated in the historical ramifications of the Northern Arapahos unjust placement on the Shoshone Reservation. They should also question and assess the encroachment of white people onto Indian land.

Fremont County should be audited on every level and every interaction that has involved tribal property in the county should be scrutinized. When dealing with the issue of tribal property rights and Indian land is questioned, all parties become complacent. White people need to mentally reverse the situation and question what their resolve would be if

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Arapaho or Shoshone people encroached on their property. The Indians of the Wind River

Indian Reservation have been treated unfairly and suffer a prejudice that is unique. The reservation has been an economically vital factor in the success of county development, yet segregation and oppression have dominated the social environment.

Implications

The ideal resolution to the problem of complacency and land boundary issues on the

Wind River Indian reservation would be a complete consensus of tribal property rights that reflect Federal Governmental maps and recognition of Northern Arapaho and Eastern

Shoshone historical contributions. Reparations could be achieved if all interested parties are informed with regard to tribal land boundaries and the governmental laws that stipulate American Indian Reservations. Historical facts clearly indicate that an injustice was made by the U.S. Government when they maliciously ordered the Arapahos onto the

Shoshone Reservation. The government made promises to both tribes that the Arapaho people would receive a reservation of their own. An unbiased assessment of the economic losses both tribes accrued because of these governmental decisions should be made.

The cultures of the Northern Arapaho and Eastern Shoshone people should be recognized for their resilience and perseverance that they embrace. The tribes should be financially reimbursed for the economic losses they have endured because of encroachment. Reparations should be made in the form of historical preservation and advancement of the Eastern Shoshone and Northern Arapaho people. All decisions thus far concerning the Wind River Indian Reservation have clearly stripped land away from Indian

29 | P a g e entitlement. The unjust circumstances that have been allowed to occur must be rectified for future generations.

The attitudes and opinions of white community members and officials on the state and local levels are oppressive in nature, and they devalue American Indian existence in

Wyoming. The Indians of the Wind River have provided the white community with various kinds of economic sustenance for over a century; including the relinquishing of land for the cities of Riverton and Lander and currently casino tourism (Eastern Shoshone and

Northern Arapaho, 2008). Fremont County has surpassed most many other counties in population, but the county only uses the reservations population to increase budgetary endeavors. Fremont County will continue to monopolize the Eastern Shoshone and

Northern Arapaho people because that is how it has been for the last hundred years. The white people will continue to encroach on Indian land and future generations will have more obstacles to overcome. County officials will not want to relinquish tribal lands that have already been encroached on. They have earmarked tribal land for future endeavors and continue to keep Indians and white people segregated (Fremont County Planning

Commission, 2004). The issue of tribal property rights in Fremont County can no longer remain dormant, and white people need to be held accountable for any illegal behavior that has infringed on the rights of the Northern Arapaho and Eastern Shoshone people.

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References

Anderson, J. D. (2011). The History of Time in the northern Arapaho Tribe. Ethnohistory, 58, 2 229-261.

Antell, J. Blevins, A. Jensen, K. & Massey, G. (1997). Residential and Household Poverty of American Indians on the Wind River Indian Reservation.

Eastern Shoshone Tribe and Northern Arapaho Tribe of the Wind River Reservation. (2008). Statement of Legal Counsel Regarding the Tribes’ Authority to Regulate Air Quality (December 17, 2008).

Fowler, L. (2003). The Four Hills of Life: Northern Arapaho Knowledge and Life Movement. [Review of the book The Four Hills of Life: Northern Arapaho Knowledge and Life Movement] Ethnohistory, 50, 4, 728-729.

Fowler, L. (1978). Wind River reservation Political Process: An Analysis of the Symbols of Consensus. American Ethnologist, 5, 4, 748-769.

Fremont County, Map. (2011) Map of Land Zoning in Fremont County, Wyoming. Office of Assessor, Fremont County, Wyoming. County Court House, located in Lander, Wyoming.

Fremont County Planning Commission: Fremont County Wyoming Land Use Plan (2004).

Frosch, D. (1020, December 19). Custer’s Last Stand Was Only the Beginning. The New York Times, pp. A.22.

Kearney M. S. (2005). The Economic Winners and Losers of Legalized Gambling. National Tax Journal, 58, 2, 281-302.

Markley, E. R. (1997). Walk Softly, This is God’s Country: Sixty-Six years Among Shoshone and Arapahoe Indians 1883-1949 Wind River Reservation. Letters and Journals of Reverend John Roberts. Lander, WY: Mortimore.

Murphy, J. C. (1969). The place of the Northern Arapahoes in the relations between the United States and the Indians of the Plains 1851-1879.Annals Of Wyoming, 4113.

National Atlas. (2011). [interactive map displaying federal lands]. Retrieved from http://www.nationalatlas.gov/mld/indlanp.html.

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Northern Arapaho Tribe v. States of Wyoming and Governor Jim Geringer (2004). United States Court of Appeals Tenth Circuit Nos. 02-8026, 02-8031 (Gaming Law Review App. Ariel IP: 129.82.28.195).

Oxford University Press. (2008). The State of the Native Nations: Conditions Under U.S. policies of Self-Determination. The Harvard Project On American Indian Economic Development.

Professor: Reservation poverty discourages political involvement. (2007). News from Indian Country, 21, 4, 14.

Sherow, J. E. (2010). Storms of the Grasslands: Indian peoples and the Colorado Gold Rush of 1859. Journal of the West, 49, 2, 15-22.

Stamm, H. E. (1999). People of the Wind River: The Eastern Shoshones 1852-1900. University of Oklahoma Press: Norman, Publishing Division of the University.

Tefft, S. K. (1971). Task Experience and Intertribal Value Differences on the Wind River Reservation. Social Forces, 49, 4, 604-614.

United States Census Bureau. (2010). Population Demographics Fremont County, Wyoming [Data file]. Rettrieved from http://quickfacs.census.gov/qfd/sattes/56/56013.html. van Wormer, K. (2007). Human Behavior and the Social Environment, Macro Level: Individuals and Families. 198 Madison Avenue New York, NY: Oxford University Press.

Wilmot, K. A., & Delone, M. A. (2010). Sentencing of Native Americans: A Multistage analysis Under Minnesota Sentencing Guidelines. Journal Of Ethnicity In Criminal justice, 8, 3, 151-180

Yellowbear v. State of Wyoming, No. 06-246, 174P.3d 1270, January 14, 2008.

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Scott Cheney Mentor: Dr. Lisa Hunt Major: Anthropology

Research Topic: A Medieval Perspective A Digital and Iconographic Exploration

During the years of the Second Bulgarian Empire, Bulgaria was characterized by war and vast holdings that rivaled the powers of Rome and Byzantium, and provided refuge to the relatively young religion of Christianity.1 Following the fall of the empire in the 17th century and the subsequent Ottoman invasion out of Turkey, religious art retained the traditional late Byzantine forms. Church murals and religious icons are still used in

Orthodox religions today, and their medieval predecessors can be found upon on the walls and vaults of the many medieval chapels that dot the landscapes of Eastern Europe. Many of the frescoes in the more distant monasteries have not survived well over time and are currently suffering from neglect (Fig. 1). Often, physical and chemical restoration is the only aid, but these techniques require the funding and experience that can be difficult to

1 Nicolas Zernov, Eastern Christendom (New York: Putnam Publications, 1961), 108-110; R. Crampton, A Concise History of Bulgaria (Cambridge: Cambridge University Press, 2007), 9-28; Frederick Chary, The History of Bulgaria (Santa Barbara, CA: Greenwood, 2011), 3-22.

33 | P a g e find in Bulgaria.2 Thus, documentation of these images is paramount, as is research into alternate forms of restoration and conservation. This study will explore the application of iconography and digital reconstruction to damaged murals, in the effort to virtually preserve fragments of a neglected history.

The small monastery chapel of Sveta Bogoroditsa in Iskrets, dedicated to Saint Mary the Mother of God, or the Holy Theotokos, is not far from the village of the same name, situated near the Iskur Gorge in the low hills of the Balkan range of western Bulgaria. At one point in time this small chapel was part of a larger monastery compound built in the fourteenth century during the Second Bulgarian Empire, which was destroyed during the

Ottoman period and the chapel was restored in the sixteenth and seventeenth centuries.3

Dating to 1602, the frescoes of this chapel follow the traditions seen in other orthodox chapels throughout medieval Eastern Europe and Russia.4 Except for a report done on the chapel and the frescoes by Dora Kamenova in 1977, there has not been significant data recorded about the Iskrets monastery chapel. Unfortunately the frescoes, as well as the building as a whole, are quickly deteriorating. This paper will demonstrate the utility of digital preservation and documentation of images and meanwhile argue for a strong cultural connection to the local Bulgarian community through the application of iconographic analysis.

In the past, Adobe Photoshop has been utilized to restore and virtually conserve renaissance paintings and antique photographs through digital images and Adobe’s Color

2 Vasilev, personal communication. 3 Ibid; Tatiana Nikolaeva Nikolova-Houston, Margins and Marginality: Marginalia and Colophons in South Slavic (Austin: University of Texas at Austin, 2008), 84. 4 Dora Kamenova, Mural Paintings of the Iskrets Monastery (Sofia: Bulgarski Khudozhnik, 1977), 162-164.

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Cloning process (Adobe). This process can copy the same RGB color from other sections of the image to cover and fill spots, cracks, and empty areas within images. This paper proposes that this method can be implemented to start conservation practices on medieval frescos using extant digital photographs from the database of The Balkan Heritage Field

School. The digitally restored images will then be compared to other frescoes from the region and to religious images from Eastern Europe and Russia in order to re-create the iconographic program of a wall of the monastery chapel. This work will be supplementing the existing image database of the Balkan Heritage Field School, and this research will explore a form of art conservation that can be used when other options are limited or when one is exploring the restoration potential of a medieval fresco, as is the case of The Balkan

Heritage Field School.

In the Sveta Bogoroditsa chapel of the Iskrets Monastery, the presentation of the religious images follows the traditions that one would see in medieval chapels throughout western Bulgaria in the simple single-apse churches surmounted with barrel vaults. The walls are divided into five registers (Fig. 2). The walls and the apse are unified by the lowest register containing a simple decorative pattern of red criss-crossing lines forming a diamond shape on a beige colored background. On the north and south walls the eye level register holds the full-length portraits of six to eight saints belonging to the Bulgarian

Orthodox religion, some of which still have their names inscribed above their heads for identification. In the third register, before the wall meets the barrel vault, there is a succession of busts of sixteen to twenty martyrs. Unlike these registers, many of the upper registers contain significant narrative, and traditional, scenes.

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On the eastern wall flanking the apse opening, the second register includes a full- length depiction of a warrior archangel to the left and a high priest to the right of the apse, emphasizing the role of guardianship that the priest possesses which is a scene that is echoed in several of the frescoes. The third register holds the scenes of “The Annunciation” on the left and right of the apse with the archangel Gabriel on the left and the image of Mary on the right. In the top register of the eastern wall, there is a narrative that depicts a priest, possibly Melchezidech, holding out a banner and is surrounded in a half circle of eight to nine holy men. Flanking this central image are two badly damaged panels, each holding one of the four evangelists, which are similar in style and composition to the panels in the top register of the opposite wall which will be examined later in this study; the damage to these images has erased any narrative inscriptions as well as the heads of the evangelists. In the apse itself, there are six church doctors depicted in the second register, and above these is the image of “Christ administering the First Eucharist,” again echoing the priestly theme. In the vault of the apse, located directly above the altar, is the image of “The Mother of the

Sign,” which contains a large mural figure of the Virgin Mary holding out her arms with a medallion of a child Christ, Christ Emmanuel, on her chest. In the zenith of the vault are the traditional images of “Christ as Final Judge,” “Christ as Ancient of Days,” and another badly damaged image in a medallion. Radiating from the zenith of the vault there are images from the cycles of the infancy and the passion of the Christ, such as “The Nativity” and “The

Crucifixion,” presented in narrative registers along the length of the vault.5

The western wall of the chapel holds the narrative images of the namesake of the chapel, Saint Mary, the Holy Mother of God, and forms the case study of this paper (Fig. 2).

5 Vasilev, personal communication.

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The second register frames the doorway of the chapel, with images of St. Michael and St.

Gabriel to the left and right of the door respectively, echoing the depictions of the warrior archangel and the high priest on the eastern wall. Two seraphim flank the archangels and a later inscription from 1845, written in Old Church Slavonic, is placed above the doorway of the chapel in the central panel of the register. Unlike the male saints and martyrs on the north and south walls, the register above the doorway holds the busts of the female martyrs of Saints Lisaveta, Anna, Marina, Catherine, Barbara, Natalia and Petka, as well as one unknown saint at the beginning of the register. These martyrs are encased in red borders with the background sub-divided into gold and blue. The main register shows three panels depicting the life of Mary; from left to right they are: the Presentation of Mary at the Temple, the Dormition of Mary, and the Entombment of Mary. These images are all of near equal size, roughly four feet wide. The top register also holds three full-length figures: the central panel holds the image of the Assumption of Mary and is flanked by the images of two evangelists, again echoing the panels of two other evangelists on the eastern wall.6 This top tier has sustained a considerable amount of damage; the upper halves of the two evangelist images have been lost, as well as almost the entire upper half of the central narrative. Additionally, there are numerous large cracks in the church walls that continue through the images, and many gouges and divots mar the surface of the plaster. The register below has also sustained some damage in the form of large cracks in the first two images coming from the upper tier with large portions lost or significantly damaged. While

Adobe Photoshop will be useful to correct these images virtually, the lost iconography must be recovered by comparing the existing portions of the images to extant iconography found

6 Ibid

37 | P a g e in chapels from the region. This damage necessitates both the software and iconography to ascertain the missing parts; the software of Adobe will be useful in removing the smaller pockmarks and cracks seen in the narratives while the iconographic research will be of great assistance, since nearly 50% of the top tier has been lost. Moreover, this conservation will be of assistance in connecting the themes of the chapel to the regional history.

The top two registers narrating the life of Mary contain traditional compositions. In the main register, all three of the panels are unified by a constant blue background situated behind a series of buildings, painted in either red, white or yellow, with simple blue shadows. The buildings vary in size and exhibit triangular roofs, clear lines and small windows. Dark shading is used in the windows and short, diagonal lines are used to show recession. The background yields to the illusion of a continuous cityscape to unify the three episodes of Mary’s earthly life. Throughout the images the coloring in the robes of the figures is very similar, with the men clad in red tunics beneath flowing white robes and the women in plain red robes over white tunics. This commonality is broken up most notably by the depictions of St. Mary, who is shown in a royal purple robe in the first two scenes and in stylized bandages in the last narrative; this shift emphasizes the figure of Mary, who is also typically located in the center of the scenes. The coloring of the robes typically creates a pattern, of red then white, and the curved lines in the robes creates a rhythm that effectively leads the eye to the central figure of St. Mary. Additionally, this patterning of robes is occasionally broken by other hierarchical figures such as Christ in the central narrative who is depicted in a gold robe, and the figure of the high priest Zacharias, who is shown in a flowing white robe with a long white beard.

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The narratives begin on the left with the “Presentation of Mary to the Temple.” In this image there are nine to ten individuals. At the far left is a huddled group of witnesses with three full bodies and several heads behind them moving left to right along a stylized walkway. In the center of the image, Saints Joachim and Anne, father and mother of the

Virgin Mary, present the young girl to the high priest Zacharias to the right.7 Joachim and

Anne face one another and, with their outstretched arms, they gesture toward Mary to the right. In the center is the small figure of Mary, and with her arms she addresses the priest

Zacharias, who is ensconced in a curved white throne.8 Saints Mary and Zacharias are the only two figures depicted on raised red square platforms, elevating them separately from the others on the pavement. This hierarchy is further enforced not only by the throne surrounding the priest, but also his placement directly in front of the largest building in the background, presumably the temple (Fig. 7).

Iskrets’ “Presentation of Mary to the Temple” shares many similarities with the central image of a 1598 Russian veneration icon now in the Moscow Museum (Fig. 8). In both fresco and icon, a group of figures in the left side of the icon appear as witnesses to the holy scene and the background is made up of neutral-colored buildings, with a large white temple located to the right. Like Iskrets, the central character in the image is that of the young Virgin Mary, who stands on a raised platform draped in the royal color of purple, unlike anyone else in the image. Additionally, behind the young virgin Joachim and Anna

7 Alfredo Tradigo, Icons and Saints of the Eastern Orthodox Church (Los Angeles: Getty Publications, 2006), 98; Nikodim Pavlovich Kondakov, Icons (New York: Parkstone International, 2008), 225; Georgi Geroz, Icons From Melnik and the Melnik Region, Bulgaria (Sofia: Pensoft, 2007), 25-26; Henry Maguire, The Icons of Their Bodies (Princeton: Princeton University Press, 1996), 154-157; Simon Marsink, The Power of Icons (Ghent: Snoeck Publishers, 2006), 118-119; Maria Vassilaki, Mother of God (Milan: Skira Editore, 2000), 72-74; 131. 8 Ibid

39 | P a g e present their daughter to the temple. In both images, Zacharias is in front of the largest building in the background and is placed in a stylized curved throne.

The curved throne that is depicted in both of the images harkens remarkably to the

6th century ivory throne of Archbishop Maximian of Ravenna (Fig. 3). As in the fresco and icon, this throne has a curved back and figural panels following the curve of the throne and in the paintings this throne is depicted as being made of a separate material, shown by being painted in white, and the throne of Maximian is carved from plaques of valuable ivory. Multiple images culled from both Russian and Eastern European collections contain similar curved thrones, and often for an important figure like a high priest as well as the

Madonna and Child (Fig. 4-6).9 In a comparable image from Novgorod, a priest is ensconced in a curved throne participating in the veneration of the holiest symbol of the orthodox faith: the cross. In other comparable images from Russia and Greece, the virgin Mary with the Christ child sit in a curved throne symbolizing strength and power. The placement of the throne around Zacharias and in front of the largest building in both of the images serves to assert its importance and perhaps the divine authority invested in the figure of

Zacharias. Moreover, the placement of the throne emphasizes the important role of the priest; as is also seen around the apse, the iconographic features emphasize the important and divine pastoral role taken on by the monks of Iskrets to the surrounding community.

The “Dormition of the Virgin” is in the center of the register and directly over the only doorway of the chapel (Fig. 9). At the center of the image Mary, with her eyes closed, lies upon a raised funeral bier, which is draped in a red and gold cloth stylized with small

9 Kondakov, Icons, 126; Marsink, The Power of Icons, 98-99; Vladimir Ivanov, Russian Icons (New York: Rizzoli International Publications, 1988), 105; 111.

