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The Virginia Engineering and Science Research Journal

Highlighting Undergraduate Research Volume Volume I, I, Issue Issue 11 Spring Spring 2010 2010




The Virginia Engineering and Science Research Journal

Inaugural Issue Spring 2010

Highlighting Undergraduate Research

The Spectra Journal is published by the University of Virginia School of Engineering and Applied Science Paper copyrights are retained by the authors, however, The Spectra retains the right to non-exclusive use in print and electronic formats for all papers submitted for publication in The Spectra. Front Cover: Murine Retinal Tissue, Courtesy of Katelyn Mason

Executive Board

Primary Editors



Christopher Belyea Editor-in-Chief

Matthew Brodt Chas DeVeas Elizabeth Dobrenz Patrick Gildea Atul Kannan Hannah Meredith

Ryan Clairmont Charlie Cox Ian Czekala Ian Davey Todd Gerarden Noah Goodall Sarah Grigg Carolyn Pelnik Peter Sahajian Jack Valentine

Justin Sinaguinan Layout Editor

Wyatt Shields Associate Editor Eric Fried Secretary and Publicity Chair Garrett Wheaton Treasurer and Fundraising Chair

Author Photos Courtesy of Adrianne Castro

The Spectra was printed by Bailey Printing. An online version is available at For more information please contact




“The University of Virginia prides itself on undergraduate research and, in the spirit of our founder Thomas Jefferson, on student self-governance. It is here in The Spectra that you find both.”

Christopher Belyea is a fourth-year chemical engineering and pre-medicine student from Ashburn, Va. During his time as a Jefferson and a Rodman Scholar at U.Va., he has served as the co-President of the Rodman Scholars Program. He has been involved in cardiovascular research at the University of Virginia Hospital and has received a Harrison Undergraduate Research Award for study in Denmark. During the summer of 2009 he conducted biochemical engineering research as a Merck Engineering & Technology Scholar at Merck’s BioVentures research labs. Christopher is a member of the Raven Society, Tau Beta Pi Engineering Honor Society and ODK. He will be attending medical school next year under an Army HPSP full scholarship.


Dear Reader, Welcome to the inaugural issue of The Spectra Engineering and Science Research Journal. Recognizing the integral importance of research at the undergraduate level, the founding editorial board has worked hard to provide you this forum in which the best of the engineering and science research at the University of Virginia can be featured. The idea for The Spectra was developed by a group of undergraduate engineering students in the autumn of 2009. The goal of our founding editorial board was to increase the exposure of U.Va.’s undergraduate research by creating a new University publication: The Spectra. Our hope has been to provide a publication platform which cultivates undergraduate engineering research, applied science research and engineering design. Over the course of this past academic year we have created a peer review research journal which is student-led, student-driven and student-defined. The entirety of the journal’s operation, from the competitive selection of the featured articles, to the peer review and editing process, to the physical layout of the journal, has been in the hands of our student editorial board. It is a journal by students, for students. The articles presented within this publication issue represent an array of fascinating undergraduate research at U.Va. This research has taken our featured authors to the high desert plains of Chile to study one of the largest engineering marvels of modern times, to laboratory work which has helped to elucidate the healing potential of adipose stem cells, and to research into the material science of aerospace engineering. We believe these articles provide an informative window into some of the best of U.Va.’s undergraduate research. The development of this journal would not have been successful without the incredible guidance and support of many in the University community. Upon learning of the idea for The Spectra, James H. Aylor, dean of the School of Engineering and Applied Science, has provided his full support for The Spectra initiative. We have been very grateful for both Dean Aylor’s and our faculty advisory board’s guidance in undertaking this project. I would like to give due credit to the generous financial support of engineering alumnus Linwood A. “Chip” Lacy Jr. and the U.Va.’s Office of the Vice President for Research, without whose funding this publication would not have been possible. Lastly, the SEAS Communications Office has served as an incredible resource for aiding us through the publishing process. The University of Virginia prides itself on undergraduate research and, in the spirit of our founder Thomas Jefferson, on student self-governance. It is here in The Spectra that you find both. The sharing of ideas and discoveries through technical writing is paramount to advancing our human faculty in the engineering and sciences. The creation of The Spectra will serve to highlight and foster undergraduate research excellence at U.Va. for years to come. Nil sine magno labore,

Christopher M. Belyea Founder, Editor-in-Chief

Table of Contents II


Foreword from the Editor-in-Chief

The Collaborative Engineering of the Atacama Large Millimeter Array Ian Czekala


Expression and Purification of Inclusion Membrane Protein A (IncA) from Chlamydia trachomatis using Dual-Detergent System and Secondary Structural Determination using Circular Dichroism Spectroscopy Ashley Elizabeth Keller


High Power Laser Texturing of Surfaces for Aerospace Applications Ami Patel


Role of EphrinB2 Reverse Signaling in Pathological Retinal Neovascularization Katelyn Mason


The Shady Side of Sunscreen Haarthi Sadasivam


Efficacy of Adipose-Derived Stromal Cell Homing and Role in Vascular Remodeling of Inflamed Tissue Carolyn Mulvey


Founding Staff






The Collaborative Engineering of the Atacama Large Millimeter Array

-Ian Czekala

Abstract The Atacama Large Millimeter Array (ALMA), a radio telescope array of mammoth proportions currently under construction in Northern Chile, is a modern scientific, technical, and political marvel. Through travel to the ALMA project in Chile and interviews with the project scientists and engineers, the author has attempted to holistically view the project while identifying many of the methods and design processes that underlie the successful completion of the array. ALMA represents an entirely new design and integration environment that is truly a product of the 20th century’s government-funded “Big Science”, as well as a touchstone for future large scale international scientific endeavors.


Ian Czekala

“The scientists and engineers working on the ground in the construction of ALMA have developed a unique project culture that fosters technical communication and minimizes the potential impact that bureaucratic or political inefficiencies might have on the pace of construction of the telescope.”

Ian Czekala is a fourth-year aerospace engineering and astronomy double major. He hails from Miller Place, N.Y., where he grew up and attended Rocky Point High School. At the University of Virginia, he is a Jefferson and Rodman Scholar, and greatly enjoyed serving as Rodman Scholars co-president from 2008-2009. In addition to his study of the ALMA telescope, Czekala has engaged in a couple other memorable research projects, such as an NSF-funded REU research internship at Harvard University in the summer of 2009 and his astronomy thesis with faculty of the NRAO. In Fall 2010, he will begin his Ph.D. in astrophysics at Harvard University. He is excited about the move to Cambridge, and the appeal of living at the footsteps of such a city as Boston, but he is also reluctant to leave the haven of Charlottesville after what seems like a very brief four years.