40 | P a g e white details to simulate an expensive textile. At the head of the Virgin Saint Peter holds a censor and at the foot Saint John mourns over the body.10 The other apostles also appear encircling the funeral bier.11 Located behind the bier, at the center of the image, Jesus

Christ is surrounded by a large almond-shaped mandorla and flanked by two angels, both with red robes and red wings; the upper part of the mandorla and the angel on the right are badly damaged by smoke. In Christ’s hands, he holds a small, nearly indistinguishable bundle that, according to legend, is the soul of St. Mary, which was carried into heaven by

Christ himself. This pairing creates an interesting parallel to the typical image of the Virgin

Mary holding the young figure of Christ, as well as the image of the “Mother of the Sign” located opposite, in the apse of the church, which is located roughly at the same height (Fig.

10).12 The foreground of the image, in front of the bier, contains the scene with the Jewish priest, Jephonius, who is typically depicted in Eastern Catholic images.13 According to legend, Jephonius doubted the sanctity of the Virgin and attempted to tip over the bier and his hands were swiftly cut off by the Archangel Michael, also depicted here next to the priest as a small figure with splayed white wings and a simple white robe, wielding a sword and cutting off the hands of the priest, which fall to the left of his body.14 The small scale in

10 Kondakov, Icons, 126; Marsink, The Power of Icons, 98-99; Vladimir Ivanov, Russian Icons (New York: Rizzoli International Publications, 1988), 105; 111. 11 George Ferguson, Signs and Symbols in Christian Art (New York: Oxford University Press, 1954), 121-122. 12 Kondakov, Icons, 92-93; Gerov, Icons From Melnik and the Melnik Region, Bulgaria, 104-105; Maguire, The Icons of Their Bodies, 54; Robert Nelson, Kristen Collins, Icons from Sinai (Los Angeles: J. Paul Getty Trust, 2007), 251- 253; Marsink, The Power of Icons, 58-59; Gojko Subotid, Art of Kosovo (New York: The Manacelli Press, 1998), 101- 104; Ivanov, Russian Icons, 16. 13 Geroz, Icons From Melnik and the Melnik Region, Bulgaria, 104-105. 14 Kondakov, Icons, 174; Gerov, Icons From Melnik and the Melnik Region, Bulgaria, 82-83; Maguire, The Icons of Their Bodies, 62-65; Slobodan Durčid, Gračanica (University Park: The Pennsylvania State University Press, 1979), 81; Bertrand Davezac, Four Icons in the Menil Collection (Houston: Menil Foundation Incorporated, 1992), 25-43; Robin Cormack, Painting the Soul (London: Reaktion Books, 1997), 196; 207; Marsink, The Power of Icons, 174-175; Subotid, Art of Kosovo, 88; 49-50; Helen Evans, William Wixom, The Glory of Byzantium (New York: The Metropolitan Museum of Art, 1997), 112-113; Ivanov, Russian Icons, 60-62; Vassilaki, Mother of God, 132-133; Robin Cormack, Maria Vassilaki, Byzantium 330-1453 (London: Royal Academy of Arts, 2009), Pg. 441.

41 | P a g e which the priest Jephonius is depicted contrasts greatly with the scale in which Zacharias enthroned is shown in the preceding narrative.

The image of the Dormition from a fourteenth-century monastery church in

Gracanica, Serbia, is very similar in both form and style to the Iskrets panel (Fig. 11). Both images are located centrally above the church doorway, have blue as the dominant background and foreground color, and the backgrounds both have a simulated cityscape. In both, Mary is clothed in a purple robe and lies on a bier in the center of the image upon a stylized textile. The episode of Jephonius is occurring at the foreground of the Serbian image with the figure of Saint Michael, holding a bloody sword, in the top left of the image.

The apostles lead the funeral bier in a procession to the right with three apostles acting as pallbearers, while a heavenly host surrounds the bier and follows the procession. Again,

Christ stands centrally behind Mary’s bier, surrounded by a pointed mandorla, and he holds in his arm the infant soul of Mary. A difference in the wider and more populated

Serbian composition is found directly above Christ: a pair of open doors in which contains a further host of heavenly attendants. Unlike the Bulgarian image, these figures in the

Serbian fresco are fairly muddled, and the characters of Saints Peter and John are not readily identifiable by their positions or emblems. The overall translation of the Serbian image is more imperialistic rather than somber, exemplified by the large amount of individuals behind the figure of Christ and the procession, illustrating the power of Christ and by extension the powers of heaven and the church.

Additional narratives depicting the “Dormition” from a twelfth-century mosaic in

Istanbul and a thirteenth-century Novgorod icon provide useful comparisons as well to

42 | P a g e compare the Iskrets scene (Fig. 12-13). In these two images, Mary lies upon a simulated textile, clad in a purple robe, and the Christ stands in an almond shaped mandorla directly behind the funeral bier holding the personification of Mary’s soul. In these, Saint Peter holds the censor and Saint John mourns at the feet of the Virgin. In the Novgorod image there are numerous angels and multiple deified individuals who look down upon the scene, including the twelve apostles. While the mosaic and icon lack the episode of Jephonius, they do illustrate older conventions such as the stylized textile simulating an altar cloth, the role of the priest as Peter holding the censor and the candlesticks harken to the liturgy; all of these conventions work in tandem to point to the importance of the pastoral role emphasized in the Iskrets chapel.

In the third and final image from this register, “Entombment of Mary,” thirteen individuals surround the body of St. Mary at the center (Fig. 14). At the left of the image there is a separate group of eight mourners all standing still, facing the body of the Virgin.

Again, at the head of Mary is St. Peter, carefully cradling her head as she is lowered into her tomb while at her feet St. John mourns.15 The tomb in which they are placing the body is red with short linear lines and dark shading to simulate the depth of the tomb. The body of the Virgin is wrapped in white bandages highlighted with red and forming a crossing pattern of multiple lines. The background cityscape is interrupted by a stylized mountain of earth in varying gray tones located behind the group of mourners that swoops down and under the tomb of Mary. Finally, dominating the cityscape in this image is a large white building very similar to that seen in the “Presentation;” the similar building frames the rightmost of the three images, providing a parallel end.

15 Ibid

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The less-often known scene of the “Entombment of the Virgin” is usually coupled together with the better known image of the “Dormition.”16 The Bulgarian image is comparable to an image from Italy in 1430 by the artist Francesco d’Antonio (Fig. 15). The d’Antonio panel compares to the medieval wall painting in that the figure of Mary is at the very center of the image surrounded by a group of individuals. Similar in style to the

Bulgarian images is the dominant color of red in the apostles clothing and the figure of

Mary in a purple robe to reiterate her religious royalty; additionally, the background of the

Italian predella is done in hues of brown illustrating the feeling of an earthly environment in which they are lowering Mary’s body, similar to the large grey hill and grey foreground that is in the Bulgarian image. It is important to note here that this Italian image is comparable with “Dormition” images seen from Eastern Europe with the figure of Christ standing among the apostles behind Mary, holding Mary’s soul in his arms. Also, the figure of Mary in the Italian image is garbed in a full robe contrasting the burial cloth made up of crossing linear lines that is seen in the Iskrets painting. And, while it does appear that Mary is lying on a funeral bier in the Italian image, the bier is not covered in a stylized textile like the Bulgarian image or the other comparable images that have been examined and leads to the appearance of Mary being lowered into a grave or casket. This lack of textile as well as the earthy background is overall more similar to the scene of the “entombment” rather than the narrative of the “Dormition;” nevertheless, the iconographic features present emphasize the pastoral care of Mary in death by the apostles and the priests.

16Ferguson, Signs and Symbols in Christian Art, 121-122; Vassilaki, Mother of God, 132-133; Cormack, Vassilaki, Byzantium 330-1453, 441.

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The top register of the western wall also houses three panels, however, as stated previously, they have sustained considerable damage. These three images share a continuous dark blue background and floor and, in the first and last image of the two evangelists, there is what appears to be a cityscape or some kind of architecture to make up the background of the images, but this linking feature cannot be seen in the central image of the “Assumption of Mary.” Like the rest of the wall, these three panels maintain the canon of only a few colors to make up the scenes. While the damage is extensive to the point where iconographic analysis is limited, certain features of the Adobe software can help read what identifying features may be present.

According to Kamenova’s initial iconographic map, the “Assumption of Mary” is in the center of the top register (Fig. 16). In this scene three clear figures remain in addition to partial fragments of three other figures. In the center of this image is a mandorla, on the center axis of the western wall, with the bottom portion of a figure that is dressed in a purple robe over a white undergarment; with the appearance of purple robes in the previous register, this figure framed in the illuminated mandorla is Mary making her ascension into heaven, where her corporeal body will reconnect to her soul.17 Following traditional iconography, the three full figures on the ground are the apostles, cowering from what occurs above them and two red rays of light come from the mandorla towards the three apostles on the ground. Framing the mandorla, there appear to be two angels on either side, similar to the angels seen in the “Dormition” narrative flanking Christ: the figure to the right of the mandorla is better preserved, the figure is wearing a gold robe

17 Ferguson, Signs and Symbols in Christian Art. 121-122; Gerov, Icons From Melnik and the Melnik Region, Bulgaria, 126-127; Cormack, Painting the Soul, 196; Marsink, The Power of Icons, 174-175; Subotid, Art of Kosovo, 88; Helen Evans, Byzantium Faith and Power 1262-1557 (London: Yale University Press, 2004), 284-286.

45 | P a g e under a red one and has red wings; the figure to the left of the mandorla has been nearly completely lost, leaving behind the hem of a red robe and the tips of red wings. Behind the central image of the Virgin there appears to some kind of an earthen mound or even an architectural structure painted in white.

Similar to the Iskrets image is a comparable wall painting from the fourteenth- century Gracanica monastery in Serbia (Fig. 17). Although the Iskrets painting is badly damaged, the connection in overall style between the two images is striking even though the composition is markedly different. In each image the background is dominantly blue and the narratives in both images are divided by red borders and the drapery seen on the figures is very similar to the style of the Iskrets images in that the drapery follows the movement of the bodies and the highlighting is done in a stark-white, highlighting the folds and movements of the fabric. Also harkening to the Iskrets images, the male figures appear to have more attention and detail in their folds when compared to the figure of Mary who is clad in a simple purple robe. In both images Mary is elevated above the rest of the scene and is encased in a mandorla with two angels flanking her. Below the figure of the ascending Mary in both images are multiple individuals, presumably the apostles; yet in the

Serbian image the apostles are looking into an empty tomb while in the Bulgarian image they are cowering on the ground; overall, the Serbian image looks like a compilation of

“The Entombment” and “The Assumption.”

Since the damage to the Iskrets image is undeniable, the narrative being presented in the top register is somewhat obscure; however, by comparing what is present in Iskrets to other images, iconography can suggest ways to fill in the gaps.. When comparing the

46 | P a g e image to other like images, such as the 1715 Bulgarian image of “The Transfiguration” from

Bozhkov or even the 13th Century Greek manuscript image of “The Transfiguration” from the “Tetraevangelion” shown by Evans, we see many compositional elements that are shared with Iskrets (Fig. 18-19).18 While the subject of both of these images is the transfiguration of Christ, in both of these images the central figure is elevated above the rest of the individuals and is encased in an almond shaped mandorla with rays of light coming from the mandorla towards the figures on the ground below. Additionally, the central figure is flanked by angels and the apostles on the ground. All of these elements are seen in the composition of the assumption at Iskrets and, though the central figures of the scenes are different, the subject of transfiguration/assumption is the same and one can conclude that in the Iskrets image, the central figure of Mary would have been flanked by angels and the apostles would serve as witnesses to the scene.

The two figures that flank “The Assumption,” dubbed by Kamenova as “Saint

Evangelists,” have sustained considerable damage with nearly the entire top halves being lost (Fig. 2). In both of these images a male figure sits upon a raised dais with their feet resting on raised platforms. Due to the damage, narrative inscriptions and heads of the evangelists have been lost; however, with the aid of research and digital conservation efforts, it is possible to suggest the identity of the figures. The figure present at the right of the top register is somewhat easier to decipher when one examines the traditional elements that tend to appear. In the image, the evangelist has what appears to be a codex sitting next to him on the dais and also appears to be holding something in his hand. After

18 Atanas Bozhkov, Bulgarian Icons (Sofia: Bulgarski Houdozhnik Publishers, 1987), 93; Evans, Byzantium Faith and Power (1261-1557), 284.

47 | P a g e the digital conservation, it became clearer that the item that the individual is holding is triangular in shape and is void of a center and the triangular shape acts as a frame; additionally, the software helped illustrate that the individual is sitting on a dais and his feet rest on a raised platform. This scene is reminiscent of an icon of the evangelist Luke from Bulgaria in the eighteenth century (Fig. 20). Like the Iskrets painting, this individual is seated and his feet rest on a raised platform. In the 18th century image, Luke is painting an icon of Mary holding the Christ child, illustrating the traditional story of the first icon of

Mary. The overall triangular shape that is made in the comparable image by the painting easel is comparable to the shape seen in the Iskrets image. The open codex that is sitting next to the evangelist in the Iskrets painting may be equally defining of Luke. It is tradition that Saint Luke is shown either painting the first icon of Mary, or writing his gospel.19 As is seen from a Macedonian manuscript, the figure of Luke is again seated with his feet resting on a raised platform (Fig. 21). In both of the comparable images, the figure of Luke is writing his gospel, illustrated as either a scroll or an open book, and sitting in front of the individual is a podium upon which Luke can write his gospel. These features, the possibility of a painting easel or a writing podium and the open manuscript, as well as the tradition of the figure of Luke often accompanying images of Mary, all work in tandem to support the claim that the right figure in the top register from Iskrets monastery chapel is indeed that of the evangelist Luke.

The other evangelist, however, does not provide us with these traditional elements.

The images of the four evangelists are typically shown writing their gospels and as such,

19Ferguson, Signs and Symbols in Christian Art, 234; Maguire, The Icons of Their Bodies, 8-9; Cormack, Painting the Soul, 45; Subotid, Art of Kosovo, 213; Evans, Wixom, The Glory of Byzantium, 103-104.

48 | P a g e this obscure figure at Iskrets could be any of the remaining three evangelists: Matthew,

Mark or John. Following the traditions of more western programs, the figure of John the

Evangelist is traditionally shown alongside Mary, and this tradition is exhibited by a fourteenth-century image from Turkey (Fig. 22).20 In the manuscript illumination, the seated figure has an open manuscript on his lap and is sitting next to a desk. One interesting comparison that can be made between the Turkish and the Bulgarian images is that of a curved throne; While there are a few comparisons present, the matter of what evangelist is depicted in the first panel of the register is still obscure, but that in itself is a result that illustrates a need and outlines the possibility of more digital conservation practices.

It has taken several years and countless software products to bring the digital age into the realm of art history, but since its reluctant induction, software has been an integral catalyst in progressing art history and analysis. Software has opened the doors for the restoration of historical art pieces using digital images. As noted by Jan Blažek, digital images are comprised of thousands of pixels and digital software technology uses mathematical algorithms working on a pixel-wise basis to transform images and apply a variety of analyses to them.21 These analyses consist of infrared reflectography and wavelet transformations used to raise certain colors or hues from a piece of art that can help the researcher in discerning the image despite the previous damage; additionally, x-ray scans

20 Kondakov, Icons, 40; 52; Maguire, The Icons of Their Bodies, 16-18; Marsink, The Power of Icons, 138-139; Sarah Brooks, Byzantium: Faith and Power (New York: The Metropolitan Museum of Art, 2006), 144-145. 21 Jan Blažek et al., Fresco Restoration: Digital Image Processing Approach (Presented at “The 17th European Signal Processing Conference,” 2009).

49 | P a g e are gaining popularity because if their ability to “see through” to images that may have had additional paint or plaster layers placed on top of them over the years.22

When Wolfgang Baatz and his team underwent a restoration endeavor of the fourteenth century Neidhart frescoes, they faced a large amount of fresco panels that were in the state that the Iskrets frescoes are in today. There were numerous holes in the frescoes due to previous restoration attempts as well as considerable amounts of the images lost due to time and neglect. This team approached the project with an algorithmic technique known as the grayvalue approach; this approach can allow for the inpainting of homogeneous areas in the frescoes. However, as is noted by Baatz, this technique is not very satisfying when one attempts the continuation of the edges into the larger holes in the plaster.23 Another issue that is now facing the realm of digital restoration is the idea of

“Undo,” being able to reverse an action or even a whole series of actions within the digital process. This is a luxury that can be afforded to most digital techniques since actions are often tracked within the programs, however, techniques such as spectroscopy and x-ray study run the risk of causing irreversible damage to the artifacts and only exacerbate the situation.24 The present examination uses Adobe Photoshop in order to make the case for a more invasive and cost-efficient means of digital examination. These results may be preferred by non-profit organizations, like the Balkan Heritage Field School, who do not have the funds for in-depth digital research such as spectroscopy or x- ray analysis.

22 Blažek, Fresco Restoration: Digital Image Processing Approach; Central Science, “Digital Restoration: Introducing and Undo Function,” accessed October 29, 2011, http://cenblog.org/artful-science/2011/05/15/digital-restoration- intriducing-an-undo-function/; Carola Schönlieb, “Restoring Profanity,” Plus Magazine, 50 (2009): 3. 23 Wolfgang Baatz et al., Binary Based Fresco Restoration (Presented at “Bridges: Mathematical connections in Art, Music, and Science,” Banff International Research Station, 2009). 24 Central Science, “Digital Restoration: Introducing an Undo Function”.

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The name “Adobe” has become synonymous with digital photography. Many people, like myself, have used the simpler elements of Adobe Photoshop to manipulate light levels and color balances in order to turn a “good” photo into a “great” photo. However, another popular feature of the Adobe software is that of the color cloning and rubber stamp tools where the colors are copied and moved to damaged parts of the image in order to digitally repair antique photographs and even edit scratches and indentations in digital images of renaissance paintings. With the new Adobe Photoshop Creative Suites 5, the designers at adobe have combined these two tools and created a feature called “Content Aware.” This feature mimics textures, space, line and color balances to fill in an area, allowing one to remove or edit entire sections of a photograph with ease.25

Using the content aware feature of Adobe Photoshop CS5, I went through the six images from the upper registers of the western of the Iskrets chapel, and the software worked as well as one could hope for. As is seen in the “Presentation” narrative for example, the software was able to fill in the divots made by previous conservation efforts as well as “repair” scratches, holes, cracks and nearly anything else in between (Fig. 23).

Using modern digital means, I was able to bring forth a clearer view of the image and at an absolute fraction of the cost when compared to the price of IRR photography or physical conservation of the narratives in situ. Another interesting feature that I was able to utilize when examining the images was the act of inversion.

Inverting an image is taking the colors to a “hyper scale” and intensifying them in either a warm or cool color scheme. By highlighting shapes and shadows obscured by salt

25 Photoshop Essentials, “Photoshop CS5 New Features-Content Aware Fill,” 2011, (Accessed June 15th, 2011)

51 | P a g e or smoke damage, inversion enhanced certain elements and characteristics within the narratives like Luke’s easel to substantiate the iconography. In the last image, what I have concluded as “Saint Luke the Evangelist,” inversion helped to illustrate the cityscape comprising of the background of the image as well as the open manuscript lying next to

Luke; inversion also highlighted the shape of the easel (or writing podium) that is placed in front of the evangelist (Fig. 24). Additionally, when using inversion in the narrative of the

“Assumption,” it became clear that the two figures flanking Mary do appear to have wings coming off of their backs and that Mary is transcending into heaven and is surrounded by a holy mandorla (Fig. 25). Also, the inversion allows us to follow the lines that make up the flow of the apostles robes as well make out their facial expressions more than we could with the naked eye, lending to the interpretation that the apostles appear to be stunned by the holy event occurring before them, although more support in iconography is still needed

(Fig. 26).

When looking at the before and after images, there is no doubt that the software of

Adobe Systems is impressive. However, there are many things that it cannot do, and these issues are probably things that no software can do. For example, the Content Aware feature cannot rebuild letters or words and when there is a rough space in the image that is fairly large, then the software tends to falter and is incapable of rebuilding some fragments. Also, this software cannot simply reverse the extensive damage and obscurity caused by smoke and the natural salts found within plaster. At this moment in time, it is my assertion that software cannot replace a trained restorer who has the funds to do an in situ conservation, but what this examination provides is the first step. Combining these digital efforts with iconographic research is the first step in filling the research gaps that exist and it is the first

52 | P a g e step in the creation of a searchable and comparable database of Bulgarian iconography which is sorely needed if research of this region is to continue. When one is working to substantiate the program of a church’s decoration that has miraculously lasted through time intact, a comparable database is an absolute must. Although one can compare images from Turkey, Greece, Macedonia, Russia and even Bulgaria, however, only a few images match well in stylistic elements as well as traditional forms. When searching for a particular image or narrative on the ArtStor database, results are comprised of images from Renaissance Italy or France which are not comparable when one is researching images hundreds of miles away and spanning centuries difference. While Princeton does offer the Index of Christian Art for academic use, it is extremely cost prohibitive which limits one’s access to their resources. What is needed is a searchable, international, and accessible digital database that consists of comparable images and programs pulled from

Bulgaria as well as the rest of Eastern Europe. This would enable researchers to identify saints and compare styles and designs to assign potential artistic schools to these images which is still coming to light in the 21st century. This would most defiantly be a substantial undertaking to initiate and maintain, one that would require the cooperation of many field schools, academic and government institutions alike, but it certainly is not impossible.

Perhaps most importantly, this software and this project result in a cultural record, a documentation of images that translate a thousand different ideas to a thousand different people, and it is a record that will not easily fade away into obscurity like these frescoes are so dangerously close to doing. These walls have protected sacred images for centuries, and with a little effort they can be around for centuries to come.

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References

Baatz, Wolfgang, Peter A Markowich, Massimo Fornasier, and Carola-Bibiane Schönlieb. "Binary Based Fresco Restoration." Bridges 2009: Mathematics, Music, Art, Architecture, Culture. Banff: Banff International Research Station, 2009. 337-338.

Blažek, Jan, Barbara Zitová, Miroslav Beneš, and Janka Hradilová. "Fresco Restoration: Digital Image Processing Approach." 17th European Signal Processing Conference. Glasgow: EURASIP, 2009. 1210-1214.

Bozhkov, Atanas. Bulgarian Icons. Sofia: Bulgarski Houdozhnik, 1987.

Brooks, Sarah. Byzantium Faith and Power. New York: The Metropolitan Museum of Art, 2006.

Cahry, Frederick. The Histroy of Bulgaria. Santa Barbara: Greenwood Press, 2011.

Central Science. Digital Restoration: Introducing n Undo Function. 2011. http://cenblog.org/artful- science/2011/05/15/digital-restoration-introducing-an-undo-function/ (accessed October 29, 2011).

Cormack, Robin. Painting th Soul. London: Reaktion Books, 1997.

Cormack, Robin, and Maria Vassilaki. Byzantium 330-1453. London: Royal Academy of Arts, 2009.

Crampton, R. A Concise History of Bulgaria. Cambridge: Cambridge University Press, 2007.

Ćurčić, Slobodan. Gračanica. University Park: The Pennsylvania State University Press, 1979.

Dayezac, Bertrand. Four Icons in the Menil Collection. Houston: Menil Foundation Incorporated, 1992.

Evans, Helen. Byzantium Faith and Power. New York: The Metropolitan Museum of Art, 2004.

Evans, Helen, and William Wixom. The Glory of Byzantium. New York: The Metropolitan Museum of Art, 1997.

Ferguson, George. Signs and Symbols in Christian Art. New York: Oxford University Press, 1954.

Gerov, Georgi. Icons from Melnik and the Melnik Region, Bulgaria. Sofia: Pensoft, 2007.

Ivanov, Vladimir. Russian Icons. New York: Rizzoli International Publications, 1988.

Kamenova, Dora. Mural Paintings of the Iskrets Monastery. Sofia: Bulgarski Khudozhnik, 1977.

Kondakov, Nikodim Pavlovich. Icons. New York: Parkstone International, 2008.

Maguire, Henry. The Icons of their Bodies. Princeton: Princeton University Press, 1996.

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Marsink, Simon. The Power of Icons. Ghent: Snoeck Publishers, 2006.

Nelson, Robert. Icons from Sinai. Los Angeles: J. Paul Getty Trust, 2007.

Nikolaeva Nikolova-Houston, Tatiana. Margins and Marginality: Marginalia and Colophons in South Slavic. Austin: University of Texas at Austin, 2008.

Schönlieb, Carola. "Restoring Profanity." Plus Magazine, 2009.

Subotić, Gojko. Art of Kosovo. New York: The Manacelli Press, 1998.

Tradigo, Alfredo. Icons and Saints of the Eastern Orthodox Church. Los Angeles: Getty Publications, 2006.

Vassilaki, Maria. Mother of God. Milan: Skira Editore, 2000.

Zernov, Nicolas. Eastern Christendom. New York: Putnam Publications, 1961.

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Figure 1. Christ as Ancient of Days, Zimevitsa Monastery Chapel, Bulgaria.

Figure 2. Dora Kamenova, Mural Paintings of the Iskrets Monastery (Sofia: Bulgarski Khudozhnik, 1977).

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Figure 3. “Ivory Throne of Maximian, Bishop of Ravenna”. Ivory, Ravenna, Ca. 6th century. Ravenna Museo Arcivescovile, Ravenna, IT.

Figure 4. “Exaltation of the cross”, egg tempera on wood. Novgorod, Russia, late 15th Century. Recklinghausen, Ikonen- Museum, Recklinghausen, Germany. From: Eva Haustein-Bartsch, Icons (London: Taschen, 2008), 43.

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Figure 5. “Madonna and Child on a Curved Throne”, Tempera, gold and gesso on wood. Greece or Macedonia, 1275-1300. National Gallery of Art, Washington D.C., Andrew W. Mellon Collection. Image scanned from: Helen Evans, Byzantium Faith and Power 1261-1557 (London: Yale University Press, 2004), 286.

Figure 6. “The Virgin and Child Enthroned, with an Archangel and St. Sergius of Radonezh”. Alexandrov, Russia, first third of the 15th Century. Church of the Trinity in the Makhrishchi Monastery. Image scanned from: The History Museum, Moscow, Russian Icons (Leningrad: Aurora Art Publishers, 1988), 50.

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Figure 7. Presentation of the Virgin to the Temple, Iskrets Monastery, 1602. Photo taken by Scott Cheney.

Figure 8. “Presentation of the Virgin in the Temple and the Virgin of the Burning Bush”. Tempera and gold on wood, Moscow, Ca. 1598. Walters Art Museum, Baltimore, MD. Accession #: 37.2664.

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Figure 9. Dormition of the Virgin, Iskrets Monastery, 1602. Photo taken by Scott Cheney.

Figure 10. Mother of the Sign, Iskrets Monastery, 1602. Photo taken by Scott Cheney.

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Figure 11. “Dormition of the Virgin”. Serbia, 14th Century. Image scanned from: Gojko Subotić, Art of Kosovo: The Sacred Land (New York: Monacelli Press, 1998), 49-50.

Figure 12. “Dormition of the Virgin”. Mosaic, Istanbul, 1310-1321.

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Figure 13. “The Dormition”. Moscow, 13th Century. Tretyakov Gallery. Image Scanned from: Engelina Smirnova, The Dawn of Faith: Icons of Early Russia XI-XIII (Milan: Fondazione Russia Cristiana, 1995), Pg. 13.

Figure 14. Entombment of the Virgin, Iskrets Monastery, 1602. Photo taken by Scott Cheney.

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Figure 15. “Burial of the Virgin”. Francesco d’Antonio, Italy, 1430’s.

Figure 16. Assumption of the Virgin, Iskrets Monastery, 1602. Photo taken by Scott Cheney.

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Figure 17. Ascension of the Virgin, Gracanica, Serbia, 14th Century.

Figure 18. “The Transfiguration”. Bulgaria, 1715. Scanned from: Atanas Bozhkov, Bulgarian Icons (Sofia: Bulgarski Houdozhnik Publishers, 1987), 93.

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Figure 19. “The Transfiguration” detail from “Tetraevangelion”. Tempera and gold on parchment, Greece, 13th Century. Scanned from: Helen Evans, Byzantium Faith and Power 1261-1557 (London: Yale University Press, 2004). 284.

Figure 20. “St. Luke the Evangelist Painting an Icon of the Virgin Mary”. Bulgaria, 18th Century. Scanned from: Atanas Bozhkov, Bulgarian Icons, (Sofia: Bulgarski Houdozhnik Publishers, 1987), 24.

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Figure 21. “Saint Luke”. Parchment. Macedonia, Byzantine, late 13th-14th Centuries. Great Lavra, Mount Athos; part of a larger Gospel Book. Scanned from: Helen Evans, Byzantium Faith and Power 1261-155 (London: Yale University Press, 2004), 285.

Figure 22. “Saint John the Theologian” detail from “The Four Gospels”. Tempera on Parchment. Turkey, 1300. The J. Paul Getty Museum. Scanned from: Helen Evans, Byzantium Faith and Power 1261-1557 (London: Yale University Press, 2004), 278.

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Figure 23. “Presentation of the Virgin to the Temple”, original image vs. digitally conserved image; photo taken and altered by Scott Cheney.

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Figure 24. “Saint Evangelist”, original image vs. inverted image; photo taken and altered by Scott Cheney.

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Figure 25. “Assumption of Mary”, original image vs. inverted image; photo taken and altered by Scott Cheney.

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Figure 26. “Assumption of the Virgin”, original image vs. digitally conserved image; photo taken and altered by Scott

Cheney.

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Cameron Finley Mentor: Dr. Daniel Wall

Major: Molecular Biology

Research Topic: Myxobacteria as a Biocontrol Agent of Agricultural Plant Pathogens

STATEMENT OF THE PROBLEM

The current study aims to determine whether the family of predatory soil bacteria, myxobacteria, can be utilized as a biocontrol agent against pathogens of agricultural crops, including: cereals, potatoes, and sugar beets. A variety of bacterial and fungal pathogens were screened against a panel of myxobacteria to test in vitro for inhibition, predation, and relevant mechanisms.

INTRODUCTION

There are approximately 50,000 microbial diseases of plants in the United States alone, the large majority of which are caused by fungal infection (USDA 1960). US crop losses to plant pathogens are estimated to total over $33 billion per year (USBC 1998).

Furthermore, US farmers spend an additional $720 million per year on fungicides

(Pimentel 1997). In addition to crops, plant pathogens account for an estimated $2 billion per year in losses to lawns, gardens, and golf courses (Pimentel 2000).

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As previously alluded, the most prevalent method of eliminating microbial pests

(specifically fungi) is the use of chemical fungicides. However, these come at a hefty economic and environmental cost. Widespread utilization of chemical fungicides results in: damage to the natural (often beneficial) flora and fauna, pesticide resistance in the fungi, damage to the crops themselves, and long-term groundwater contamination (Pimental

2002). Economically, it is estimated that the destruction of natural wildlife, crop losses, and public health impacts add up to approximately $8 billion per year in the US (Pimental

2002).

Interest in an alternative to chemical pesticides has recently manifested in increased research and development of biological control agents (Whipps 2001). Agricultural biological control agents, or biocontrol agents, are naturally occurring or artificially introduced microorganisms that are encouraged to inhabit the environment of the plant, most often the rhizosphere, and protect the plant from its respective pathogens. The essential idea is to fight fire with fire. This type of biocontrol is highly specified, where the specific microbial interactions that take place can be altered by minute changes in the presence of other microbes, soil composition, and other environmental factors (Whipps

1997). There have been numerous bacterial and fungal biocontrol agents that have been shown to be active against a number of bacterial, fungal, and protozoan pathogens. In line with the diversity of known biocontrol agents, they can each deter pathogenesis in a variety of ways. Such methods include: production of antimicrobial compounds, competition for nutrients, competition for colonization sites, induction of plant resistance mechanisms, breakdown of pathogenic toxins, and parasitism of the pathogen (Keel 1997).

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Currently, the hunt for new biocontrol species goes on in an effort to find new and more effective agents capable of combating the plethora of pathogens in their specific environments. One family of bacteria that has not yet been fully confirmed as a biocontrol agent is myxobacteria. Myxobacteria are highly ubiquitous soil microbes that act as microbial predators in the environment. The key to Myxobacterial predation is the production of antibiotics and hydrolytic enzymes that neutralize and digest the prey, respectively. Myxobacterial antibiotics have been shown to have activity against fungi, gram-positive and gram-negative bacteria, and even insects (Reichenbach 2005). A previous study has shown that different strains of Myxobacteria effectively control certain fungal pathogens of strawberries (Bull 2002). However, work has yet to be done on pathogens of corn, barley, or sugar beets, amongst which some have no biological control strategies developed (Schwartz 2005). Additionally, it has yet to be shown whether

Myxobacteria can actually treat infected plants.

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MATERIALS AND METHODS

Materials

Biocontrol Bacteria Pathogenic Bacteria Bacteria and Species Strain Species Strain Myxococcus xantus DK 1622 Erwinia carotovora G-424 fungal strain used in this M. xanthus DK 1622 4530KO E. carotovora 054-5 M. xanthus ER 5014 B P. aeruginosa Sba100715 study are listed in Table M. xanthus DW 1034 Xanthemonas campestris Sba100715 M. xanthus DK 6204 Pathogenic Fungi 1. Cultures of M. xanthus DK 8601 Species/Designation Strain M. xanthus A23 Alternaria solani/F1 AS12B myxobacteria were M. xanthus A47 Cercospora beticola/F2 08-128 M. xanthus DK 801 C. beticola/F3 |09/1-34 routinely cultured on CTT M. xanthus DK 816 Fusarium culmorum/F4 F03-47 M. xanthus DK 836 F. equiseti/F5 F04-24 media (Hodgkin) or PT M. xanthus DK 897 F. graminiarum/F6 F04-22 Corallococcus coralloides CCc127 F. oxysporum/F7 F04-30 media (Burchard). All M. flavescens AJ 122298 Rhizoctonia solani/F8 L8 Pseudomonas aeruginosa PAK other bacteria were Table 1. Strain List. Biocontrol Bacteria: DK 1622 is a standardized lab strain. DK1622 4530KO-DK8601 are all genetic derivatives of DK1622. A23-DK897 are all cultured on LB media environmental isolates. Pathogenic Fungi: Designation refers to short-hand reference names to be used throughout the paper. (Bertani). Fungi were cultured on Potato Flakes Agar, PFA (Rinaldi). For plant work, a commercial strain of Beta vulgaris was planted in FertiLome Ultimate Ready to Use Potting Mix.

Effects of myxobacteria on pathogenic bacteria

Two swarming tests were conducted, whereby pathogenic bacteria were cultured in

LB, concentrated to OD 10 and spread on a plate, which was subsequently spotted with 3-4,

10 μL samples of myxobacteria at KLETT 1000. Plates were incubated for a period of 3-5 days at 33°C, after which, swarm diameter of myxobacteria spots were measured. The first test was conducted on CTT plates with all four pathogenic bacteria. The second test was

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conducted on TPM plates and, due to culturing difficulties, with only P. syringae and X.

campestris.

Effects of myxobacteria on pathogenic fungi

10 μL volumes of myxobacteria at KLETT 1000, as well as P. aeruginosa at OD 1,

were spotted individually on CTT plates - or PT for C. corralloides and M. flavescens - and

left to grow for 3 days at 33°C. Subsequently, sets of two strains of fungi were paired (by

rate of growth) to each myxobacteria strain by placing a 7mm diameter plug of fungi 1 cm

away from the edge of the myxobacteria spot. Growth of fungi was monitored and

photographed at several times over the course of 3 weeks.

Determination of contact dependent inhibition of fungi

Non-motile myxobacteria, DK 6204 and DK 8601, were

streaked on CTT in a zig-zag pattern according to Figure 1 and

incubated for 3 days at 33°C. Subsequently, three, 7mm plugs of

F. culmorum were plated according to Figure 1 and kept at room

temperature. Growth of the fungi was monitored and

photographed over the course of 10 days.

Figure 1. Not to scale.

Examining the ability of myxobacteria to kill pathogenic fungi

7 cm diameter circles of sterilized nitrocellulose paper were individually centered

on PFA plates, to which one 7mm plug of fungi (one strain per plate) was added. After the

fungi had grown to the diameter of the filter paper, it was transferred with the fungi to a

75 | P a g e new TPM plate. Finally, three to four strains of myxobacteria were spotted in quadrants of the filter disc in volumes of 10 μL KLETT 1000. Plates were monitored over the course of ten days for phenotypic effects and further examined and photographed under the microscope at 10x magnification.

Developing in vitro model of sugar beet infection

Sugar beet seeds were soaked in distilled water for 16 hours, removed from antifungal coating, and subsequently placed in sets of five seeds directly on TPM plates; pushed into the agar of TPM plates; or performing an agar overlay of seeds on TPM plates.

Each of these conditions were replicated and either exposed to direct sunlight or enclosed in the dark. Each of these were monitored to see how many seeds germinated in each condition.