Figure 1: Artist’s renderings of the Atacama Large Millimeter Array (ALMA) on the Zona de Chajnantor, Chile, at 16,500 feet of altitude. When finished, the array will consist of 66 radio antennae working in concert to observe cosmic sources of sub-millimeter emission. Image Credit: (ESO, 2009).

The Atacama Large Millimeter Array (ALMA), a radio telescope array of mammoth proportions currently under construction in northern Chile, is a modern scientific, technical, and political marvel. Through travel to the ALMA project (Fig. 1) in Chile and interviews with the project scientists and engineers, the author has attempted to survey the methods and design processes that underlie the successful completion of the array. The author has explored U.S. and Canadian participation by working through ALMA’s North American hub, the North American ALMA Science Center (NAASC), which is located in Charlottesville, Virginia. In December 2009, the author traveled to the ALMA headquarters in Santiago, Chile, and the ALMA build site in the northern Chilean desert at 16,500 feet of altitude. At both the National Radio Astronomy Observatory (NRAO) headquarters in Charlottesville and at the ALMA project in Chile, the author conducted 18 formal interviews with international project scientists and engineers, and spoke informally with several more.

Astronomy is a rapidly evolving field in which new technology drives new discoveries. For modern astronomers, the romantic scene of an astronomer squinting through the eyepiece of a telescope, backdropped by a chill starry sky and working until early morning, is a thing of the past. Galileo’s optical telescope of four centuries ago has been surpassed by electronic detectors and powerful computers. The ALMA project is enormous; it supersedes all previous ground-based telescopes in cost and scientific capability (Wootten, 2006) and its construction requires a substantial investment from many countries (Barish, 2008). The project is a collection of 66 radio antennae that will act in concert as a single large telescope. The telescope combines the resolution and clarity of a large interferometric array with the brightness-sensitivity of a single-dish telescope (Brown, Wild, & Cunningham, 2003). ALMA will meet the scientific requirements of astronomers to observe the earliest and most distant galaxies, search dusty star-forming regions for inchoate stars and planets, and image remnants of the formation of our 3

own solar system that orbit in the icy reaches beyond Neptune (Brown et al., 2003). Currently, the observatory is on-target to begin full science operations in 2013. A confluence of astronomical ambition, technical prowess, and political savvy has informed the decision to construct ALMA in Chile. Since 1998, the United States, the European Southern Observatory (ESO) (consisting of 14 European nations), Japan, Canada, Taiwan, Chile, and other parties have been involved in ALMA’s development (Wootten, 2003). ALMA represents an entirely new design and integration environment that is truly a product of the late 20th century’s government-funded “big science.” Within this environment, the scientists and engineers have developed a unique project culture that has proven successful in keeping the construction moving inexorably forward. Literature Review Western physical sciences, particularly physics and astronomy, underwent a fundamental paradigm-shift in the 20th century. Before World War II, science was typically carried out in a university environment with the aid of private funding. For example, the California Institute of Technology constructed and maintained a several-decade monopoly over the world’s largest telescope, the Palomar 200-inch (Florence, 1994), which only astronomers closely affiliated with the university could use. However, military spending associated with World War II helped cultivate many fields of science through government funding, especially high-energy particle physics, which had applications for nuclear technology (McCray, 2004). The wide-spread increase of government scientific funding also included funding for astronomical science. Observatories such as the Gemini telescopes and the Kitt Peak National Observatory represent large government-funded endeavors that embodied “big science,” compared to the “small science” carried out at universities prior to the war (McCray, 2004). Scientific endeavors were now so complex that they were best carried out in groups. Once a device used intimately by an astronomer, the modern telescope could now be considered a “discovery factory.” In the 4

March 2010 newsletter of the American Astronomical Society, president John Huchra stressed that with the advent of large-scale, “big science” astronomical endeavors that closely mimic the field of high-energy physics, “[A]s a field we are now aspiring to systems of facilities and programs that exceed any individual nation’s ability to support. Cooperation is essential for the science”(Huchra, 2010). In response to criticism of the cost of other large-scale telescope collaboration concepts, such as the California Extremely Large Telescope and Giant Magellan Telescope, Richard Ellis of the California Institute of Technology has admitted that, “We are really going to have a hard time building even one of these [large telescope projects].” To pay for such massive telescopes, the merging of private and public sources of financing is often required. ALMA’s taxpayer-based funding from many different countries is a product of the late 20th century --in the past, most ground-based telescopes have been financed by private observatories and universities (Overbye, 2003). ALMA is a collaboration between the astronomical communities of NRAO, ESO, the National Astronomy Observatory of Japan (NAOJ), and the Academica Sinica of Taiwan. With Japan’s entry to the project as an equal third partner and other late additions to the collaboration, ALMA has become one of the first truly global projects in the history of fundamental science (Europe, 2001). Parts for the telescope are designed and tested in laboratories around the world, including Charlottesville, Virginia (Wilson, 2007). The ALMA telescope array draws its design from previous telescope concepts originating in the United States, Europe, and Japan (Vanden Bout, 2005). Throughout the 1980s and 1990s, the teams developing the American Millimeter Array (MMA), the European Large Southern Array (LSA), and the Japanese Large Millimeter Array (LMA) converged upon the Zona de Chajnantor in Chile as an excellent site to build their respective telescopes. The Chajnantor site may hold unique astronomical merit: its flatness at high altitude, aridity, and stable air make it optimal for observing in the millimeter wavelengths (Otarola et al. 2005). Not surprisingly, scientists and their sponsoring governments were

reluctant to build substantially redundant instruments so close to one another (Vanden Bout, 2005), and thus the collaboration began (ALMA Memorandum, 1999). Each community has established procedures. The collaboration between the European Southern Observatory (ESO) and the North American Radio Observatory (NRAO) began in 1997 with the signing of an agreement to merge the MMA and the LSA. On February 25th, 2003, Japan agreed to merge its LMA with ALMA in the form of the Atacama Compact Array (ACA). The burden of the funding is split roughly three ways between these partner countries. Once completed, the array will be run by the ALMA Observatory, which will be established as a legal en-

factors at the construction site of a telescope array. There are a number of associated consequences. There is no “ALMA” “Our biggest problem is that there is no ALMA,” said Paulo Cortes, an ALMA Test Scientist (Cortez, 2009). ALMA comprises three main partner organizations: the NRAO, ESO, and NAOJ, and each maintains control of roughly a third of the project. Essentially, all of the partner countries have a say in where the funds go while the over arching organization, the Joint ALMA Observatory (JAO), has little spending authority. This funding structure has manifested itself in the procurement of the 66 antennae for the array. Within this complicated departmental structure, project managers have made sure that their subordinates exchange information with the other departments of the project through daily teleconferences and shift overlaps (Peck, 2009).

Antenna Mitosis The ALMA antennae (Figs. 2 and 3) are possibly the best sub-millimeter antennae ever constructed. They will gather vast data to an incredible precision (Mangum et al., 2006). Construction of the 66 planned antennae is rather circuitous and subject to political influence by each of the partner countries. The antenna acquisition process best illustrates some of the unique political challenges an international scientific endeavor faces. To meet the scientific demand for high-quality data, each antenna must be capable of surviving strong winds and temperatures between -20 and +20 degrees Celsius, while aiming Figure 2: MELCO antennae at the Operational Support Facility precisely enough that it could target a golf ball 15 km (OSF), made by the Japanese electronics company Mitsubishi. away. The surface of the dish must be accurate to betPhoto by the author. ter than 25 micrometers --the thickness of a human hair. Since these specifications represented a major tity in Chile, funded by the partners of the project advance in technology at the time of the conception (Kurz & Shaver, 1999). of the ALMA project, the ALMA consortium solic Within the history of the astronomical com- ited prototype antennae bids from many different munity, ALMA represents the first telescope of its manufacturers. The most prominent bids were from kind both by technical specifications, but more im- Vertex and AEC, representing the National Radio portantly, by the international agreements that were Astronomy Observatory (NRAO) and the European necessary to build it. ALMA is a multinational scien- Southern Observatory (ESO), respectively (Layton, tific endeavor comprising varying cultures that sig- 2006). nals the arrival of political, scientific, and technical Under pressure from each partner country to 5

attract funds to national manufacturers, what began as a competition for the single best antenna ended in the acceptance of three antenna designs into the array; the Vertex and AEC designs were initially accepted and the Japanese MELCO antenna was accepted later. Richard Murowinski, ALMA Project Engineer, describes the difficulty of synchronizing antennae within the array: “This can only be achieved with the perfect synchronization of the antennae and the electronic equipment: a precision much better than one millionth of a millionth of a second between equipment located many kilometres apart” (ALMA Newsletter V. 3, 2009). Quality testing showed that all the antennae had passed the required specifications and were accepted by the project (Mangum et al., 2006); however, the production speed of manufacturers has varied. Once an antenna is fully tested at the OSF, it is transferred to its final location at the Array Operations Site, at 16,500 feet of altitude. Adrian Russell, North American ALMA Project Manager, remarked on the activity at the build-site, “A Japanese antenna with North American electronics was carried by a European transporter! Now the real work begins and the exciting part is just beginning” (Russell, 2008). Russell’s words illustrate the close collaboration in the ALMA project while hinting at the complicated chain of supply that dictates the pace of progress.

that are available to all involved. The need to document so much of the work can be both a blessing (Marti-Canales, 2009) and a curse (Webber, 2009). Javier Marti-Canales, the lead systems engineer for ALMA, contends that in such a large, international project (which involved many for whom English is a second or third language) rigorous documentation is essential to having all of the pieces fit together once they are brought to Chile to be assembled. By making sure the design road-map is sound before beginning construction, the project will avoid many unnecessary delays due to incorrectly specified parts at the time of integration (Marti-Canales, 2009). Alternatively, John Webber, the correlator subsystem manager, contends that the extensive amount of documentation necessary for ALMA acts to slow the design and construction process of ALMA because so much of his and his engineers’ time is spent writing about what they have just done, rather than beginning the next step of their work. He favors a design setting in which all of the engineers are in the same building, and when minor design changes need to be made, they are done face-to-face rather through writing (Webber, 2009). Joseph Schwartz, deputy computing information technologist for ALMA, spoke about the ease with which the software community for ALMA was able to communicate across continents and timeDocumentation Overhead zones, even though much of the rest of the project After an engineering project surpasses a criti- has suffered from the difference in time zones (Webcal level of subscription, efforts to keep members of ber, 2009). ALMA engineers and scientists have the project correctly informed about project develop- adapted bug-tracking software from traditionally ments evolves from a convenience to a nuisance. As a open-source software-based projects, such as Bugzilproject like ALMA scales up from a national project la and JIRATicket, to track and resolve bugs in both to an international project, the range of talent that it the software and hardware of ALMA. These tracking can attract also broadens. Thus, within the small ra- mechanisms help reduce the redundancies of workdio astronomy community, an influential project like ers’ efforts, and try to eliminate people solving the ALMA has the potential to literally attract the best same problem twice. astronomers in the world (Cortez, 2009). However, the increase in talent can be quick- The Soul of a New Machine ly offset by many of the difficulties that arise when Like any scientific endeavor worth pursuing, working internationally. When pieces of a telescope the ALMA project faces the struggle of doing things are manufactured all over the world (Wilson, 2007), for the first time. Grueling, 14-hour workdays are not there must also be detailed records of the design deci- uncommon for scientists and engineers, even though sions that led to the current technical specifications the official workday is a more standard length. As6

tronomers, who, in the author’s experience, often work long hours to complete a project, appear to work as if their jobs depend on it for the ALMA project. Their desire for high performance under pressure is reminiscent of Tracy Kidder’s report on the construction of a new computer design in a high-pressure environment, The Soul of a New Machine (Kidder, 1981). The most prevalent reason why so many astronomers (as opposed to just engineers) are working on the construction of ALMA, is that they want to gain a thorough understanding of the instrument and will be better able to write quality observational proposals once the telescope officially begins operation (Corder, 2009; Cortez, 2009; Sheth, 2009). However, it is the collective opinion of the scientists working on the project (Barkats, 2009; McMullin, 2009; Sheth, 2009) that while one is not working on ALMA, one is not publishing papers. In a publish-or-perish world, this drought in scientific publications may significantly impede a scientist’s academic career.

is not a formal board member. However, Chilean nationals play a significant role in the construction of ALMA in various capacities. Chile has excellent electrical and engineering education, mostly due to the large copper mines up in its northern deserts (Peck, 2009). This has made it easier to recruit technical talent to construct the array, but talented radio astronomers are not yet in abundance in Chile. Therefore, these specialists need to be imported (Simon, 2009). The focus of the ALMA team is to complete the array on time so that science observations may begin; whether the talent needed to do this comes domestically or internationally is of secondary concern to the project. In order to improve the local communities surrounding the ALMA project, the partners plan to contribute $700,000 annually to Chile for the duration of the project (ALMA partners, 2003) to finance local projects and develop science at the national level.