Examining protection of plants in vitro

A thin line of different myxobacteria was laid across the center of TPM plates using a microcaliper. Then, three seeds that had been soaked in distilled water for 16 hours, removed from their coating, and placed evenly on one side of the myxobacteria line. Three,

7mm plugs of each fungi were evenly distributed on the opposing side of the line. Sugar beet and fungal growth were monitored over 2 weeks.

Determining pathogenesis in vivo

16 planter pots were filled with 40g of soil each and planted with two seeds

(previously soaked in distilled water and removed from coating). Potters were kept in indirect sunlight and watered with 25 mL of distilled water every three days. Four days

76 | P a g e after the seeds had germinated, two to four fungal plugs of either C. beticola, F. equiseti, or

R. solani were inoculated 1 cm below the soil at varying distances, ranging from the edge of the potter to directly on the plant. This test was replicated three times with the various conditions.

RESULTS

Effects of myxobacteria on pathogenic bacteria

Swarm diameters of the first test on CTT plates were inconsistent and non- reproducible. However, the second test on TPM plates yielded myxobacteria swarms, as can be seen in Figure 2.

Myxobacterial Growth on Bacterial Lawns- 10 days

M. flavescens DK 897

DK 836

DK 816 DK 801 X. campestris A47 A23 P. syringae Myxobacteria DK 1622 DW 1034 ER 5014B 0 5 10 15 20 25 30 35 Growth Diameter (mm) Figure 2.

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Effects of myxobacteria on pathogenic fungi

Photographs were taken over several days (see

Figure 3 for example). The patterns of growth were

categorized and ranked in a 4 scale system, where “-“

denotes no inhibition, “+” denotes contact dependent

inhibition, “++” denotes a zone of inhibition, and “+++”

denotes that the myxobacteria have actively swarmed out to

the fungal plug. See Figure 5 for the results of each Figure 3. F. graminearum shows “+” ranking and A. solani shows “++”. myxobacteria and fungi pairing.

Determination of contact dependent inhibition of fungi

Fungal growth was monitored and

photographs were taken (see Figure 4). Notice

that the aerial hyphae grow to the edge of the

streak, but not over, and that they reach

further on the side of the streaks than they do

into the peaks.

Figure 4.

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Figure 5

Examining the ability of myxobacteria to kill pathogenic fungi

Of all the fungal plates, only two - F. equiseti

vs. A47, DK801, DK 816, & DK 836 and F.

oxysporum vs. DK 897, CCc127, & AJ 122298 -

showed any significant phenotypic change. This

was denoted by brown discoloration and leveling

of the aerial hyphae (see Figure 6). Upon

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Figure 6 examination under the microscope, myxobacteria colonies were found to be persisting (see

Figure 7).

Figure 7.

Developing in vitro model of sugar beet Figure 8. infection

Growth of sugar beets under different Seeds Germinated Out Of 5 conditions occurred as shown in Figure 8. Results Light Dark

show that seeds germinate ideally when placed Direct placement 4 2 directly on the plate in sunlight. Insert 0 0

Overlay 0 0

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Examining protection of plants in vitro

This test yielded poor results. On TPM plates, myxobacteria did not grow robustly enough to protect the sugar beets from the one fungus, R. solani, that could grow on the media. In that case, R. solani was able to arch over the myxobacteria streaks. The other fungi, like myxobacteria, were unable to grow on the nutrient starved media.

Determining pathogenesis in vivo

In all replications of this test, sugar beets grew healthily regardless of the concentration and proximity of fungal pathogens.

DISCUSSION

Although agricultural biocontrol is a new and growing field, myxobacteria have yet to be fully examined as a candidate for the inhibition of plant pathogens. In testing, I have determined that myxobacteria can predate on two bacterial pathogens. I have also shown that each of the eight fungal pathogens is inhibited to some degree by at least 3 of the strains tested. Furthermore, the disparity between DK 1622 and the 4530 knock-out against F. equiseti suggests that the myxalamid produced by this gene may play a mechanistic role in fungal inhibition. The test of contact dependency seems to show that myxobacteria can inhibit the growth of fungi by using both diffusible elements and an unknown contact dependent mechanism. Further work will need to be done to confirm and characterize this. It is also interesting to note that while myxobacteria do not seem to strongly predate on fungi, they can persist in an environment where no nutrients are available, except those contained in the fungal organism. In determining experimental

81 | P a g e methods, I have found a quick and easy way to germinate and infect sugar beet seeds on a petri dish. This will likely prove useful in future experiments. While infection modeling has largely been a failure up to this point, it is likely due to the high specificity of microbial plant interactions based on environment (Whipps 1997). Future testing will center on determining exact experimental conditions in which we can attain a working in vivo model for infection and biocontrol.

ACKNOWLEDGEMENTS

I would like to personally thank Dr. Daniel Wall for his gracious support and guidance in my educational endeavors and my first real step into academic research, as well as Yao Xiao for her continual advice and friendship as a mentor. I also thank Dr. Gary

Franc for supplying me with pathogens, seeds, and direction. Furthermore, I thank Ms.

Susan Stoddard and Ms. Zackie Salmon for their ongoing work towards my scholarly future.

This research would not be possible without a grant from the TRIO McNair Scholars

Program and the US Department of Education.

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Literature Cited:

Studies on Lysogenesis: Mode of Phage Liberation by Lysogenic Escherichia Coli." Journal of Bacteriology 62.3 (1951): 293-300.

Bull, CT, KG Shetty, and KV Subbarao. "Interactions Between Myxobacteria, Plant Pathogenic Fungi, and Biocontrol Agents." Plant Disease 86 (2002): 889-96.

Burchard, Robert P. "Gliding Motility Mutants of Myxococcus Xanthus." Journal of Bacteriology 104.2 (1970): 940-47.

Hodgkin, J., and D. Kaiser. "Cell-to-Cell Stimulation of Movement in Nonmotile Mutants of Myxococcus." Proceedings of the National Academy of Sciences 74.7 (1977): 2938-942.

Keel, C., and G. Defago. Multitrophic Interactions in Terrestrial Systems. AC Gange and VK Brown ed. London: Blackwell Science, 1997. 27-46.

Pimental, David, ed. Encyclopedia of Pest Management. 1st ed. Vol. 1. CRC, 2002.

Pimental, David, Lori Lach, Rodolfo Zuniga, and Doug Morrison. "Environmental and Economic Costs of Nonindigenous Species in the United States." BioScience 50.1 (2000): 53-65.

Pimentel, D., ed. "Environmental and Socio-economic Costs of Pesticide Use." Techniques for Reducing Pesticide Use: Economic and Environmental Benefits. Chichester (UK): John Wiley & Sons, 1997. 51-78.

Reichenbach, Hans, and Gerhard Hofle. "Production of Bioactive Secondary Metabolites." Myxobacteria II. Ed. Martin Dworkin and Dale Kaiser. Washington, D.C.: American Society for Microbiology, 2005.

Rinaldi, Michael G. "Use of Potato Flakes Agar in Clinical Mycology." Journal of Clinical Microbiology 15.6 (1982): 1159-160.

Schwartz, Howard F., and David H. Ghent. "Helminthosporium Leaf Blight." High Plains IPM Guide (2005).

(USBC) US Bureau of the Census. 1998. Statistical Abstract of the United States 1996. 200th ed. Washington DC: US Government Printing Office

(USDA) US Department of Agriculture. 1960. Index of Plant Diseases in the United States. Crop Research Division, ARS. Washington DC: US Department of Agriculture.

Whipps, John M. “Developments in the Biological Control of Soil-Borne Plant Pathogens.” Advances in Botanical Research 26 (1997): 1-134.

Whipps, John M. "Microbial Interactions and Biocontrol in the Rhizosphere." Journal of Experimental Botany 53.Roots Special Issue (2001): 487-511.

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Nathan Fletcher Mentor: Dr. John Oakey

Major: Chemical and Petroleum Engineering

Research Topic: High Aspect Ratio Inertial Focusing for Complex Fluid Enrichment

Abstract High aspect ratio inertial focusing channels offers high throughput portable devices that can remove colloidal-size particles from a continuous media for the preparation of samples or membranes filtration are in need for point of care operations in a compact and lightweight format. This research attempts to increase concentrating efficiency by studying particle behavior in the channel expansion that follows a high aspect ratio inertial focusing channel. The particle behavior in the channel expansion depends on the geometry of the inertial focusing portion of the channel. The inertial focusing geometries studied were two high aspect ratio channels one tall and the other wide and a staged channel. We found that the staged inertial focusing devices create one focusing position near the channel wall, which in general had provided the narrowest particle streak. This shows that the staged devices have greatest potential for high efficiency particle separation. With a straight tall IFD we were able to attain a 16.7 fold increase in particle concentration when starting with a less than 0.1%volume concentration solution and a 6 fold increase the a particle solution with an initial concentration of around 1%volume. This shows the viability of using IFDs for particle separations and given the prior stated information we should be able to achieve even higher concentration efficiencies by using a staged IFD.

Introduction Recently, much more effort has been directed at making lab equipment more portable and less expensive in order to bring medical testing to the point-of-care(POC). POC operations must be completely self-sufficient, as opposed to a Centralized testing model, in

84 | P a g e which blood is sent to a lab for analysis. As such, laboratory capabilities must be brought to the point of care. The theme of this research is the development of portable (compact and lightweight), high-throughput microfluidic device platforms for the separation of colloidal-sized particles - debris, bacteria, cells - from a continuous media. These platforms are of great interest for use in point-of-care instruments as sample preparation devices, which is an important step in cellular assays. This technology could also be of potential interest for use in biological laboratories, or as components in water filtration units. These devices will effectively serve as membrane-less filters, able to remove small particles from fluids without the use of a physical, size-selective filter. With no physical membrane to clog or foul, these devices are robust and ideal for use in remote or harsh settings. The devices that are the subject of this project are microfluidic devices that use inertial forces to order cells into narrow streamlines. This ordering occurs passively, with no moving parts or active actuation, at very high flow rates. As such, despite the approach being microfluidic in nature, milliliters of fluid per minute may be processed by a single channel, and channels may be easily parallelized for even higher throughput. This is a very compelling enabling technology as it will enable low-overhead, autonomous tools that can be easily integrated into hardware systems for sample preparation, biological sample screening or membrane-less filtration of on-board water supplies.

Background Microfluidics is often associated with viscosity-dominated hydrodynamics, but a recently introduced technique called “inertial focusing” represents a counter-intuitive phenomena that focuses cells and particles in a predictable, reliable manner[1]. It is currently understood that this behavior arises due to the effects of inertia, which rarely apply to the behavior of microfluidic devices. At Figure 1. Particles focused and ordered by microfluidic increasing Reynolds Numbers (Re), inertial focusing. Fluorescent streak images and high speed however, counterintuitive behavior camera images show lateral focusing in two dimensions. begins to emerge. A transition to focusing and ordering, as seen in Figure 1, occurs at Re approaching 1, where inertia must be considered, yet flow conditions are well below the turbulent transition. As such, the interactions between particles, fluids and microchannel surfaces become more complex, yet predictable and useful. While each set of conditions requires a unique analysis, the

85 | P a g e important physical forces at work in inertial focusing devices (IFDs) are, i) lift forces that drive fluids away from walls and the flow centerline[2], ii) shear forces, iii) Stokes drag on particles being subjected to Dean flow[3], or inertial convection[4], and iv) interacting wakes surrounding rotating particles that result in long range interparticle repulsions[5]. IFDs have been developed and demonstrated for lateral passive particle focusing in the absence of a sheath fluid, the concentration of particulate suspensions and size-based fractionation of a variety of complex fluids[6].

Methods and Materials Device design: Three different device designs were used to study the effect of channel geometry on particle focusing behavior. Two straight, high aspect ratio channels were used, one 50µm in width and 30µm in depth, the other 30µm in width and 50µm in height. The third was a staged inertial focusing device that employs curvature and dean flow in the first portion of the channel to achieve a single focusing position in the straight portion of the channel[3]. The staged device was 50µm in depth, the focusing portion of the curvature was 100 µm in width and the straight protion of the channel was 30µm in width. All three IFDs terminated in an expansion, which branched into three outlets, each if which captured a varying amount of the total flow. All focusing measurements were made in these expansions. Device Fabrication: Standard soft lithography techniques with PDMS were used when fabricating the devices. The PDMS then was bonded to glass using oxygen plasma treatment[7]. Experiments: All channels were operated using a syringe pump (Chemyx) at 80 µL/min; then particle distributions in channel expansions were measured using time-lapse fluorescence microscopy. 9.9 µm dragon green florescent spherical polystyrene beads (Fisher Scientific, Fluoro-MaxTM) were used to examine focusing behavior. The fluid density was modified (OptiPrep® Density Gradient Medium) to equal the density of the polystyrene beads at 1.05g/cm3, which ensure experimental consistency by preventing particle sedimentation and therefore local variation in the feed particle solution. Long exposure fluorescence images were taken using a Retiga 2000R camera and Qimaging software. Particle concentration experiments were only performed on the straight high aspect ratio channels with 50µm width and 25µm depth using a flow rate of 20µL/min. Two different initial concentrations were used in the device. First a concentration 1.5 million beads per mL was used for the feed. A hemocytometer was then used to calculate the concentration of the effluent particle solution. Then a higher concentration solution, 1.116x107 beads per mL was made to simulate a second pass through the device. Data Analysis: For the channel expansion experiment a one-dimensional fluorescent intensity distribution was obtained in the channel expansion were by image profiling with ImageJ64. The full width-half maximum of particle streaks were found by the fitting the light intensity data to a Gaussian distribution curve for each intensity profile. Channel

86 | P a g e width and particle stream position measurements were acquired and normalized with ImageJ64, as well. All measurements in the channel expansions were taken at the same three normalized channel widths (channel width divided by the particle width) for all three device geometries.

Results: Channel Geometries and Expansions: When using inertial focusing to concentrate a particle suspension, it is desirable to minimize the ratio of the section of fluid containing particles (streak width) to volume occupied by entire fluid (channel width) is as small as possible. This minimizes the amount of fluid that exits through the particle outlet. Channel expansions can be used to decrease that ratio. As a microfluidic channel expands, the average fluid velocity decreases and, although inertia ceases due to Re dropping below unity, the particles maintain a constant lateral position that is dependent on their initial position as they enter the expansion. As the channel expands, the drop in fluid velocity is uniform, except very near the channel wall, where the change in velocity is not as dramatic. Therefore, particles in streamlines closer to the expansion wall will also slow less and incur less interparticle collisions due to the decrease in fluid velocity, which in turn decreases particle streak width. With this action any deviations of the particle from the focusing positions will be amplified as the stream lines diverge down the expansion. This action allows for more accurate particle streak width measurements. Initial particle position is determined by its inertial focusing position and that is determined by the geometry of the focusing portion of the channel. The geometry of the focusing portion of the device can be used to manipulate the initial particle position in the expansion Figure 2. The three channel types give three distinct particle paths through focusing positions. High aspect and streak behaviors in the channel expansion. (Top) Comparison ratio channels are used in inertial focusing of particle streak broadening as the expansion widens for the differing geometries. (Bottom) devices to eliminate all focusing positions except two[1]. In straight high aspect ratio channels that are short and wide the two focusing positions are in the same vertical plane and centered horizontally in the channel. This creates one particle stream that lies in the center of the expansion, as shown in by the

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red particle streak in Figure 2. High aspect ratio channels that are tall and narrow contain two focusing positions that are in the same horizontal plane with one focusing position next to each wall. In the channel expansion two particle streams are created that are closer to the channel wall (Figure 2: yellow streams). Staged Inertial focusing devices reduce the number of focusing positions to one by focusing first in an asymmetric curvature section of channel, and subsequently in a tall, Wide Channel Expansion narrow straight channel. In curved Fluid Outlet channels, dean flow biases the particles to one side of the channel so when the particle suspension reaches the straight portion of the Particle Outlet channel the particles fall into only Fluid Outlet the focusing position on the corresponding side of the channel[1]. This creates one particle Concentrator Feed Concentrator Product streamline in the expansion that is near the wall, which is the blue streak in Figure 2. The arrangement of the staged channel allows for the one particle stream to enter the faster streamlines on the side of the channel expansion. Analysis of fluorescence intensity data provides the full width-half max streak width in the channel expansion of each of the channel geometries. This streak width is equal to the size of the particle stream section of the channel expansion. This information can be viewed in the chart of Figure 2. The staged IFD has the lowest particle streak width when the normalized expansion is 20 and 52. It also shows the steadiest increase in streak width as the channel Figure 3: The expansion and outlets of the wide straight channel 6 broadens. The tall straight channels that was used to concentrate the 1.5*10 bead per mL particle solution. (Top) Center is the particle solution before and after display the smallest amount of being concentrated. The concentration ability of the device at two streak broadening between 20 and different initial concentrations (Bottom) 36 normalized channel widths and

88 | P a g e the largest amount of streak broadening between 36 and 52. This channel geometry also has the smallest absolute single streak width for the normalized channel width of 36. The wide straight channel, conversely displays the largest increase in streak width between the normalized channel widths of 20 and 36; also the smallest increase in streak width between 36 and 52. Streak broadening starts high and tapers off for this geometry in this section of the expansion. This data does show that staged IFDs do in general provide the smallest gross particle streak area as the single particle streak travels down the side of the expansion. Differences between trends for the three channel geometries are attributable to differences in the point at which interparticle collisions initiate for each channel geometry. We speculate that for wide, straight channels the interparticle collisions first occur between the normalized channel widths of 20 and 36. For tall straight channels the collisions occur between the normalized channel widths of 36 and 52. For staged channels, the earlier inflection in channel width indicates that collisions must occur at a narrower channel width than in tall straight channels The difference in the collision onset is a function of both particle spacing by inertial focusing and the velocity profile in the channel expansion. Concentration Factior: We demonstrate particle concentration in a focusing channel that measured 50µm in width by 30µm in depth with an expansion at the end. As seen in Figure 3, this device was capable of increasing the particle solution from a concentration of 1.5x107 beads per mL (less than 0.1% by volume) to 2.5E107 beads per mL (approximately 1% by volume), which is a 16.7 fold increase in the particle concentration. It also increased a 1.7x108 (approximately 1% by volume) to 1.3x108 beads per mL (approx. 5% by volume), A six fold increase in the particle solution concentration. This data shows that the concentrating ability is inversely proportional to the initial concentration of the particle solution as well as the fact that the concentrating ability approaches some maximum concentration in an asymptotic fashion.