Conclusion The astronomical community, although relaChilean Involvement tively small compared to other scientific fields such as Although Chile, the host country for ALMA, physics, has had one of the longest histories of intercontributes land for the construction of the telescope national collaboration (Huchra, 2010). The ALMA (Kurz & Shaver, 1999) and receives 10% of the total telescope represents the merger of several design conobserving time, it does not financially contribute on cepts of the United States, Europe, and Japan into the level of the United States, Europe, or Japan and the world’s premier sub-millimeter astronomical facility and one of the largest astronomical collaborations to date. ALMA scientists and engineers working to construct the array have developed a unique project culture that has driven the progress of the array forward despite challenges such as three different types of antennae and a large amount of necessary documentation. The power of “big science” will create intriguing new scientific discoveries as well as myriad ramifications associated with a massive international project. The methods used range from the everyday, such as team manager Richard Prestage’s efforts to speak clearly and ask questions for understanding of his team (many for whom English is a second language) (Prestage, 2009), to the technical, such as the JIRATicket bug system used to track and resolve Figure 3: MELCO antennae at the Operational Support Facility software bugs in the performance of the telescope (OSF), made by the Japanese electronics company Mitsubishi. Photo by the author. during commissioning. ALMA represents the most 7

significant “big science” astronomical project to date. The success of the telescope and the experiences of the scientists and engineers working on the project will serve as an important touchstone for the future of large international scientific collaborations. References ALMA Memorandum of Understanding. (1999). ALMA Newsletter V. 3. (2009). ALMA Observatory.

European Southern Observatory. Retrieved March 30, 2010, from jpg Europe, Japan and North America prepare for joint construction of “ALMA” in Chile. (2001). Retrieved from en/newsroom/press-releases/119-europe-japanand-north-america-prepare-for-joint-construction-of-qalmaq-in-chile

ALMA partners granted site to build and oper- Florence, R. (1994). The Perfect Machine: Building ate telescope in Chile. (2003). Retrieved from the Palomar Telescope. New York, NY: Harp erCollins. press-releases/132-alma-partners-granted-siteto-build-and-operate-telescope-in-chile Mangum, J.G., Baars, J.W.M., Greve, A., Lucas, R., Snel, R.C., Wallace, P., Holdaway, M. (2006). Barish, B. (2008). ILC NewsLine - 8 January 2009 Evaluation of the ALMA Prototype Antennas. - Feature 1. Retrieved from http://www.linAstronomical Society of the Pacific. (118:847) (1257-1301). ftr1.html Huchra, J. (2010). President’s Column. AAS NewsletBarkats, D. (2009). ALMA Interview. ter, (151), 2. Bout, P. A. V. (2005). Origins of the ALMA Project in the Scientific Visions of the North American, European, and Japanese Astronomical Communities. European Space Agency, 23(577), 23–27.

Kidder, T. (1981). The Soul of a New Machine (1st ed.). United States: Little, Brown and Company. Kurz, R., & Shaver, P. (1999). The ALMA Project. European Southern Observatory, 7K(96).

Brown, R. L., Wild, W., & Cunningham, C. (2003). ALMA – the Atacama large millimeter array. Elsevier, 555–559.

Layton, L. (2006). ESO to produce ALMA antennae. Astronomy.

Corder, S. (2009). ALMA Interview.

Marti-Canales, J. (2009). ALMA Interview.

Cortez, P. (2009). ALMA Interview.

McCray, W. P. (2004). Giant Telescopes: Astronomical Ambition and the Promise of Technology. Cambridge, Massachusetts: Harvard University Press.

ESO. (2009, December). alma-chajnantor-scene1.jpg (JPEG Image, 1280x720 pixels) - Scaled (56%).

McMullin, J. (2009). ALMA Interview. Russell, A. (2008). The Future Evolution of ALMA. Charlottesville, VA. 8

Schwartz, J. (2009). ALMA Interview. Sheth, K. (2009). ALMA Interview. Simon, R. (2009). ALMA Interview. Webber, J. (2009). ALMA Interview. Wilson, T. L. (2007). The Atacama Large MillimeterArray (pp. 591–593). Cardiff.

Acknowledgments The author graciously acknowledges support from the David A. Harrison III Undergraduate Research Grant and the NRAO student travel fund for the ALMA project. The author would especially like to thank Professor Kelsey Johnson of the University of Virginia and Dr. Alison Peck of the ALMA project for their incredible support throughout the duration of the ALMA research project.

Wootten, A. (2003). Atacama Large Millimeter Array (ALMA). (In Society of Photo-Optical Instrumentation Engineers (SPIE) Conference) (It 4837, 110–118). Wootten, A. (2006). Revealing the Molecular Universe: One Antenna is Never Enough. In ALMA: Exploring the Outer Limits of the Millimeter Sky. Presented at the Astronomical Society of the Pacific Conference Series. Otarola, A., Holdaway, M., Nyman, L., & Radford, S. (2005, January). Atmospheric Transparency at Chajnantor: 1973-2003. NRAO ALMA Memo. Overbye, D. (2003). Astronomy’s New Grail: The $1 Billion Telescope. The New York Times. Peck, A. (2009). ALMA Interview. Prestage, R. (2009). ALMA Interview.


“Inclusion Membrane Protein A (IncA) of Chlamydia trachomatis is an essential protein that helps govern intracellular interactions of the bacterium with the host cell, and it is being studied in this report through biophysical characterization with precluding expression and purification protocols.” -Ashley Keller

Abstract Inclusion Membrane Protein A (IncA) of Chalmydia trachomatis was expressed and purified using a dual-detergent system, and analysis of purified protein using circular dichroism (CD) spectroscopy indicates an alpha-helical super-secondary structure, supporting known helical motifs found in primary structure bioinformatic analysis. The incA gene was inserted into the PET28b vector and cloned using Polymerase Incomplete Primer Extension (PIPE) PCR and was expressed in BL21(DE3)RIL E. coli cell strain. IncA was found to express in monomeric and dimeric forms. Four detergents (DM, FC-10, FC-12, and DDM) were screened for optimal extraction and purification of IncA sample. The FC-10 extraction and FC-12 purification dual-detergent system returned the purest IncA. This dual-detergent system yielded a maximum IncA concentration of 206 µM. CD analysis of the purified IncA revealed all-around alpha-helical super-secondary structure.


Ashley Elizabeth Keller

Expression and Purification of Inclusion Membrane Protein A (IncA) from Chlamydia trachomatis using Dual-Detergent System and Secondary Structural Determination using Circular Dichroism Spectroscopy

Ashley Keller is a third-year biochemistry major and biology minor. She currently works in Professor Linda Columbus’ lab on membrane proteins of pathogenic bacteria. Specifically, she investigates the biophysical properties of the Inclusion Membrane Protein A in Chlamydia trachomatis. She has worked this past year on expression and purification protocols for IncA, and her future aims are to investigate the protein’s topology in a detergent environment through electron paramagnetic resonance and nuclear magnetic resonance studies. She recently received a Harrison Undergraduate Research Award for her work on IncA, and she wishes to use this grant to advance her research goals this summer. Ashley is a member of Chi Omega sorority, and she enjoys volunteering her time with Madison House Adopt-A-Grandparent program. After graduation, she wishes to attend graduate school and study immunology. She would like to one day have a lab of her own and teach future scientists.