Conclusion We have demonstrated passive particle enrichment in straight and staged (curved-straight) inertial focusing channels. We have investigated the influence of geometry on the enrichment factor and find relationships between overall device performance and channel geometry. These differences are attribuatble to interparticle spacing and lateral positioning, which directly affect the ratio of clean fluid to particle-laden fluid in the effluent stream. We found that tall straight channels achieved a concentration factor of 16.7 in one pass and 6 fold in a second pass, which is capable of a high enough separation efficiency to be used in many sample preparations. Staged IFDs, however, display the greatest potential to provide higer per pass concentration efficienty because they create the smallest streak area.

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Acknowledgement This work was supported by the McNair Scholars Program - United States Educational System and Wyoming IMBRE. I thank to my faculty advisor Dr. John Oakey and graduate student mentor Kaspars Krutkramelis for guidance, assitance, and the learning experience as well as my fellow undergraduate researcher Amy Reece for assistance on experiments.

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References:

1. Di Carlo, D.; Irimia, D.; Tompkins, R. G.; Toner, M. Continuous inertial focusing, ordering, and separation of particles in microchannels Proceedings of the National Academy of Sciences of the United States of America 2007, 104, 18892. 2. Saffman, P. The lift on a small sphere in a slow shear flow Journal of Fluid Mechanics 1965, 22, 385. 3. Berger, S. A.; Talbot, L.; Yao, L. S. Flow in curved pipes Annual Reviews in Fluid Mechanics 1983, 15, 461. 4. Edd, J.; Di Carlo, D.; Humphry, K.; Koster, S.; Irimia, D.; Weitz, D.; Toner, M. Controlled encapsulation of single-cells into monodisperse picolitre drops Lab on a Chip 2008, 8, 1262. 5. Matas, J.; Glezer, V.; Guazzelli, E.; Morris, J. Trains of particles in finite-Reynolds- number pipe flow Physics of Fluids 2004, 16, 4192. 6. Di Carlo, D.; Edd, J.; Irimia, D.; Tompkins, R. G.; Toner, M. Equilibrium separation and filtration of particles using differential inertial focusing Analytical chemistry 2008, 80, 2204. 7. Duffy, D. C.; McDonald, J. C.; Schueller O. J. A.; Whitesides, G. M. Rapid Prototyping of Microfluidic Systems in Poly(dimethylsiloxane)Analytical Chemistry 1998, 70, 4974

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Joshua Heyer Mentor: Dr. Jacqueline J. Shinker

Major: Geography

Research Topic: An Investigation of Climatic Controls during Low Stream Flow Years in the Laramie River Watershed

Introduction

The upper Laramie River watershed is a tributary of the North Platte River located

in the drought-prone intermountain west (Figure 1).

Laramie River Watershed

Figure 1. The Laramie River Watershed is located in southeast, Wyoming. The Laramie River has a drainage area of 1,790 square miles and is a tributary to the North Platte River. Lower-than-normal stream -flow years were selected from the USGS gage site in Bosler, Wyoming, which is depicted on the map as a red star.

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This watershed provides important water resources used for agriculture, municipalities, energy production, and tourism in Wyoming, Colorado, and Nebraska (Shinker et al. 2010).

Low stream flow inhibits the water available for these uses. Water provided by western

U.S. rivers, like the Laramie River, is a highly valued commodity for growing populations in this region (Barnett et al. 2008; Milly et al. 2008). The majority of water resources in the west (50 – 80%) are provided by snowmelt (Stewart et al. 2004).

Variability in precipitation, especially during snow-dominant months, leads to variations in stream flow and subsequent variations in late season (e.g. growing season) water availability. Superimposed on the natural variability of snow precipitation is anthropogenic climate warming, which has triggered changes in the hydrologic cycle and reduced runoff in some regions, such as the intermountain west (Barnett et al. 2008; Milly et al. 2008, Pederson et al. 2011). Stewart et al. (2004) observed shifts towards earlier snowmelts in the western U.S. from 1948-2000 reducing summer runoff, and increasing the length of summer drought conditions with consequences for water supply management.

Regionally, the North Platte river basin has experienced a 10.5-day advance in the timing of peak runoff over the last 92 years (1916-2007), coupled with a 2.21°C increase in mean

March, April, May (MAM) spring temperatures (one and a half times faster than the mean annual temperature increase over the same period). This coupled advance in spring runoff and increasing spring temperatures is equivalent to an advance of spring by 4.75 days per

1°C over the 92-year record (Shinker et al. 2010). The vulnerability of the Laramie River watershed to lower-than-normal stream flows as a function of earlier peak runoff compounded by lower-than-normal snow precipitation and increasing spring temperatures complicates water management responsibilities in this region. Since low

93 | P a g e stream flow and low precipitation years are interrelated, have existed in the past, and water resources will likely be impacted by continued increasing temperatures, a study of the climate controls leading to drought conditions will add to our understanding of this hazard.

The vulnerability of the west to drought is compounded by the heterogeneous climate of the intermountain west, which is a function of the varied topography influenced by large and small spatial scale climatic controls in the atmosphere and at the surface

(Shinker and Bartlein 2010). The diverse topography of the western U.S. contributes to climate variations, which can lead to drought, and makes it necessary to investigate stream flow responses to the climate within individual basins. The occurrence of past droughts in the western U.S. has been documented through a variety of proxy records including tree- ring and lake-level reconstructions. Tree-ring records in the western U.S. show evidence of a mega-drought lasting from AD 900 to 1300 (Cook et al. 2004). For example, from AD

1440 to 1470, sustained low snowpack conditions persisted in the Upper Colorado River watershed (Cook et al. 2004; Pederson et al. 2011). Evidence from past lake shore levels also display rapid hydroclimatic shifts in the intermountain west, leading to long-term droughts lasting centuries to sometimes millennia (Shuman et al. 2010). Knowing that long-term droughts have occurred in this region in the past and will likely occur again, justifies the importance of this study. Additionally, understanding the hydroclimatic controls of recent droughts (e.g. last 100 years) is important when dealing with the impacts this hazard has on society (Stewart et al. 2004; Miller and Piechota 2008). Miller and

Piechota (2008) studied the effect of recent increasing temperature trends on precipitation, and the seasonal timing of peak stream flows in the Colorado River

94 | P a g e watershed. Like Stewart et al. (2004), they observed earlier peak stream-flow rates

(January through March) leading to decreased stream-flow trends during traditional peak stream-flow months (April through July) (Miller and Peichota 2008).

The above studies only provide information on the occurrence of drought (via proxy records, e.g. Cook et al. 2004 and Shuman et al. 2010), or the timing of drought conditions using temperature, precipitation, and stream-flow records (e.g. Miller and Piechota 2008), and do not consider the atmospheric and surface controls within the climate system that lead to and enhance drought conditions. This study investigated climate controls during low stream flow and low precipitation years in the upper Laramie River watershed. To accomplish this, it is important to understand how climate controls connected to drought conditions vary spatially (e.g. local-to-regional) and temporally (e.g. monthly-to-seasonal) in the atmosphere and at the surface. This spatial and temporal approach was applied to a variety of climate variables that encompass atmospheric mechanisms controlling the suppression of precipitation (e.g. atmospheric ridges, sinking motions, low specific humidity in the atmosphere) and surface conditions (e.g. precipitation and soil moisture).

Studying spatial and temporal variations of climate controls regulating drought conditions provides a more comprehensive representation of how low precipitation and low stream flow years take place in the upper Laramie River basin.

Data

Precipitation data and temperature data were obtained from division 10 in

Wyoming, from the National Oceanic and Atmospheric Administration (NOAA). Stream- flow data was provided by the United States Geologic Survey (USGS) National Water

Information System. The stream-flow data was gathered from United States Gage Site

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06661585, located along the Laramie River near Bosler, Wyoming. Climate variables representing atmospheric and surface conditions were obtained from the National Center for Environmental Prediction and National Center for Atmospheric Research (NCEP-NCAR)

North American Regional Reanalysis (NARR) dataset (Messinger et al., 2006). Five lower- than-normal precipitation and steam-flow years were selected from 1979-2009. This timeframe was chosen because climate data provided by the NARR dataset is only available during these years. Based on the five selected years, temporal (seasonal-annual) data of precipitation and stream-flow amounts were gathered.

The NARR is a gridded forecasting model (32-km gridded spatial scale) initiated with previously measured climate data. The output from the NARR forecasting model include a suite of climate variables, many unobservable, that can be used to analyze climate processes over a variety of time scales. For the purpose of this study, the atmospheric circulation variables that are analyzed include geopotential height at the 500 millibar (mb) level (providing information on the shape of the middle part of the troposphere), and omega at the 500 mb level (a measure of vertical velocity of rising and sinking motions in the atmosphere associated with enhancing or suppression of precipitation respectively).

Additionally, the atmospheric variable specific humidity was analyzed at the 850 mb level

(moisture available in the atmosphere available for uplift by omega). Climate variables analyzed that represent surface conditions include the surface soil moisture (moisture at the surface), precipitation rates (representing actual precipitation that has reached the surface). In addition, air temperature was observed at the surface. The spatial (local- regional) distribution of both atmospheric and surface climate data was displayed via composite-anomaly maps constructed using NARR data.

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Methods

Average annual stream flow from 1979-2009 was determined at the United States

Gage Site 06661585, near Bosler, Wyoming (see Figure 1). Based on the average stream flow, five lower-than-normal stream flow years were selected. Next, a time-series plot of precipitation data from climate division 10, including the North Platte River headwaters, was constructed for the years 1979-2009. Lower-than-normal precipitation years were selected from this time-series plot and compared to stream-flow data obtained from the

Bosler Gage Site. The precipitation time series plot captures precipitation data from the winter and spring seasons (snow-dominant seasons). This data was used to verify lower- than-normal stream-flow amounts during the five selected years controlled by lower-than- normal snow precipitation. A time-series plot of temperature data from climate division 10 was constructed for the years 1979-2009. The temperature data is used to verify higher- than-normal temperatures during the snow-dominant seasons of the selected low stream flow years.

After determining five precipitation years lower than the long-term mean (1979-

2009), composite anomalies of mechanistic climate variables were calculated. A composite anomaly represents several cases (e.g. multiple dry years) that are averaged together and compared to a long-term mean (e.g. 1979-2001) (Yarnal, 1993). Once composite-anomaly values were calculated, the values were plotted on maps. Composite-anomaly maps were used to illustrate the connection between large-scale atmospheric circulation patterns and climate responses at the surface (Yarnal, 1993; Shinker, 2006). These maps provide information on the spatial (e.g. local-to-regional) variations of the identified climate controls. Temporal analysis (e.g. monthly-to-seasonal-to-annual) of stream flow and

97 | P a g e precipitation is assessed, to establish how stream flow and precipitation amounts vary during the selected dry years in relationship to climate controls. By investigating the temporal variations of stream flow and precipitation amounts in relation to the spatial variations of selected climate controls, a better understanding of how low stream flow and low precipitation years in the upper Laramie River watershed respond to the climate system is provided.

Results

Reduced snow runoff has been observed in many regions of the intermountain west, enhancing drought conditions and complicating water management responsibilities

(Stewart, et al. 2005; Barnett et al. 2008; Milly et al. 2008, Pederson et al. 2011). Our results, like previous studies captured lower-than-normal stream-flow years in the Laramie

River watershed regulated by reduced snow runoff. However, our results, unlike other studies, provides context for the atmospheric circulation and surface conditions associated with recent dry conditions.

Selected Dry Case Years

The selected lower-than-normal stream-flow years were 1981, 1989, 1994, 2001, and 2002 (Figure 2). The time series plots capture precipitation (Figure 3) and temperature anomalies (Figure 4) during the snow dominant seasons (i.e. DJF winter season and MAM spring season) for the selected stream-flow years.

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Selected Low Stream-Flow Years

Year Annual stream Gage Peak stream Peak stream flow

flow cfs height (ft) flow cfs date

1981 48.9 2.49 4616 Jun. 12, 1981

1989 73.2 1.682 2536 Jun. 09, 1989

1994 49.8 2.242 3826 May 17, 1994

2001 59.1 2.782 4786 May 22, 2001

2002 17.4 6,9 Mar. 31, 2002

1979-2009 159.6

Figure 2. Lower-than-normal stream flow years in the Laramie River watershed (base period 1979-2009). Five lower-than-normal annual stream flows were selected from the Bosler stream flow gage site: 1984, 1989, 1994, 2001, and 2002. The average annual stream flow during these five years was 49.7cfs, compared to the long-term-mean of 159.6cfs. Note, during the five years there was a trend towards earlier peak stream-flow. For example, 2002 had a peak stream-flow at the end of March, which is a very early peak stream-flow for the Laramie River.

99 | P a g e

During 1981, 1989, 1994, 2001, and 2002, suppressed lower-than-normal precipitation amounts contributed to lower-than-normal stream flow. The year 2002, a year of very low stream flow, experienced lower-than-normal precipitation during both the winter and spring season.

However, the year 1981 had higher-than-normal precipitation during the spring season, experiencing low stream flow as a function of higher-than-normal temperatures and lower-than- normal winter precipitation (Fig 4). This illustrates the effect both precipitation and temperature can have on stream flow in the Laramie River watershed.

Figure 3. Precipitation anomaly time series from 1979-2010 for winter (blue) and spring (green) months (base period 1895-2011). During the five lower-than-normal stream-flow years, lower-than-

normal precipitation was observed during either the DJF (winter) or MAM (spring) seasons in Division 10, Wyoming. This reflects the important relationship between snow precipitation and stream flow in the Laramie River watershed.

100 | P a g e

The seasonal temperature time series depicts higher-than-normal spring temperatures during the years 1981, 1989, and 1994, which melted snow away quicker and resulted in lower- than-normal stream flow. Higher-than-normal spring temperatures combined with lower-than- normal snow precipitation controlled low stream flow during the years 1981, 1989, and 1994.

The years 2001 and 2002 did not have higher-than-normal temperatures. As a result stream flow during 2001 and 2002 was a function of lower-than-normal snow precipitation and not higher- than-normal temperatures.

Figure 4. Temperature anomaly time series from 1979-2010 for winter (blue) and spring (green) months (base period 1895-2011). Higher-than-normal DJF (winter) or MAM (spring) temperatures were observed during 1981, 1989, and 1994. Higher-than-normal spring temperatures enhanced spring snow melting and caused earlier peak stream flow. Earlier spring snow melt controls lower- than-normal stream flow during the late spring and summer seasons. The year 1981 illustrates this point with both winter and spring temperatures that were higher-than-normal resulting in low stream flow, despite higher-than-normal spring precipitation occurring (i.e. Figure 3). 101 | P a g e

Composite-anomaly Map Interpretations

The controls of climate anomalies (e.g. drought) at the surface can be observed via composite-anomaly maps (Yarnal, 1993; Shinker, 2006). Our study illustrates climate controls at the surface via composite-anomaly maps for the selected lower-than-normal stream-flow years

(Figure 5). The composite-anomaly maps display anomalous surface precipitation, surface temperature, and surface soil moisture during low stream-flow years. Lower-than-normal precipitation rates were observed for the spring, illustrating the connection between snow precipitation and stream flow. Surface soil moisture was lower-than-normal for both the winter and spring. Overall, conditions were dry at the surface. Enhancing dry conditions, higher-than- normal temperatures were observed during the spring season. The observed higher-than-normal temperatures during the spring enhanced snow melt, contributing to earlier peak stream flow and lower-than-normal stream flows later in the year.

Composite-anomaly maps for surface climate controls DJF(Winter) MAM(Spring)

Precipitation Rate

Surface

Low High

Low High

Air Temperature Low High

102 | P a g eLow High

Surface

Surface Soil Moisture

Content

Low High

Low High

Figure 5. Composite-anomaly maps for surface climate controls during the winter (left column) and spring (right column) seasons (base period 1979-2001). Precipitation rates (top maps) show lower-than-normal precipitation values in green-to-red and higher-than- normal precipitation values in blue-to-purple. Air temperature (middle maps) show lower- than-normal temperatures in blue-to-purple and higher-than-normal in green-to-red. Surface soil moisture (bottom maps) show lower-than-normal soil moisture in green-to-red and higher-than-normal soil moisture in blue-to-purple.

The heterogeneous climate of western U.S. is a function of the varied topography influenced by large and small spatial scale climatic controls in the atmosphere and at the surface

(Shinker and Bartlein 2010). During the selected lower-than-normal stream-flow years in the

Laramie River watershed, climate controls in the atmosphere regulated conditions observed at the surface. Composite-anomaly maps for the selected low stream flow years 1981, 1989, 1994,

2001, and 2002 illustrate the atmospheric controls of geopotential height (500mb), omega

(500mb), and specific humidity (850mb) (Figure 6). Geopotential height (500mb) was higher- than-normal during the winter and spring season, indicating a consistent ridge in the atmosphere consistent with stable atmospheric conditions that would lead to dry conditions at the surface.

Omega (500mb) during the winter and spring seasons was dominated by sinking motions.

Sinking motions in the atmosphere suppress the uplift of moisture for cloud development and precipitation, further enhancing dry conditions at the surface. Specific Humidity (850 mb) was

103 | P a g e

lower-than-normal during the winter season and average during the spring season. Specific

humidity is the amount of moisture available in the atmosphere for uplift, which enhances cloud

development and precipitation. While the spring season did not have anomalous specific

humidity, it did have sinking motions in the atmosphere. Therefore, regardless of moisture

availability in the atmosphere (via specific humidity), the mechanism for precipitation (rising

motions) were not present during the spring season. Overall however, moisture available

(850mb) was lower-than-normal. The atmospheric composite-anomaly maps demonstrate

sinking motions suppressing moisture available in the atmosphere, and enhancing drought

conditions at the surface.

Composite-anomaly maps for atmospheric controls

DJF(Winter) MAM(Spring)

Geopotential Height (500 mb)

Low High

Low High

Rising Sinking

Rising Sinking

Omega (500 mb)

Low High Low High 104 | P a g e

Figure 6. Composite-anomaly maps for atmospheric climate controls during the winter (left column) and spring (right column) seasons (base period 1979-2001). Geopotential height (top maps) illustrates higher-than-normal troposphere heights in green-to-red and lower-than-normal heights in blue-to-purple. Omega (middle maps) illustrates sinking motions in the troposphere in green-to-red and rising motions in blue-to-purple. Specific Humidity (bottom maps) illustrates lower-than-normal moisture available in the troposphere for uplift in green-to-red and higher-than-normal moisture available in blue-to-purple.

Discussion

Previous drought events observed in the western U.S. via tree ring records have been discussed as a function of precipitation and temperature anomalies (Cook et al. 2004;

Pederson 2011). Lower-than-normal snow precipitation amounts reduce spring runoff, while higher-than-normal temperatures enhance snow melting. Mountain snowpack is the largest natural reservoir of surface water for stream flow in the western U.S. (Stewart et al.