Chlamydia trachomatis are obligate bacteria that require the intracellular environment provided by a host cell in order to effectively reproduce and survive. However, Chlamydia are unique bacteria in that their residency within the host cell is so secretive as to be undetectable by the host cell exocytic machinery. Outer membrane proteins of individual Chlamydia modulate initial entry into the host cell through interaction with host cell outer membranes, resulting in the phagocytosis of the bacterium within an endocytic membrane of host cell composition.1 Once the bacterium is inside the host cell, it becomes reproductively-capable in a form known as the reticulate body. This reticulate body is metabolically active and begins formation of the inclusion membrane, a unique endocytic vesicle modified and maintained by intracellular Chlamydial communities.2 The inclusion modification involves fusion with other Chlamydial endocytic vesicles causing a concentration of reticulate bodies within a single large vesicle. The inclusion is an evolutionary trademark of the extensive success of Chlamydial survival, hallmarking an approximate

2,000 million-year existence within the host cell.1 The development and modification of the inclusion membrane is mediated in part by the Inclusion Membrane Protein A (IncA) which is embedded within this inclusion membrane. IncA is stipulated to play a vital role in promoting homotypic fusion of

Figure 1. Heptad Repeat Sequences and Transmembrane Domains in IncA Primary Structure: Primary structure of IncA is shown with heptad repeat sequences highlighted in bold and transmembrane domains boxed in gray and black. The heptad repeat sequence follows the pattern of seven amino acids (a-b-c-d-e-fg) with the first and fourth amino acids being a leucine (L) or nonpolar amino acid. This pattern is seen throughout the IncA sequence with slight variations as to the position of the fourth nonpolar residue. The italicized residues represent the first and fourth amino acid residues of characteristic nonpolarity. IncA has conserved heptad repeats toward the C-terminal end, and these repeats are seen in SNARE primary structure as well. The heptad repeats code for the coiled-coil alpha-helical secondary structure.


endocytic vesicles of individually-housed Chlamydia into one large inclusion body vesicle.3,4 Although the IncA structure has not formally been determined, bioinformatic analysis of its amino acid sequence has revealed characteristic leucine zipper peptide sequences that encode coiled-coil domains (Figure 1).5,7 These domains are thought to facilitate IncA-IncA interaction between vesicular membranes through dimeric fusion of coiled-coil domains. IncA is known to homo-dimerize in detergent solution (Keller, A. 2009), and IncA-IncA interactions have been observed.6 Thus, IncA is a major player in fusion of vesicular membranes, promoting inclusion membrane composition diversification through host-cell component and Chlamydial component membranous fusion. The fusion of host cell and Chlamydial membranes diversifies the inclusion membrane through incorporation of host cell phosphoro/glycerolipids that can aid in the inclusion stealth and non-detection within host environment. The inclusion membrane “mimics” host cell intracellular membrane vesicles, bypassing detection by monitoring endosomal proteins. The maintenance of the inclusion “niche” is crucial to the intracellular survival of Chlamydia and requires extensive interaction with host cell cytoplasmic proteins. It is proposed that IncA plays a significant role in this interaction, preventing the inclusion from being “digested” by the host cell endocytic and lysosomal pathways.2 Eukaryotic SNARE proteins (soluble N-ethylmaleimide-sensitive-factor attachment protein receptors) are known to facilitate fusion of membranous vesicles in the cytoplasm of

Figure 2. 11 Vamp 4 SNARE Motif: This is a fragment of the Vamp 4 SNARE protein found in eukaryotic cells. Alpha-helical chains are in gray, and leucine residues representing the location of heptad repeats are highlighted in dark gray. These heptad repeat sequences are located primarily at the interfacing domains of the chains where coiled-coil interactions keep the protein fragment together and facilitate membrane-fusion interactions. IncA and SNARE proteins could have possible evolutionary linkages through conservation of heptad repeat sequence encoding characteristic coiled-coils. (Visualized using Educational PyMol)


eukaryotic cells, and it is postulated that IncA interacts with these SNARE proteins through similar, conserved coiled-coil domain entailment (Figure 2).5 This interaction could signal the inclusion as a necessary endosomal vesicle, cleverly bypassing host cell defenses. The inclusion mechanism of Chlamydia allows the bacteria to proliferate inside the host cell without any host cellular triggering or awareness of the Chlamydial presence.8 This stealth also makes the bacterium a somewhat tepid invader, where its habitation of the host cell does not often result in that cell’s destruction. Nevertheless, Chlamydia infection is the number one cause of sexually-transmitted disease, and the trachoma condition resulting from Chlamydia trachomatis infection is still a serious prenatal transmissive disease worldwide.9 Understanding of IncA interaction with itself and possible cyoplasmic proteins is a necessary step in understanding overall mechanisms of chlamydial stealth invasion and possible future novel treatments. This paper focuses on the expression and purification strategies necessary to successfully isolate IncA protein from E. coli BL21(DE3)RIL cell strain. A novel dual-detergent strategy is developed for the extraction and subsequent elution of IncA from a cobalt IMAC (Immobilized Metal Affinity Chromatography) column. In addition, the elucidation of the secondary alpha helical structure is determined using circular dichroism (CD) spectrum. Successful purification of IncA and determination of alpha-helical structure through CD analysis are critical steps towards characterizing the IncA protein biophysically. The next step after succinct purification of IncA is determination of its structure through NMR (Nuclear Magnetic Resonance), and the determination of IncA structure will help elucidate its function within the inclusion body, further bringing to light the mechanism by which Chlamydiae avoids host cell detection. This understanding of IncA function within the inclusion body can aid in drug targeting research and overall targeting methods for other obligate bacteria that exclusively bypass host cell defenses.