2004; Mote et al. 2005; Barnett, et al. 2008; Pederson et al. 2011). Our results revealed lower-than-normal snow precipitation regulating lower-than-normal stream-flow years in the Laramie River watershed during all of the case years study (1981, 1989, 1994, 2001, and 2002). During the selected five years climate controls in the atmosphere and at the surface suppressed snow precipitation. The suppressed snow precipitation amounts regulated low stream-flow years. In addition, higher-than-normal temperatures were observed during the spring, and contributed to drought conditions.

Our study illustrates the climate controls in the atmosphere and at the surface via composite-anomaly maps that regulated low snow precipitation amounts. In the atmosphere, higher-than-normal troposphere heights (Geopotential heights at 500mb) indicating the presences of a ridge, vertical sinking motions (Omega 500mb) suppressing the uplift of moisture, and lower-than-normal atmospheric moisture availability (specific humidity at 850mb) controlled the drier-than-normal conditions at the surface seen in our

105 | P a g e case years. At the surface, surface-soil moisture was lower-than-normal, snow precipitation was lower-than-normal, and temperatures were higher-than-normal. Our study identified these climate controls in the atmosphere and at the surface suppressing snow precipitation and regulating the five lower-than-normal stream-flow years.

Higher-than-normal temperatures melted away snow faster and contributed to lower-than-normal stream flow during the late spring and summer season (e.g. growing season). The composite-anomaly map of spring temperatures illustrated higher-than- normal temperatures throughout the Laramie River watershed during the low-stream-flow years. In relation to our study, other researchers have observed springtime warming enhanced by anthropogenic forces throughout the western U.S., which has changed the hydrologic cycle, reduced runoff, and could have considerable consequences for regional water supplies (Stewart et al. 2005; Barnett et al. 2008; Milly et al. 2008; Shinker et al.

2010; Pederson et al. 2011; and Shuman 2011). Widespread warming observed throughout the western U.S. since 1916 has contributed to drought conditions in the intermountain west (Hamlet et al. 2005). Observed trends towards earlier peak stream flow could cause a 10% - 50% decrease of average spring-summer stream-flow amounts, which will enhance summer drought conditions in the western U.S. (Stewart et al. 2005).

While our study only investigates temperature variability and not trends, our results illustrated that years with higher-than-normal spring temperatures coupled with reduced snow precipitation in the Laramie River are associated with earlier spring snow and contribute to summer drought conditions. Together, climate controls in the atmosphere and at the surface regulated low stream-flow years in the Laramie River watershed,

106 | P a g e limiting important water resources used for agriculture, municipalities, energy production, and tourism in Wyoming, Colorado, and Nebraska.

Acknowledgments

I would like to thank the McNair Scholars Program at the University of Wyoming for funding this project and for their support. I would like to thank Jacqueline J. Shinker for the comments and help that improved this paper. Other funding came from the USGS, NOAA, and NCEP-NARR.

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Bibliography

Barnett, T. P., D.W. Pierce, H.G. Hidalgo, C. Bonfils, B. D. Santer, T. Das, G. Bala et al,. 2008. Human-induced changes in the hydrology of the Western United States. Science 319:1080- 83.

Cook, E.R., C. Woodhouse, C.M. Eakin, D. M. Medo, and D. W. Stahle. 2004. Long-term aridity changes in the western United States. Science 306:1015-18.

Hamlet, F. A., W.P. Mote, P.M. Clark, P.D. Lettenmaier. 2005. Effects of Temperature and Precipitation Variability on Snowpack Trends in the Western United States. Journal of Climate. 18: 4545-4561.

Mesinger, F., G. Dimego, E. Kalnay, K. Mitchell, P.C. Shafran, W. Ebisuzaki, D. Jović, D. J. Woollen, E. Rogers. B.H. Ernesto, B. EK. Michael, Y. Fan, R. Grumbine, W. Higgins. LI. H. Lin, Y. G. Manikin, D. Parrish, W. Shi. 2006. North American Regional Reanalysis.

Miller, W.P., and T.C. Piechota. 2008. Regional analysis of trend and step changes observed in hydroclimatic variables around the Colorado River Basin, Journal of Hydrometeorology, 9, 1020-1034.

Milly, P.C.D., J. Betancourt, M. Falkenmark, R.M. Hirsch, Z.W. Kundzewicz, D.P. Lettenmaier, and R.J. Stouffer. 2008. Stationary is dead: Whither water management? Science 319:573- 74.

Mote, P. W., A. F. Hamlet, M. P. Clark, D. P. Lettenmaier. 2005. Declining Mountain Snowpack in Western North America. 39-49.

Pederson, G. T., S. T. Gray, C. A. Woodhouse, J. L. Betancourt, D. B. Fagre, J. S. Littell, E. Watson, B. H. Luckman, L. J. Graumlich. 2011. The Unusual Nature of Recent Snowpack Declines in the North American Cordillera.

Shinker, J. J., and P.J. Bartlein. 2010. Spatial variations of effective moisture in the western United States. Geophysical Research Letters, 37.

Shinker, J.J., B. Shuman, T.A. Minckley, A.K. Henderson. 2010. Climatic Shifts in the Availability of Contested Waters: A Long-Term Perspective from the Headwaters of the North Platte River. 1-14.

Shinker, J.J., P.J. Bartlein, B. Shuman. 2006. Synoptic and dynamic climate controls of North American mid-continental aridity. Quaternary Science Reviews, 25:1401-1417.

Shuman, B., 2011. Recent Wyoming temperature trends, their drivers, and impacts in a 14,000-year context. Climatic Change, DOI 10.1007/s10584-011-0223-5.

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Shuman, B., P. Pribyl, T. A. Minckley, and J. J. Shinker. 2010. Rapid hydrologic shifts and prolonged droughts in Rocky Mountain headwaters during the Holocene. Geophysical Research Letters, 37: L06701.

Stewart, I, T., D. R. Cayan, and M. D. Dettinger. 2004. Changes in snowmelt runoff timing in the western North America under a ‘business as usual’ climate change scenario. Journal of Climate 62: 217–232.

Stewart, I. T., D. R. Cayan, and M. D. Dettinger. 2005. Changes toward Earlier Streamflow Timing across Western North America. Journal of Climate. 18: 1136-1155.

Yarnal, B., 1993. Synoptic Climatology in Environmental Analysis; A Primer. Belhaven Press.

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Kimberli Ogg Mentor: Dr. David Jones and Dr. Mary Hardin-Jones

Major: Communication Disorders

Research Topic: Oral Pressure Requirements for Common Blow Toys

Abstract

Oral-motor exercises (OMEs) [MHJ: Implies that the reader has knowledge. Maybe say

“Despite the lack of empirical support, OMEs are a common treatment strategy”] to be applied as a treatment method to improve speech production by allegedly increasing muscle tone/strength of the oropharyngeal musculature. One particular oral-motor approach involves blowing tasks using common novelty horns. Although many speech pathologists have accepted this approach as a means to improve oropharyngeal strength, little is known about the aerodynamic requirements associated with blowing these horns.

This study investigated the intraoral pressures required to blow 10 novelty horns included in the Horn Hierarchy program, a common OME regimen. Subjects included 8 normal- speaking children ages 4-5 years. Each child was asked to blow 10 different horns from the

Horn Hierarchy program. Intraoral pressure (IOP) was measured via a small polyethylene tube placed beside the horn mouthpiece; the tube was coupled to a pressure transducer.

The IOP signal was amplified and digitized to a laboratory computer. The results from this

110 | P a g e investigation showed that the intraoral pressures for only two of the ten horns were close to the normative mean for children, suggesting that the majority of horns included in the

Horn Heirarchy program do not provide the necessary resistances for a speech muscle- training regimen. Further, the intraoral pressures required to blow the horns did not increase systematically across the hierarchy. These results indicate that common novelty horns may not provide the aerodynamic resistance needed to improve speech production.

Oral Pressure Requirements for Common Blow Toys

Oral-motor exercises (OMEs) are treatment practices used by speech-language pathologists to enhance speech production by allegedly increasing the strength of the oropharyngeal musculature. Some of the most frequently used oral-motor exercises include tongue “push ups”, pucker-smile alternations, tongue wags (lateralizations), “big smile” exercises, tongue-to-nose-then-to-chin movements, cheek puffing, blowing kisses, and tongue curling (Forrest, 2002; Lof & Watson, 2008). One of the most popular oral-motor exercises is blowing activities, which frequently involve the use of common novelty horns

(Lof & Watson, 2008). These tasks have been used with children who have delays in articulation development unrelated to structural or neurological problems, children who experience dysarthria (muscle weakness), and children with structural deficits such as cleft palate (Lof & Watson, 2008; Forrest &, Iuzzini, 2008). Supporters of oral-motor exercises argue that such exercises increase strength of the speech musculature and improve both muscle tone and range of motion (Beckman, 1986; Rosenfeld-Johnson, 1999; Bahr 2001).

However, opponents have argued that such activities are inappropriate when you consider that 1) most children with speech disorders do not demonstrate deficits in muscle strength,

111 | P a g e

2) typical blowing tasks offer little resistance to the speech musculature, and 3) these

“exercises” are not conducted during speech. There is a scarcity of literature regarding oral motor exercises, but the findings that have been published agree that oral motor exercises do not facilitate speech production (Lof, 2003; Ruscello, 2008). Despite the lack of empirical support, a large number of speech language pathologists (SLPs) report using oral motor exercises--blowing tasks in particular-- as an intervention for children who demonstrate developmental articulation problems (Lof 2003; Ruscello 2004; Clark 2005).

There are a variety of horn programs available to SLPs that, according to the manufacturers, are designed help a child progress to better speech. One such program developed by Rosenfeld-Johnson is the Horn Hierarchy program. This program involves 12 horns that reportedly require increasingly greater pressure as a child progresses from one horn to the next. According to Rosenfeld-Johnson, use of this program can improve articulation by increasing muscle strength; however, no data have been reported describing the amount of oral pressure required to blow these simple horns. Further, there are no data to demonstrate that the horns are arranged according to a hierarchy of increasing pressure.

The current study investigated the oral pressures required to effectively blow 10 novelty horns included in the Horn Hierarchy program. During this investigation, the following questions were addressed: 1) Is the intraoral pressure required to blow horns in the Horn Hierarchy program similar to the average speech pressure of normal speaking children? 2) Does the intraoral pressure required to blow the horns increase systematically along the hierarchy? The hypothesis of this study was that the majority of

112 | P a g e these horns would require oral pressures that are lower than the mean oral pressure exhibited by children. The investigators speculated that if little pressure was required to blow the horns, it would be unlikely that these horns would offer the resistance necessary to strengthen muscles, and thus, would probably have no impact on articulation performance.

Methods

Participants

Participants included 8 children with normal speech, ages 4-5 years, who were identified through local Laramie daycare centers. Potential participants with a history of chronic or acute respiratory illnesses, cleft palate, neurological impairment, or cognitive impairment were not included in the study. In addition, children with velopharyngeal inadequacy (inability to seal off the nasal cavity from the oral cavity – the closure required to make speech sound oral) were excluded since they might have difficulty generating the pressure necessary to blow these horns. As an added control, the potential participants were engaged in conversation to informally assess their speech and ensure normal development.

Procedure

Following informed parental consent and child assent, each participant was scheduled for a single one-hour session in the Speech Physiology laboratory at the

University of Wyoming. A parent or caregiver accompanied the child to the session and was present throughout the data collection process. Each participant was provided with his/her own horn kit. During the session, each child was asked to blow 10 of the 12 different horns

113 | P a g e from the Horn Hierarchy program. Two of the horns were excluded due to horn designs that precluded assessment of oral pressure. Each of the participants was instructed to blow each horn and to sustain the sound at a soft level for as long as possible. A minimum of 10 blowing trials were obtained for each horn.

Intraoral pressure (IOP) was measured via a small polyethylene tube placed beside the horn mouthpiece; the tube was coupled to a pressure transducer, which was calibrated to 10 cm H20. The IOP signal was amplified and digitized to a laboratory computer using a data acquisition system (WINDAQ). A microphone was positioned approximately 3” from the horn to record sound onset and duration. Oral secretions often collected in the oral end of the tube; when this occurred, the tube was cleared or replaced, and the task continued.

Results

Mean onset pressures of the ten horns were calculated to address the first question of whether or not the pressures produced compare to normative mean speech pressures in children (see Table 1). The results indicate that only two of the ten horns (horns 6 and 8) were close to the 6.2 cm H20 mean speech pressure reported for the production of the bilabial consonant /p/ (Stathapoulos & Weismer, 1985). Three of the ten horns (horns 1, 3,

5) were slightly below the comparative mean, and the remaining five (horns 4, 7, 9, 11, 12) were considerably higher (see Figure 1).

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Table 1. Mean Onset Pressure and Standard Deviation (SD)

Horn Mean Onset Pressure (SD)

1 2.43 (0.69)

3 3.04 (1.26)

4 12.9 (4.77)

5 4.16 (1.51)

6 5.59 (2.33)

7 14.9 (4.00)

8 5.37 (1.91)

9 24.8 (4.39)

11 21.9 (6.90)

12 31.4 (5.09)

*Mean for normal children’s speech = 6.2 cm H20*

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Figure 1. Graphical Depiction of Horn Data Compared to Normative Child Speech Pressures.

Mean Onset Pressure of Horns Compared to Child Normative Pressure

35

30

0) 2 25

20

15 Intraoral Pressure (cm H (cm Pressure Intraoral 10

5

0 0 2 4 6 8 10 12 Horn #

The second question dealt with the claim of the horn set following a hierarchical order. A one-way analysis of variance (ANOVA) was performed to examine differences in onset pressure by horn, and revealed a significant horn effect (F = 294.829; p<.001). Post- hoc analysis (Tamhane’s T2) revealed significant difference in sound onset pressure for 39 of the 45 pairwise comparisons. There was no significant difference in resistance between

Horns 1 and 3, Horns 4 and 7, Horns 5 and 6, Horns 5 and 8, Horns 6 and 8, and Horns 9 and 11.

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Discussion

When blowing novelty horns, how do the pressures produced compare to normative speech pressures in children? Results from this investigation suggest that the average onset pressures generated by blowing these horns are either much lower or considerably higher than those utilized in normal speech production. Three of the ten horns (horns 1, 3, 5) required oral pressures below the mean oral pressure used by children during speech.

This raises the question of whether these horns provide adequate resistance to the oral musculature for a muscle training regimen. In their article, Lof and Watson summarized treatment management studies done by Clark, Duffy, and Hodge (year of publication?), in which they stated, “If strengthening of the articulators is necessary then traditional muscle strengthening principles would need to be followed, such as using sets of multiple repetitions against resistance until failure” (2008). They concluded that if these principles weren’t followed, then the OMEs probably wouldn’t build muscle strength, contrary to the expected goals of these exercises.

Fifty percent of the horns used in this study (horns 4, 7, 9, 11, 12) produced pressures greater than the 6.2 cm H20 norm reported for children. Although it is tempting to speculate that these horns will provide the necessary resistance for oral muscle strengthening, there is no evidence to support this notion. Moll (1965) used cinefluorography (the process of making motion pictures of images of objects by means of

X-rays) to study velopharyngeal closure in 10 adults with normal speech and reported differences in closure for blowing and speech. Velopharyngeal closure is the process of the soft palate rising up to the posterior pharyngeal wall to separate the nasal cavity from the

117 | P a g e oral cavity, which allows for the build-up of pressure in the oral cavity during speech.

Specifically, Moll observed greater velar elevation and greater pharyngeal wall activity during blowing than speech. He concluded that the mechanism involved in velopharyngeal closure might be different for speech than that of blowing and argued that while tasks such as blowing could be used to strengthen muscles that are weak, it was unlikely that the improvement seen during blowing would transfer to speech.

Few clinical investigations have been conducted to examine the usefulness of oral motor exercises. In two subsequent investigations by Massengill (1968) and Powers and

Starr (1974), blowing exercises were used with children and adolescents with velopharyngeal inadequacy over a period of 6 weeks. No change in velopharyngeal function was observed in either study, suggesting that the blowing tasks had no impact on strengthening the velar musculature.

In a more recent study, Forrest and Iuzzini (2008) examined the impact of oral motor exercises on articulation in a group of nine children with developmental speech delays. The study employed an alternating treatment design to evaluate changes in production of sounds targeted by oral motor exercises and articulation therapy. Each child received treatment on two linguistically different sounds in which one sound was treated with oral motor exercises and the other sound was treated with articulation therapy. The findings of their study revealed that greater speech production improvement occurred with articulation therapy compared to oral motor exercises.

Despite a lack of empirical support for oral motor exercises, a large percentage of

SLPs continue to use oral motor exercises in their clinical practice. Lof and Watson (2008)

118 | P a g e conducted a nationwide survey of 537 practicing clinicians from 48 states, and found that

85% of the respondents reported using oral motor exercises to address speech sound production problems, while the remaining 15% percent claimed they had never used these exercises. The clinicians who reported using these exercises indicated a belief (all of the clinicians?) that oral motor exercises have benefits that lead directly to speech improvement. However, the research currently available contradicts the use of oral motor exercises to improve speech sound production (Ruscello, 2008).

Do the pressures produced by the horns follow a hierarchical order? According

Rosenfeld-Johnson, the horns in the horn hierarchy should be used one at time starting with Horn 1. Each horn must be blown 25 times in rapid repetitions before moving on to the next horn. Theoretically, once these requirements are met the client should move on to the succeeding horn in the hierarchy. The results from this study demonstrate that pressure did not increase systematically along the horn hierarchy. The only significant differences seen were at the extremes, the horns at the beginning of the hierarchy showing different onset pressures than those at the end (refer to Table 1). This indicates that the

Horn Hierarchy program does not provide a systematic increase in oral pressure requirements, and should not be considered a treatment regimen for speech muscle resistance training.

Study Observations. The Horn Hierarchy kit from Talk Tools is packaged and sold as a high quality controlled therapy tool, when the reality is, most of these horns can be found in any party supply store. Throughout the study, multiple observations were made concerning the quality of the horns. Accumulation of saliva often affected the quality of

119 | P a g e sound in the horns with noise mechanisms on or near the mouthpiece (4, 5, and 8). When this occurred, the participant either blew harder in an attempt to create the sound, or stopped entirely stating the horn no longer worked or was “broken”. If this occurs with children who are given these horns as “homework”, it is unlikely many of them will faithfully practice with tools that are too difficult to use or deemed as damaged.

For three of the eight participants, a hole developed in the paper portion of Horn 12, resulting in a lack of adequate pressure to fully extend the paper tube [When this occurred, the other Horn 12 in the kit was used for data acquisition…correct?]. According to the program, these horns are to be used repeatedly until a child can complete the 25 rapid repetitions; however, these horns were useless after only 10-15 blows. Another complication for Horn 12 that arose was the amount of pressure needed to produce noise.