been efficient with other protein extractions. The following procedure was performed with each of these detergents, with only detergent concentration modulated. Cell pellets were resuspended in 30 mL lysis buffer (150 mM NaCl, 150 mM tris base pH 7.6). The cell suspension was lysed using a microfluidizer at 60 psi. The cell lysate was centrifuged at 11,000 rpm (low-speed run) for 40 minutes at 15ºC. The supernatant was centrifuged again at 30,000 rpm (highspeed run) for 1.5 hours at 15ºC. Membrane pellets from high-speed spin were resuspended overnight (15 Transformation and Clone Purification. The pET28b- hours) in 10 mL Extraction Buffer (25 mM FC-10 (10 incA construct was transformed into E. coli mM DM, 10 mM FC-12, 2 mM DDM), 150 mM NaCl, BL21(DE3) RIL cell line using a standard heat shock 150 mM tris base pH 7.6). The extraction suspension method and kanamycin for selection. Plasmid con- was centrifuged at 11,000 rpm at 15ºC for 30 minutes. structs were purified from an individual-colony using Qiagen Miniprep kit. Purified plasmid constructs IncA Purification using Imobilized Metal Affinity were run on agarose gel (1%) and constructs of cor- Chromatography (IMAC). The pET28b vector prorect base-pair length (6230 bp) were extracted and vided encodes an N-terminal hexa-histidine fusion purified from the gel using Qiagen Gel Purification followed by a thrombin cleavage site. Therefore, IncA Kit. Sequencing with forward and reverse primers was expressed with thrombin-cleavable His-tag, allowing purification using Co2+ IMAC. The column (Genewiz) confirmed the proper incA sequences. was prepared by equilibrating Co2+ chelated resin (3 IncA Expression. A single colony from transforma- mL, sepharose beads) with 60 mL of extraction buftion plate was used to inoculate 5 mL of LB (Luria- fer. The extraction supernatant was loaded onto the Burtani)/Kanamycin cultures. The tubes were incu- equilibrated resin, and the flow-through was collectbated at 37ºC and shaken at 250 rpm for 15 hours ed in two fractions (35 mL/fraction). The column was (overnight). Two 1 L beveled flasks with 500 mL of then washed with 50 mL of wash buffer (4 mM FC-12 TB (Terrific Broth) media were inoculated with the (4 mM DM, 13 mM FC-10, 0.5 mM DDM), 150 mM overnight culture (one per flask). The flask cultures NaCl, 20 mM imidazole, 150 mM tris base pH 7.6), were incubated at 37ºC and shaken at 250 rpm for and the wash flow-through was collected in three 3 hours to an optical density at 600 nm of 0.8. Ex- fractions (17 mL/fraction). IncA was then eluted with pression of IncA was induced with IPTG (500 µL, 50 mL of elution buffer (4 mM FC-12 (4 mM DM, 15 1M), and incubated overnight at 25ºC, 250 rpm for mM FC-10, 0.5 mM DDM), 150 mM NaCl, 500 mM 15 hours. Cells were harvested by centrifugation at imidazole, 150 mM Tris Base pH 7.6), and three elu6,000 rpm for 25 minutes. Cell pellets were stored at tion fractions (17 mL/fraction) were collected. These -80ºC until use. Expression was observed by compar- fraction samples were visualized using SDS-PAGE ing before and after induction samples of cell culture and Coomassie staining, and protein purity was determined by qualitatively comparing impurity peron SDS-PAGE gel. centage in each gel lane sample. Protein concentraOptimization of IncA Extraction from Cell Membrane. tion was determined using the absorbance at 280 nm Initially, four detergents were screened to determine and an extinction coefficient of 9065 M-1cm-1. optimal extraction of IncA. FC-10 (n-decylphosphocholine), FC-12 (n-dodecylphosphocholine), DM Circular Dichroism (CD) Spectroscopy. The FC-12 elu(n-decyl-beta-D-maltoside), and DDM (n-dodecyl- tion containing IncA was concentrated and dialyzed beta-D-maltoside) were chosen because they have to remove immidazole (4 mM FC-12, 75 mM NaCl, 75 Methods and Materials Clones and Plasmids. The pET28b vector (Novagen) was used as the expression system vector with T7 promoter and inducible constructs by IPTG (Isopropyl beta-D-1-thiogalactopyranoside). The incA clone was obtained from the Protein Functional Genomic Resource Center and was sub-cloned into pET28b at EcoRI/HindIII using PIPE (Polymerase Incomplete Primer Extension Method) PCR (Polymerase Chain Reaction).15


Tris Base pH 7.6). A CD spectrum of the dialyzed sample was recorded with an AVIV 410 CD spectrometer between 180-260 nm. The concentration of IncA was 81 µM (0.002 g/mL).

the wash fraction (1st fraction). The concentration of IncA in elution 1 was 160 µM. IncA extraction and purification using FC-12 buffers resulted in improved purity even further compared to the FC-10 elution fractions (Figure 5). Results The E.1 fraction had a greater intensity band Four Detergent Screen/Dual-Detergent Screen for IncA than the E.1 fraction of FC-10 sample, indicating a Extraction and Purification more robust purification. This comparison was made Four detergents (DM, FC-10, FC-12, and relative to IncA presence in the elution fractions while DDM) were screened for optimum extraction and pu- purification conditions were kept constant and only rification of IncA. The four detergents all resulted in detergent conditions modified. However, impurities IncA extraction and purification in the elution frac- were present in the E.1 fraction, clearly seen in betion but with variable yields and purity. tween dimer and monomer bands. Both flow-through IncA extracted and purified with DM resulted fractions (FT.1, FT.2) retained considerable amount in IncA monomeric and dimeric forms in the elution of protein (>50%), and the wash fraction also had fraction that was approximately 70% pure, a quali- protein but in a smaller concentration. The IncA contative estimate based on impurity and elution band centration from the FC-12 was 184 µM. relative densities. Figure 3 shows the SDS-PAGE of IncA was extracted and purified in DDM bufeach fraction of the purification in DM. IncA was observed predominately in the E.2 fraction with impurity bands present. IncA was seen less intensely in the E.3 fraction with impurities present, but these impurities were present in less significant amounts than in the E.2 fraction. Considerable amount of IncA was lost in the W.1 fraction (50% of total sample, compared to ET). A common theme seen in all four detergent screens was that a considerable amount of expressed IncA was lost in the flow-through fraction during the column run, and a considerable amount was subseFigure 3. Extraction and Purification of IncA using DM Buffers: quently lost in the wash fractions as well. However, Well 1- Protein Marker; Well 2-Low-Speed Pellet (LS P); Well 3DM screen did result in expressed and purified IncA High-Speed Supernatant (HS SN); Well 4- Extraction Total (ET); Well 5- Post-Extraction SN (PE SN); Well 6- Post-Extraction Pelwith moderate impurities (30% qualitatively delet (PEP); Well 7- Flow-Through (FT); Well 8- Wash 1 (W.1/1st termined). IncA was observed as both a dimer and fraction); Well 9- Wash 2 (W.2/2nd fraction); Well 10- Wash 3 monomer. Using DM as buffer detergent, IncA was (W.3/3rd fraction); Well 11- Elution 1 (E.1/1st fraction); Well 12Elution 2 (E.2/2nd fraction); Well 13- Elution 3 (E.3/3rd fraction). purified in total concentration of 146 µM. The top box represents the IncA dimer (60 kDa), and the bottom IncA extraction and purification using FC-10 box represents the IncA monomer (30 kDa). detergent buffers resulted in a cleaner IncA sample both in terms of impurities and relative protein reten- fers, but monomer and dimer extraction was not as tion as compared to extraction total protein amount efficient as with the other three detergents (Figure 6). (Figure 4). This was an unusual finding considering other mem IncA FC-10 extraction and purification result- brane proteins, like the Wzz of E. coli, have been efed in high purity as seen in all three elution fractions fectively extracted and purified using DDM.16 with subsequent reduction in impurity level from E.1 IncA was observed in the E.1 fraction in both to E.3 fractions. However, considerable protein was monomeric and dimeric forms, but the band intenstill lost in the flow-through fractions as well as in sity was very faint as compared to the other three 14

detergent screen yields. Impurities were present in the E.1 fraction as well, but due to the faintness of the protein bands, these impurity bands could not be relatively discerned. IncA was eluted in the flowthrough (1 and 2) and wash 1 fractions, as seen in the other three screens, so considerable protein was lost upon running the column. The concentration of IncA was 75 ÂľM. Due to the variable results in extraction and purification with each detergent, an optimized protocol using a dual-detergent system of FC-10/FC-12 buffers was explored. IncA was extracted in FC-10 buffer and purified using FC-12 wash and elution buffers.This optimized protocol resulted in high yields of highly pure IncA (Figure 7) as compared to

was also expressed and purified, but its extraction was not as great as the dimeric form. However, both dimer and monomer forms were expressed and seen in all three elution fractions with limited impurities as compared to the other four detergent screens, indicating that the dual-detergent system is optimal for IncA extraction and purification. Significant protein loss was observed in the flow through and wash fractions indicating limited column binding. A high-

Figure 6. IncA Extraction and Purification using DDM Buffers: 1st Well: Protein Marker, 2nd Well: FT 1, 3rd Well: FT 2, 4th Well: W.1, 5th Well: W.2, (Wash 3 fraction was not taken), 6th Well: E.1, 7th Well: E.3; Top box represents IncA dimer, and bottom box represents IncA monomer.

Figure 4. IncA Extraction and Purification using FC-10 Buffers: Well 1- Protein Marker; Well 2- Low-Speed Pellet (LS P); Well 3Extraction Total (ET); Well 4- Post-Extraction SN (PE SN); Well 5- Post-Extraction Pellet (PEP); Well 6- FT.1 (1st fraction/40 mL); Well 7- FT.2 (2nd fraction/40 mL); Well 8- Wash 1 (W.1); Well 9- Wash 2 (W.2); Well 10- Wash 3 (W.3); Well 11- Elution 1 (E.1); Well 12- Elution 2 (E.2); Well 13- Elution 3 (E.3); Well 14Protein Marker; Again, the top box represents expressed dimer, and the bottom box represents expressed monomer.

Figure 5. IncA Extraction and Purification using FC-12 Buffers: 1st Well-Marker, 2nd Well-FT.1, 3rd Well-FT.2, 4th Well- W.1, 5th Well- W.2, (Wash 3 fraction was not taken), 6th Well- E.1, 7th Well-E.2, 8th Well-E.3; Top box represents IncA dimer, and bottom box represents IncA monomer.

the single detergent results. IncA was purified in robust amount in dimeric form in E.1 fraction. The monomeric form

molecular weight impurity was distinctly seen in the E.1 fraction. Impurities were reduced compared to other detergent screens. The concentration of IncA using FC-10/FC-12 detergent system was 206 ÂľM. Circular Dichroism Spectroscopy of IncA Secondary structure of IncA was elucidated

Figure 7. IncA Extraction and Purification using FC-10/FC-12 Dual Detergent System: Well: Protein Marker, 2nd Well: E.1 FT Concentrated (XC) 3rd Well: E.1 FT, 4th Well: FT 1, 5th Well: FT 2, 6th Well: W.1, 7th Well: W.2, 8th Well: W.3, 9th Well: E.1, 10th Well: E.2, 11th Well: E.3; Top box represents IncA dimer, and bottom box represents IncA monomer.

using circular dichroism. IncA sample in FC-12 elution buffer was used and observed across 180-260 nm. Peak absorption readings were primarily observed at 208 nm and 220 nm, corresponding to the alpha-helical secondary structure12 (Figure 8). Al15

though the coiled-coil motif does not have a known characteristic CD spectrum, the IncA spectrum closely resembled the CD spectrum taken of GCN4 protein, which is known to have coiled-coil motifs.13

that these cellular components, along with detergent micelle conditions, make the environment optimal for IncA extraction and stabilization within a detergent micelle. IncA has differential empirical interactions with FC-10 under extraction conditions and FC-12 Discussion under elution conditions. Extraction and purification protocols for IncA A significant proportion of total IncA protein were optimized through a four-detergent screening. was lost in the flow-through and wash fractions upon The dual-detergent system (in which IncA was ex- cobalt column run. This indicates that the histidine tracted in FC-10 buffer and purified in FC-12 wash tag of IncA is not properly chelating to the cobalt and elution buffers) qualitatively produced the pur- resins, causing facile elution in flow-through and est sample of IncA and quantitatively yielded the wash fractions. The IncA structure is likely dimeric highest concentration of IncA sample (206 ¾M). The (and stable in SDS –typically a denaturing detergent) FC-10/FC-12 system empirically yielded the best as seen in the SDS-PAGE gels, and perhaps the diIncA sample of the detergent systems screened, and merization of IncA subsequently buries the histidine this empirical finding could evidence important bio- tag within the dimer interface, preventing proper physical interactions between IncA structure and de- chelation and binding to the column. The smaller tergent environment. The 10-carbon chains of FC-10 amount of monomer observed cannot account for the are an optimal environment for the initial extraction large amount of dimer lost upon insufficient binding. of IncA from the plasma membrane, and the 12-car- However, the monomer form observed in SDS-PAGE bon chains of FC-12 are a suitable environment for analysis may be due to the denaturing conditions prothe elution of IncA from the cobalt column. Perhaps vided by SDS detergent, and it is under these denaIncA has different interactions with detergent chain- turing conditions that the monomer exists. Column lengths depending on environmental conditions, environment is not denaturing, so it is likely IncA exwhich includes detergent micelles, deionized water, ists predominantly in dimeric form, which is the more and any cellular proteins or debris that survived ex- stable form. It is proposed, then, that future experitractive and column purification steps. It is possible mentation is needed in cloning more histidines (perFigure 8. Dimer Conformers Proposed for IncA Insertion into Membrane: Two possible dimer conformers are proposed for IncA insertion into the membrane. One details a C-terminal end facing the cytosol of host cell with flanking N-termini in cytosol as well. This is known as head-to-head dimerization (A). The other depicts the C-terminal region facing the inner cytosol of the inclusion body with N-termini expressed on opposing membrane surfaces. This is known as head-to-tail dimerization (B).