The level of pressure needed was too great for any of the participants to achieve [Did they not achieve this eventually?] . The lack of horn quality along with high oral pressure requirements could make it difficult for children to blow this horn for the recommended 3 second duration.

Another question that arose during this investigation is the age at which children are able to understand and complete kit instructions, even with the guidance of an adult caregiver or SLP. It was noted during the trials that only two of the eight children (both 5 years of age) were able to grasp the concept of a long, soft blow. The other six participants blew more forcefully when instructed to blow softly, even when a model was provided. In addition, they produced short, miniscule blows when asked to blow for a longer duration.

These children were unable to understand that the sound needed to be held for a specific

120 | P a g e duration to sustain sound at a minimal loudness. When the task was modeled for the child, s/he was only able to mimic the act for one trial, if at all.

It is possible that there may be a population that would benefit from the use of oral motor exercises but the current population of children receiving these therapies may not be appropriate. The fact is most children receiving oral motor exercise intervention do not suffer from muscle weakness. They have developmental speech sound problems that are unlikely to be impacted by exercises designed to strengthen the musculature. Before using these horns with any clinical population, SLPs should carefully consider the goals of treatment and expected treatment outcomes.

Future Directions. This study looked the aerodynamic qualities of a common OME. It would be beneficial to take the information from this study and integrate it into a controlled treatment study; specifically comparing this treatment against a more traditional form of articulation therapy. Employing common novelty horns in a treatment study would provide the necessary evidence to indicate whether or not they are an effective treatment practice.

Conclusion

The intent of this study was to provide speech language pathologists with information regarding the efficiency of oral motor exercises during articulation therapy.

The results from this investigation suggest that the majority of the horns in the Horn

Hierarchy program do not provide the necessary resistance for a muscle training regimen.

In addition, the intraoral pressures required to blow the ten horns did not increase

121 | P a g e systematically along the stated hierarchy. These results indicate that OMEs using common novelty horns may not provide the adequate aerodynamic resistances necessary to improve speech production.

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References

Bahr, D.C. (2001). Oral motor assessment and treatment: Ages and stages. Boston: Allyn and Bacon.

Beckman, D. (1986). Oral motor assessment and intervention. Winter Par, FL: Beckman & Associates.

Clark, H.M. (2005). Clinical decision making and oral motor treatments. The ASHA Leader, pp. 8-9.

Forrest, K. (2002). Are oral-motor exercises useful in the treatment of phological/articulatory disorders? Seminars in Speech and Language, 23, 15-25.

Forrest, K., & Iuzzini, J. (2008). A comparison of oral motor and production training for children with speech sound disorders. Seminars in Speech and Language, 29, 304- 311. doi:10.1055/s-0028-1103394

Lof, G.L. (2003). Oral motor exercises and treatment outcomes. Perspectives on Language, Learning and Education, 10, 7-12.

Lof, G.L., & Watson, M.M. (2008). A nationwide survey of nonspeech oral motor exercise use: Implications for evidence-based practice. Language, Speech, and Hearing Services in Schools, 39, 392-407. doi:0161-1461/08/3903-0392

Massengill, R., Quinn, G. W., Pickrell, K. L., & Levinson, C. (1968). Therapeutic exercise and velopharyngeal gap. Cleft Palate Journal, 5, 44-47.

Moll, K. A. (1965). A cinefluorographic study of velopharyngeal function in normals during various activities. Cleft Palate Journal, 2, 112-122.

Powers, G.L. & Starr, C.D. (1974). The effects of muscle exercises on velopharyngeal gap

and nasality. Cleft Palate Journal, 11, 28–35.

Rosenfeld-Johnson, S. (1999). Oral-motor exercises for speech clarity. Tucson, AZ: Innovative Therapists.

Ruscello, D.M. (2004). Considerations for behavioral treatment of velopharyngeal closure for speech. In K.R. Bzoch (Ed.), Communicative disorders related to cleft lip and palate (5th ed., pp. 763-796). Austin, TX: Pro-Ed.

Ruscello, D.M. (2008). Nonspeech oral motor treatment issues related to children with developmental speech sound disorders. Language, Speech, and Hearing Services in Schools, 39, 380-391. doi: 0161-1461/08/3903-0380

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Laurie Petric Mentor: Dr. Cary Heck

Major: Psychology and Criminal Justice

Research Topic: Views of the Effects of Methamphetamine on Health, Treatment Efficacy and Criminality

Abstract This study explored the views of both professionals and the general public about methamphetamine’s relation to health, treatment efficacy and criminality in Laramie, Wyoming. The professional (expert) participants included a nurse practitioner, a law enforcement officer and a drug counselor. General public participants were recruited through purchase of a randomized mailing list. All participants were given a drug severity survey, in which they rated their agreement or disagreement with four statements, each of which stated that a different drug (cocaine, alcohol, prescription drugs, or methamphetamine) causes more adverse effects than any other drug. In addition, expert participants were interviewed and asked a series of open-ended questions related to methamphetamine and other drugs. Averaged responses to the drug severity survey statements revealed that expert participants believed that alcohol causes more adverse effects than any other drug, while general participants believed that methamphetamine causes more adverse effects than any other drug. An independent samples t- test with Welch approximation was used to compare opinions between the two groups, and results indicated a significant difference in opinion between expert and general participants regarding alcohol and prescription drugs, but not cocaine or methamphetamine. Specifically, expert participants felt that alcohol and prescription drug abuse were more severe than the general participants. The results of this study may help future researchers determine whether methamphetamine use does, in fact, have a significantly greater effect on health, treatment efficacy and criminality when compared to other drugs.

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Views of the Effects of Methamphetamine on Health,

Treatment Efficacy and Criminality

According to Mosher and Akins (2007), methamphetamine has been around for centuries and has been used for many different purposes. Methamphetamine was used in

World War II by the Germans for its positive properties such as increased wakefulness, increased concentration, and increased stamina (Mosher & Akins, 2007). Today, it is known as one of the most widely used and addictive substances in America; in fact, methamphetamine is known in several countries throughout the world as being one of the most highly addictive drugs ever introduced (McKetkin, Kelly, McLaren, & Proudfoot,

2008). According to the United Nations Office on Drugs and Crime, in 2010, it was estimated that the number of people who have used methamphetamine within the last twelve months was stabilized at 53 million worldwide (United Nations Office on Drugs and

Crime, 2010). Studies have indicated that it is very likely that a person who uses methamphetamine will become physically dependent on the drug after very little use

(Wallace, Galloway, McKetkin, Kelly & Leary, 2009).

Methamphetamine can be ingested several different ways, including injection, absorption into the skin, and inhalation (Altshuler, 2005). When methamphetamine is ingested, it is processed primarily in the liver. Once it has been broken down using a quaternary level process in which several metabolites (that are not believed to have direct clinical effects) are released, the broken down compounds reach the plasmid level of the blood, spreading quickly to all parts of the body. The chemicals reach the brain and cause an explosion of dopamine while simultaneously preventing reuptake, resulting in a recycling of dopamine in the brain that can last anywhere from eight to twenty four hours

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(Cruickshank & Dyer, 2009). At this time, the user begins to experience what Cruickshank and Dyer (2009) describe as “euphoria and psychomotor activation,” or disconnect from reality and excessive psychomotor functioning. Once the compounds have passed through the body, they are excreted mainly through urination; however, at higher doses or when a using poor quality methamphetamine, compounds may be excreted through the skin creating large, open sores (Cruickshank & Dyer, 2009).

The consequences of using methamphetamine manifest in a plethora of side effects, many of which are negative. The physiological effects of using methamphetamine include elevated and extreme feelings of well-being and confidence, appetite suppression, extended periods of wakefulness, and an accelerated heartbeat (Weisheit & Wells, 2010). Other effects of using methamphetamine include hyperthermia, convulsions, cardiovascular collapse, weight loss, sleep disorders, renal pathology and dental problems. Long-term effects of methamphetamine abuse include kidney complications, lung disorders, brain and liver damage, and blood clots. At higher doses, users begin to experience symptoms of psychosis including paranoia and delusions or hallucinations. Paranoia associated with methamphetamine abuse may lead to homicidal and suicidal tendencies. Additionally, a number of injection-related health problems have been identified including Hepatitis B and

C, HIV, and various sexually transmitted infections and diseases.

It is widely known that people who use methamphetamine will become addicted after very little use. Studies have indicated that it is very likely that a person who uses methamphetamine will become physically dependent on the drug because of the positive effects that are elicited such as increased alertness, increased stamina and an increase in

126 | P a g e one’s ability to concentrate. The odds of becoming addicted to methamphetamine after one use are very high considering the physiological effects methamphetamine has on the brain.

The process of making methamphetamine is, for the most part, simple. It is a three part process that begins by combining ephedrine or pseudoephedrine, hydriodic acid, and red phosphorous and heating the mixture in various increments for twelve hours. Then, the red phosphorous is extracted from the mixture and sodium hydroxide is added to the mixture to convert it from an acid to a base. Finally, Freon is added to the mixture, enabling extraction of the methamphetamine (Manning, 1999). Once this process has been completed, several chemical compounds have covered every exposed surface in the place.

Also, fumes from the methamphetamine production process have sunken into the floor and completely saturated the carpet and other fabrics, not to mention simultaneously infiltrating the lungs of the cooks or anyone unlucky enough to be living in or close to the lab.

Many people believe that methamphetamine plays a big part in an individual’s predisposition to commit criminal acts. Whether under the influence or in search of the next high, public opinion yields to the popular belief that if you are a user, you are also a criminal. In many cases, this holds true. Methamphetamine is one of the many target drugs of the famous “war on drugs.” Law enforcement has increased their efforts to eliminate the supply of methamphetamine by stepping up patrol and increasing punishment for possession, distribution and production of meth. According to Dr. Amie Nielsen of the

University of Miami, annual federal anti drug-related spending increased from $111 million on 1970 to approximately $18 billion in 2000 (Neilsen, 2010).

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In addition to being a national problem, methamphetamine abuse is also a major problem in Wyoming (United Nations Office on Drugs and Crime, 2010). The Crime in

Wyoming report, in 2010, reported 240 out of 452 controlled substance arrests were related to methamphetamine. This includes manufacture, sale, possession, use, transportation, and conspiracy regarding methamphetamine. Also, over nine thousand grams of methamphetamine were seized and almost 1,500 grams were purchased in the state last year.

According to the Cheyenne Meth Initiative of Laramie County, at least one out of every 200 Wyoming citizens has used meth in the last 30 days (Cheyenne-Laramie Meth

Initiative, 2009). Also, during 2002 in Wyoming, methamphetamine was involved in more than 82% of federal drug arrests. Obviously, methamphetamine plays a major role in the

Wyoming drug world. This leads one to question why Wyoming has such a major problem with methamphetamine.

During his research, Nick Reding, the author of Methland: The Death and Life of a

Small Town, discovered that methamphetamine flourished where economy died, usually in rural areas. This was due to several chain reactions, beginning with people using the drug to work more hours after wage cuts. Eventually, these people would lose their jobs because their employers went bankrupt and had to close down. Once the now unemployed methamphetamine users were without a job, they turned to the only thing they did have: methamphetamine. These people now had the time to devote to methamphetamine production, distribution and of course, use. Basically, Reding attributed the rise of methamphetamine in rural areas to the economy being compromised. In addition, he

128 | P a g e credited methamphetamine abuse in rural areas to those trying to make up for lost wages working for days on end under the influence of meth’s positive properties.

Laramie, Wyoming is not much different from the town that Reding researched. It is a small town in rural Wyoming, a state that is known for containing more methamphetamine users than anywhere else in the country. Similarly, the economy in

Laramie, Wyoming is rather poor; in 2008, the state saw a recession that cost citizens

21,000 jobs.

Popular public opinion claims that people who use meth have weak moral fiber and next to no will power. Many people believe that, even though it is not easy, if a person really wants to quit using a drug, they can just stop taking it or check into a treatment facility and get the problem taken care of there. The Wyoming Meth Project, which was designed in order to deter the public from using methamphetamine and to show current users exactly what they’re getting themselves into, is an example of public opinion in its prime. The

Wyoming Meth Project utilizes billboards and television commercials to display the nasty side of methamphetamine. In these commercials, the public is “exposed” to methamphetamine in its true form, with commercials enacting children beating their parents in order to steal money to pay for the drug and friends high on meth leaving one of their own to die of a meth overdose in the gutter.

However, according to a study conducted by Mark Anderson of the University of

Washington that focused on the Montana Meth Project, similar to the Wyoming Meth

Project, government intervention and informational programs such as these only have a temporary effect on prevention of methamphetamine use. In the following study, public attitudes towards drug use will be compared to expert testimony to attempt to provide

129 | P a g e either a base of support for the public opinion or evidence on the contrary. It is hypothesized that public views of methamphetamine abuse will differ from those of expert participants. Specifically, the general public will believe that methamphetamine abuse is more problematic overall when compared to other drugs.

Method

Participants

Participants of this study included three expert participants and forty general participants. The three expert participants included a nurse practitioner, sheriff and drug counselor. Recruitment of the expert participants began with an introductory email or phone call asking them if they would like to participate in the study. If they agreed, a time to meet and interview was set up. General participants included a random sampling of two hundred citizens of Laramie, Wyoming. Recruitment of the general participants began by obtaining a randomized mailing list of 200 citizens of Laramie, Wyoming.

Materials

Materials included a Drug Severity survey which consisted of four questions pertaining to four known substances of abuse in Laramie, Wyoming: methamphetamine, alcohol, marijuana and prescription drugs. Participants were asked to rate their agreement with four separate statements on a Likert scale, where 0 indicated strong disagreement and

4 indicated strong agreement. Each statement stated that a different drug (i.e. alcohol, cocaine methamphetamine or prescription drugs) causes more adverse effects than any other drug.

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Procedure

Expert participants were scheduled for individual interviews. Expert participants were given five minutes to complete the Drug Severity survey. Once the survey was completed, expert participants partook in a thirty to forty five minute interview session during which they were asked a series of open ended questions about their perceptions of drug abuse in Laramie, Wyoming.

General participants were mailed a packet consisting of an informational letter, informed consent form and the Drug Severity survey. General participants were asked to sign the informed consent form, fill out the survey utilizing the Likert scale, and mail both materials back to the researcher in a prepaid envelope.

Results

Data analysis of the surveys consisted of averaging answers for each statement on the survey and determining which drug all participants, both expert and general, felt is the most harmful overall. Also, in addition to calculation of means, an independent samples t- test was run in order to determine if there was a significant difference in opinion between expert and general participants.

Analysis of the Drug Severity survey indicated that expert participants believe that alcohol (M = 4.0, SD = .00) caused more adverse effects than prescription drugs (M = 3.33,

SD =.58), cocaine (M = 2.67, SD = 1.53), and methamphetamine (M = 2.67, SD = 1.53) (See

Table 1). These means suggest that expert participants felt that alcohol, not meth, was the most problematic drug out of the four drugs mentioned. All three of the expert participants selected “4-strongly agree” to the statement: “Alcohol causes more adverse effects than any other drug.” Similarly, all three expressed in their interviews that alcohol has more adverse

131 | P a g e health effects when compared to any other drug. However, all three expert participants also expressed in their interview that determining which drug was the worst over all was a hard question to answer and that all drugs have similar risk associated with abuse.

Analysis of the drug severity survey indicated that general participants felt that methamphetamine (M = 3.41, SD = .832) causes more adverse effects than alcohol (M =

2.81, SD = 1.05), prescription drugs (M = 2.16, SD = .73), and cocaine (M = 2.14, SD = 1.11)

(See Table 1). These results suggest that the opinion of Laramie citizens trends in the direction of methamphetamine being the most problematic drug. Overall, meth was rated the highest in terms of agreement that it causes the most adverse effects when compared to other drugs. In addition, public opinion yielded the same neutrality regarding severity of cocaine and prescription drug abuse whereas alcohol almost reached the same level as methamphetamine, indicating a borderline agreement that alcohol abuse was as severe as methamphetamine abuse.

After running an independent sample t-test with Welch approximation, results indicated that expert and public opinion differed in the areas of alcohol, t (36) = .590, p <

.001, and prescription drugs, t (2.56) = 3.31. p = .05) abuse. Results also indicated no significant difference in opinion regarding cocaine, t (2.175) = .59, p = .64, or methamphetamine, t (2.10) = -.82, p = .50. These results show that both groups held different views pertaining to the severity of alcohol and prescription drug abuse, but held the same opinions regarding cocaine and methamphetamine.

Discussion

According to results obtained by expert participants, the belief that alcohol is the biggest problem drug in Laramie, Wyoming is common throughout all three areas of focus.

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When controlling for the fact that Laramie is home to the University of Wyoming, expert participants testified that this has nothing to do with the prevalence of alcohol abuse. In addition, expert participants, on average, said that alcohol was a major part of rural culture, especially among youth. Essentially, alcohol consumption is viewed as a rite of passage in many rural cultures.

Some things that may have hindered the outcome of results are not enough participants, both expert and general, and the broadness of the survey.

Although two hundred general participants were initially selected to participate, only forty responded. The opinions of forty people could not possibly capture the scope of the entire community as a whole. To account for this, perhaps for future studies, the researcher could set up a booth or table at a public event in several small communities and obtain data in that setting. This strategy would have the potential to yield higher response rates and would cost a great deal less in terms of postage or other materials.

In terms of the survey being too broad, if the questions asked were specified to include aspects of health, treatment efficacy and criminality instead of general harm of abusing the substance, perhaps we could have learned about which facet of the three causes more ill repute in the public mind. Just asking which drug out of marijuana, alcohol, cocaine or methamphetamine is the most harmful drug overall does not narrow down to the three variables being studied. This was made clear when interviewing the expert participants. Questions similar to those on the survey yielded difficult answers due to the fact that experts could not place blame on only one drug.

Finally, future studies should include collection of demographic data from all participants in an attempt to identify differences in opinion in terms of age or sex. Also,

133 | P a g e employment history would be relevant in that the researcher could identify general participants as having average knowledge of drug abuse or above average knowledge. For example, if a general participant was in fact a doctor, that participant would possess far greater knowledge of the physiological side effects of drug abuse than would a general participant with experience in retail.

In conclusion, it is apparent that methamphetamine is a major problem drug in the state of Wyoming as well as worldwide. This is due primarily to the fact that it is known as one of the most widely used and addictive substances in the world. In 2010, the United

Nations Office on Drugs and Crime estimated that over 53 million people have used methamphetamine. Results of this study indicate that although many people use this drug, a lot is still unknown about the potential effects that methamphetamine has on health, treatment efficacy and criminality.

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References

Altshuler, S. J. (2005). Drug endangered children need a collaborative community response. Child Welfare League 171-190. Anderson, M.D. ( 2010). Does information matter? The effect of the Meth Project on meth use among youths. Retrieved 7/4/2011 from: http://www.dmarkanderson.com /Meth_ Project.pdf Cheyenne-Laramie Meth Initiative. (2009). Meth Facts. Retrieved 7/11/2011 from: http://www.cheyennemeth.org/ Cruikshank, C., & Dyer, K. R. (2009). A review of the clinical pharmacology of methamphetamine. Addiction, 104, 1085-1099. McKetkin, R., Kelly, E., McLaren, J., & Proudfoot, H. (2008). Impaired physical health among methamphetamine users in comparison with the general population: the role of methamphetamine dependence and opioid use. Drug and Alcohol Review, 27, 482- 489. Mosher, C. J., & Akins, S. (2007). Drugs and drug policy: the control of consciousness alteration. Sage Publications, Thousand Oaks, California. p 28-35. National Drug Intelligence Center. (2001). Wyoming Drug Threat Assessment. Retrieved 7/4/2011 from: http:/ /www.justice. gov/ndic/ pubs07/712 /meth.html. Neilsen, A.L. (2010). American’s attitudes towards drug-related issues from 1975-2006: The roles of period and cohort effects. The Journal of Drug Issues, 10(2). 461-494. United Nations Office on Drugs and Crime. (2010). 2010 World Drug Report. United Nations Office on Drugs and Crime; 2010. Retrieved 7/31/2011 from: http://www.unodc.org/documents/wdr/WDR_2010/World_Drug_Report_2010_lo- res.pdf Wallace, C., Galloway, T., McKetkin, R., Kelly, E., & Leary, J. (2009). Methamphetamine use, dependence and treatment success in rural and regional North Coast of New South Wales, Australia. Drug and Alcohol Review, 28, 592-599. Weisheit, R. A. & Wells, L. E. (2010). Methamphetamine laboratories: The geography of drug production. Western Criminology Review, 11, 9-26.

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Table 1

Means and Significance Test for Expert and General Participants by Drugs

Expert General n = 3 M SD n = 37 M SD DRUG Cocaine 2.67 1.53 2.14 1.11 Alcohol 4 .00 2.81 1.05 RX Drugs 3.33 .58 2.16 .73 Meth 2.67 1.53 3.41 .83

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Kevin Schilling Mentor: Dr. Patrick Johnson

Major: Chemical and Petroleum Engineering

Research Topic: Improving Magnetic Nanoparticles

Abstract

DL-Cysteine covered magnetic Fe3O4 nanoparticles (Cys-Fe3O4) have been fabricated via a sonochemical approach using iron pentacarbonyl (Fe(CO)5) in conjunction with a small surfactant,

DL-Cysteine, which when bound to magnetic Fe3O4 nanoparticles, the resulting complex has the desired behavior and solubility properties in aqueous and non-aqueous solutions. The aim is to find an easy and simple method to achieve well-dispersed Cys-Fe3O4 nanoparticles. We found that different conditions such as sonication time and concentration of DL-Cysteine affect the magnetic properties of Cys-Fe3O4 colloidal suspensions. Bare Fe3O4 and Cys-Fe3O4 were characterized with TEM, SEM, DLS, and DL-Cysteine concentration effects (color progression chart) to investigate the difference of coated and uncoated magnetic nanoparticles. We report an optimal concentration of cysteine and iron pentacarbonyl. Some solvents such as DI-water and n-pentane were used to determine which effectively dried the particles without enhancing further oxidation.

Materials

The chemicals used in this work were iron pentacarbonyl (Fe(CO)5) (pure), DL-

Cysteine (granular, 97% pure), n-pentane (99%/volume), de-ionized water (DI-water), ferrous (Fe2+) and ferric (Fe3+) salts. All of these materials were used as given, there was no further purification.

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Introduction

Magnetic nanoparticles have various applications that they can be used for; for example, drug delivery, catalysis, biotechnology and biomedicine. In drug delivery, the magnetic particles can be used to cure cancer like in magnetic hyperthermia. There are various catalytic applications to aid in the recovery of used enzymes. In this experiment, magnetic nanoparticles are formed from two different materials but use the same method.

Iron pentacarbonyl is a chemical that is used widely within the iron oxide, magnetic nanoparticle realm. It is relatively inexpensive; however, it is toxic because when reacted it forms carbon monoxide which is hazardous.

Today, humans are producing excess amounts of carbon dioxide and releasing it into the atmosphere. The excess carbon dioxide is a large component of the greenhouse gases causing the temperature on the surface of the Earth to increase. The ocean absorbs some of the excess carbon dioxide due to the equilibrium reactions that occur on the ocean surface.

Carbon dioxide is absorbed into the water and then it is converted into its carbonate and bicarbonate forms so that it can be neutralized in the ocean. With an increase of carbon dioxide, there is also an increase of the carbonate and bicarbonate forms. These forms are highly reactive with calcium to form calcium carbonate which is commonly known as baking soda. Ocean animals that have shells or have an exoskeleton are most susceptible to this because the carbonate molecules are degrading the shells which are primarily composed of calcium and thus killing the ocean life. Most of the carbon dioxide produced and released into the atmosphere is from industrial waste. Most of these plants have an air cleaning facility where most of the harmful gases are contained and eliminated. If there

138 | P a g e was a way in order to capture some of the carbon dioxide leaving, then the overall output of carbon dioxide into the atmosphere could be minimized.

There are a few enzymes that can convert soluble carbon dioxide to the carbonate form. The carbonate form can then be isolated and neutralized. These enzymes could be placed in a solution where carbon dioxide could be bubbled in so the enzymes could do the reaction. The main problem is that when the reactions have been completed, the enzymes are lost in the process. So if there is a way to recover the enzymes, it could potentially lower the capital cost for the materials. If enzymes were bound to magnetic nanoparticles, then they could, in fact, be recovered. There is a problem that arises when using the magnetic nanoparticles, however.

DL-Cysteine Iron Pentacarbonyl Figure 1-The chemical structure of the primary compounds being used throughout the experiment.

When using the term “nanoparticles,’ these are on the order of 10-9 meter or nanometer (nm) scale. So since these are very small, the magnetic nanoparticles have paramagnetic behavior. This means that these nanoparticles have random magnetic fields unless an outside magnetic field or a large temperature change is administered. With the paramagnetic behavior the random magnetic direction causes them to magnetize with one

139 | P a g e another, forming large magnetic aggregates. These large aggregates can be too big to efficiently bind to substrates or to be placed in the bloodstream. More uniform dispersion is desired for these clusters that are resulting from the experiments. In order to achieve a uniform dispersion, a small molecule surfactant was used. DL-Cysteine (C3H7NO2S), an amino acid, was chosen as the chemical to coat the particles and form smaller clusters. The

DL-Cysteine has some advantageous properties when using it. It is relatively inexpensive and small enough to coat the nanoparticles to prevent the magnetic aggregation and increase uniform dispersion. The molecule has a sulfur group that can easily bind to the surface of the nanoparticle in a neutral pH. This sulfide bond is strong due to its affinity for iron. An important factor to using DL-Cysteine is that it contains an amine (NH2) and carboxyl (COOH) group on the other side of the molecule from the sulfur group. Figure 1 shows the chemical structure of the compound. With these groups, a biological component like an enzyme, antibody, or another compound could bind readily to either group.

To make the magnetic nanoparticles two different materials were used but they were created by the same method. The two materials are ferrous (Fe2+) and ferric (Fe3+) salts combination or iron pentacarbonyl (Fe(CO)5). Both of these are used widely within the iron oxide, magnetic nanoparticle realm and is relatively inexpensive. However,

Fe(CO)5 is toxic because when reacted, it forms carbon monoxide which is hazardous but it can be isolated and neutralized. Figure 1 shows the chemical structure of iron pentacarbonyl.

When forming the colloid complexes, a sonochemical approach was chosen. By using a sonicator with a sonication bath, ultrasonic waves can provide the energy to break

140 | P a g e the bonds. The ultrasonic sound waves create nano-bubbles within the solution. They form around the bonds of the chemicals in the solution. The bubbles burst, providing enough energy to break the bonds and then let them reform to the desired products.

Hypotheses

The hypothesis for this experiment was that when the magnetic Fe3O4 nanoparticles are functionalized with DL-Cysteine, aggregation is reduced and the newly formed complexes will retain their magnetic properties in order to be applied to biological components. In conjunction, an optimal concentration of DL-Cysteine added and sonication time will be discovered to fabricate the best Cys-Fe3O4 colloid complex.

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Procedure

Figure 2-A schematic of the process of creating Cys-Fe3O4 and Fe3O4 magnetic nanoparticles. Three major steps that occurred to making them are fabrication, washing and centrifugation, and drying.

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Figure 3-Schematic of the supernatant liquid drying and preservation step.

To begin the fabrication step of Cys-Fe3O4, seventy milliliters of DI-water was added into a clean, round bottom flask. DL-Cysteine, in granular form, was added into the water solution. A different amount was added for each experiment. Then the round bottom flask was lowered into the bath and started sonicating. One milliliter of iron pentacarbonyl was then injected into the solution. The round bottom flask was then sealed and a miniature absorber column was attached to absorb and neutralize the carbon monoxide being released. After the entire sonication time had expired, the new colloid complex solution went through the washing and centrifugation step.

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The newly created solution was added into several vials. These vials then were centrifuged at 3700 revolutions per minute for forty-five minutes. Due to the centrifugal forces, most of the magnetic nanoparticle clusters would be pulled to the bottom, forming a magnetic nanoparticle pellet. The supernatant fluid consisted of the smaller nanoparticles and the salts that formed or not reacted during the experiment. The supernatant fluid was then poured into a separate beaker thus leaving the pellet at the bottom of the vial. More

DI-water was added to the pellet to essentially wash the nanoparticles by collecting all of the extra non-reacted salts and smaller nanoparticles. The vials were re-centrifuged where the supernatant fluid was poured into the same beaker leaving a pellet. The pellet and beaker filled with supernatant fluid was then ready to be dried in a vacuum.

N-pentane, an organic, non-polar solvent was used to dry the pellet. This was chosen because since it is non-polar, it would not react with any substituents in the pellet but it would collect the non-polar salts. N-pentane is also volatile so it will evaporate easily. Thus it was added to the pellet, and then shaken by hand for ten minutes. The supernatant fluid was then properly disposed in a container. One more washing occurred to ensure that most of the salts were collected. Then the vial with the pellet was set in a vacuum chamber to dry. The beaker containing the supernatant fluid from the washing and centrifugation step was also placed into the vacuum chamber to dry. This was performed to ensure that no other contaminants interfered with the drying step and to prevent any further oxidation of the magnetic nanoparticles. After both systems were completely dried, the mass was measured. The powders resulting were contained and sealed for further experimenting or characterizing of their properties.

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Results

During the experimentation process, the round bottom flask where the reaction occurred took place had its picture taken after 90, 180 and the final 300 minutes of its reaction. The pictures were then gathered and put in a spreadsheet to see the progression of colors during the experiment. As the sonication time and the concentration of DL-

Cysteine increased, the solution became darker. This is due to the fact that smaller clusters are forming during the sonication. When increasing the sonication time, it allows the solution to be broken down further. As Figure 4 shows, the uncoated Fe3O4 nanoparticles at 90 minutes appears to have sank to the bottom, thus larger clusters are aggregating and precipitating out of solution. When increasing the sonication time, these larger clusters are broken down into smaller ones thus preventing them from aggregating thus staying in solution and creating the dark color. As the concentration of DL-Cysteine increases, it allows to coat more of the clusters and thus preventing them from aggregating, because the ratios are increasing thus smaller clusters is theoretically being totally coated. After more sonication, smaller clusters are being formed and the DL-Cysteine then still coats them and keeps them uniformly dispersed within the solution.

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Figure 4- This is a chart that shows the color progression of each flask with separate amount of DL-Cysteine added into the system.

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Table 1-The percent yield and molar ratio for each amount of DL-Cysteine added into the system for separate experiments.

DL-

Cysteine Molar Ratio (Iron

DL-Cysteine Molarity Pentacarbonyl: DL-

Mass [g] [M] Cysteine) % Yield

0 0.00000 0.000 17.17%

0.016875 0.00196 53.141 18.41%

0.03375 0.00392 26.570 26.33%

0.0675 0.00785 13.285 40.57%

0.135 0.01569 6.643 36.22%

0.27 0.03139 3.321 22.30%

During the washing and centrifugation step, the excess cleaning water was poured into a separate beaker to let dry. Once the pellet and the remnants in the water were dried and weighed, a percent yield was found. This was based on the conservation of matter, which means that the goal was trying to collect everything that reacted. Table 1 shows tabulated values of the masses that were chosen to determine highest percent yield. The highest percent yield achieved was 40.57% when 0.0675 grams of DL-Cysteine was added with 1 milliliter of iron pentacarbonyl. When considering the percent yield, both the remnants of the reaction and the centrifuged pellet were considered. A theoretical molar ratio was calculated in that for 0.0675 grams of DL-Cysteine, there were 13 iron

147 | P a g e pentacarbonyl for every DL-Cysteine molecule. Thus DL-Cysteine would coat to make smaller clusters and help create more uniform dispersion. However, Figure 4 depicts the pellet that was collected along with the total percent yield calculated versus the amounts of

DL-Cysteine added. A similar trend between the pellet mass and percent yield was discovered.

Figure 5-A depiction of how the mass of separate substituents were determined.

Mass of MNP w.r.t. % Yield

50.00% 0.5

40.00% 0.4

30.00% 0.3

% Yield % 20.00% 0.2

10.00% 0.1

0.00% 0 0 0.05 0.1 0.15 0.2 0.25 0.3

Mass of DL-Cysteine Added (g)

Amount ofMNP Amountretained (g) [not including the including [not supernatant iquid] % Yield MNP

Figure 6- A graphical result of the percent yield and the amount of magnetic nanoparticle pellet attained versus the amount of DL-Cysteine added.

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(a) (b)

Figure 7- Transmission Electron Microscopy (TEM) pictures of (a) magnetic Fe3O4 nanoparticles created from Fe(CO)5 and (b) DL-Cysteine coated magnetic Fe3O4 nanoparticle complex.

TEM, DLS, and SEM tests were performed to characterize coated and non-coated magnetic nanoparticles. A concentration of 0.135 grams of DL-Cysteine was used in comparison to the uncoated Fe3O4. When coating the nanoparticles, small, more uniform clusters resulted. When looking at the TEM charts of Figure 7, large aggregated clusters can be seen when using bare Fe3O4 but when they are coated smaller clusters result. Figure

8 shows that the coated nanoparticles result in more dispersed clusters, where a light halo of coating surrounds the dark Fe3O4, can be seen.

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Figure 8-TEM pictures of DL-Cysteine coated magnetic Fe3O4 nanoparticle complexes.

Figure 9- Dynamic Light Scattering (DLS) tests of (a) magnetic Fe3O4 nanoparticles created from Fe(CO)5 and (b) DL-Cysteine coated magnetic Fe3O4 nanoparticle complex.

When coating the nanoparticles, the particle size diminishes and becomes more precise. In Figure 9, DLS calculated the average diameter of the clusters and when looking at bare Fe3O4, the average diameter was 475.73 nm whereas; the size of Cys-Fe3O4 became

170.31 nm. Thus smaller clusters resulted when using DL-Cysteine to coat the particles.

Also the range of sizes calculated was very tight with Cys-Fe3O4. The range for the bare

Fe3O4 was from 390 to 730 nm and the range for coated Fe3O4 was from 165 to 175 nm.

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Smaller, consistent clusters were discovered when using DL-Cysteine to coat the nanoparticles. This led to the fact that more uniform dispersion and less aggregated clusters were resulting.

Figure 10- Scanning Electron Microscopy (SEM) pictures of DL-Cysteine coated magnetic

Fe3O4 nanoparticle complexes.

The SEM charts of 0.135 gram Cys-Fe3O4 show that individual clusters were being created during the reaction. Figure 10 shows small polyps on the surface of the sample.

These polyps demonstrate that they are not clustering together but instead being dispersed and individualized within the experiment.

Conclusion

In summary, fabrication of DL-Cysteine coated and uncoated magnetic Fe3O4 nanoparticles has been achieved using Fe(CO)5 via the sonochemical method. When coating these nanoparticles, (1) the size of the clusters is decreased, (2) the solutions

151 | P a g e become darker, and (3) small clusters can be seen; therefore, the hypothesis was correct;

DL-Cysteine coated the outer surface and aided in uniformly dispersing the particles in solution. An optimal concentration of 0.0675 grams of DL-Cysteine was discovered based on percent yield calculations with a theoretical molar ratio of 13. An optimal sonication time of five hours was discovered to create the most uniformly dispersed but not create too long of a reaction time. Therefore, DL-Cysteine is a great small chemical surfactant to use in coating magnetic Fe3O4 nanoparticles to achieve uniform dispersion and aid in the progression into biological applications.

Future Work

Figure 11-Reaction schematic of further bioprogression into immobilizing enzyme of antibodies.

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With DL-Cysteine having the carboxylic group and the amine group on the end of the molecule, it has a great potential for biological applications. Figure 11 depicts a schematic in which the DL-Cysteine coated complex can be beneficial for binding enzymes and antibodies onto it. In the future, another chemical like glutaraldehyde can bind onto the cysteine. Glutaraldehyde has a great ability to bind onto enzymes and antibodies due to its oxygen group on the end. Then an enzyme like carbonic anhydrase can be immobilized onto the complex so that it can be used in large scale processes to minimize carbon dioxide waste. The goal is to have the ability to recover the enzyme so if it were to be immobilized with a magnetic nanoparticle then the recovery can be achieved by using a strong magnet to collect to enzymes after the reaction occurs. This would also pertain to an antibody like one that specifically targets cancer. So it can enter the body’s affected areas, prevent to cancer cells from metastasizing, and then be recovered using a magnet. Therefore by taking the step into immobilizing subjects with magnetic nanoparticles, more possibilities are available in the biomedical world.

Acknowledgements

I acknowledge the financial support from the McNair Scholars program through the

Department of Education. I would like to thank my faculty mentor, Dr. Patrick Johnson for giving me this opportunity to perform research and being there to help with the research aspects. I would like to also thank my graduate mentor, Joo Seob Lee who helped me tremendously in choosing a topic, show how the experiments were run, help with TEM,

DLS, and SEM, and be there for whenever I had any questions.

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