haps eight as opposed to the six cloned) into the tag to see if greater histidine count can prevent its occlusion in dimeric structure. Also, the C-terminal Histag was cloned effectively into the incA construct, but it did not express as a tag (data not shown). The presence of impurities in all detergent screens of IncA extraction and purification characterizes IncA extraction as inefficient. Although these impurities are significantly reduced after the IMAC chromatography, they are still present in elution fractions and an additional chromatography step, such as size-exclusion, may be necessary for future structural studies. It is possible that these impurities are necessary stabilizing proteins and constructs that somehow interact with IncA as it binds to the column, but it is also possible that these impurities have chelating effects and bind personally to the column and elute effectively with imidazole. If the latter is the case, it will be difficult to produce a truly pure sample of IncA merely from a cobalt run and other purification strategies need to be explored. Perhaps hydrophobic interaction chromatography should be tried as well. The circular dichroism spectrum confirmed the predicted secondary structural characteristics of IncA, namely the alpha-helical motif. IncA is a membrane protein that is inserted into the host membrane through its transmembrane regions. These transmembrane regions characteristically have conserved secondary structures that allow for the polar peptide backbone of amino acid chains to associate in a thermodynamically advantageous way that reduces polar-nonpolar associations. These “thermodynamic� structures typically are helices, pleated sheets, and random loops and coils. The transmembrane regions of IncA have alpha-helical secondary structure which allows it to effectively embed within the hydrophobic membrane while polar groups are exposed to the outer cytosol. Also, the all alpha-helical structure indicated by the CD spectroscopy is consistent with the known leucine-zipper motifs in which two different alpha helices coil upon each other forming a coiled-coil structure. This structure could promote IncA dimerization in which monomer interfaces could interact via alpha helices and dimerize through coiled-coil interactions, which are known to be stable in the typically denaturing detergent SDS.14 There-

Figure 9. Dimer Conformers Proposed for IncA Insertion into Membrane: Two possible dimer conformers are proposed for IncA insertion into the membrane. One details a C-terminal end facing the cytosol of host cell with flanking N-termini in cytosol as well. This is known as head-to-head dimerization (A). The other depicts the C-terminal region facing the inner cytosol of the inclusion body with N-termini expressed on opposing membrane surfaces. This is known as head-to-tail dimerization (B).

fore, the dimer form of IncA is perhaps stabilized by coiled-coil interactions of leucine heptad repeats in the C-terminal domain (Figure 1).Two possible dimer conformers are proposed (Figure 9), and currently, site-directed spin labeling experiments are being designed to test these two dimerization possibilities. Conclusion IncA is a dynamic and vital protein to the health and maintenance of the inclusion body for Chlamydia trachomatis. A complete structural knowledge of IncA would promote a deeper understanding of its functional homotypic interactions as well as potential interactions with host cytosolic proteins. Perhaps, IncA interactions would resemble host-cell SNARE interactions and confer evolutionary linkage. If so, an understanding of IncA structure and function could generalize eukaryotic and prokaryotic mechanisms of membrane fusion and maintenance --powerful knowledge for understanding intracellular membrane interactions. References 1. Fields, K.A. and Hackstadt, T. 2002. The Chlamydial inclusion: escape from the endocytic pathway. Annu. Rev. Cell Dev. Biol. 18: 221245. 2. Wyrick, P.B. 2000. Intracellular survival by Chlamydia. Cellular Microbiology. 2: 275-282. 17

3. Hackstadt, T., Scidmore-Carlson, M.A., Shaw, E., and Fischer, E.R. 1999. The Chlamydia trachomatis IncA protein is required for homotypic vesicle fusion. Cellular Microbiology. 1: 119-130.

with multiple topologies. Embo J. 26: 9-18.

12. Alliance Proteins Laboratories, Initials. (2009). Circular dichroism. Retrieved from http://www. Rockey, D.D., Scidmore, M.A., Bannantine, J.P., ondary. and Brown, W.J. 2002. Proteins in the chlamydial inclusion membrane. Microbes and In- 13. Lumb, K.J., Carr, C.M., and Kim, P.S. (1994). fection. 4: 333-340. Subdomain folding of the coiled coil leucine zipper from the bZIP transcriptional activator 5. Delevoye, C., Nilges, M., Dehoux, P., Paumet, F., GCN4. Biochemistry. 33: 7361-7367. Perrinet, S., Dautry-Varsat, A., and Subtil, A. 2008. SNARE protein mimicry by an intracel- 14. Schuette, C.G., Hatsuzawa, K., Margittai, M., lular bacterium. PloS Pathog. 4: e1000022. Stein, A., Riedel, D., and Kuster, P. Konig, M., Seidel, C., and Jahn, R. (2004). Determi6. Delevoye, C., Nilges, M., Dautry-Varsat, A., and nants of liposome fusion mediated by synaptic Subtil, A. 2004. Conservation of the biochemiSNARE proteins. PNAS. 101: 2858-2863. cal properties of IncA from Chlamydia trachomatis and Chlamydia caviae. The Journal of 15. Klock, H.E., Koesema, E.J., Knuth, M.W., and Biological Chemistry. 279: 46896-46906. Lesley, S.A. (2007). Combining the polymerase incomplete primer extension method for clon7. Bannantine, J.P., Griffiths, R.S., Viratyosin, W., ing and mutagenesis with microscreening to acBrown, W.J., and Rockey, D.D. 2000. A secondcelerate structural genomics efforts. Proteins. ary structure motif predictive of protein local71: 982-994. ization to the chlamydial inclusion membrane. Cellular Microbiology. 2: 35-47. 16. Guo, H. Lokko, K., Zhang, Y, Yi, W., Wu, Z., and Wang, P.G. (2006). Overexpression and charac8. Dautry-Varsat, A., Balana, M.E., and Wypolsz, B. terization of Wzz of Escherichia coli O86:H2. 2004. Chlamydia- host cell interactions: recent Elsevier Protein Expression and Purification. advances on bacterial entry and intracellular 48: 49-55. development. Traffic. 5: 561-570. 9. World Health Organization. (2010). Chlamydia trachomatis. Retrieved from http://www.who. int/vaccine_research/diseases/chlamydia_trachomatis. 10. Dautry-Varsat, A., Subtil, A, and Hackstadt, T. 2005. Recent insights into the mechanisms of Chlamydia entry. Cellular Microbiology. 7: 1714-1722. 11. Zwilling, D., Cypionka, A, Pohl, W.H., Fasshaur, D., Walla, P.J., Wahl, M.C., and Jahn, R. (2007). Early endosomal SNAREs form a structurally conserved SNARE complex and fuse liposomes 18

Acknowledgements I would like to acknowledge Dr. Linda Columbus for her immense support and for giving me the opportunity to conduct research in her lab. I would also like to thank our lab technician, Iza Bielnicka and the graduate students for their guidance and daily support.


High Power Laser Texturing of Surfaces for Aerospace Applications “The project goal was to produce a black surface, created by the close placement of microscopic holes or lines that would trap incident light rays. During experimentation, the production of white surfaces was also discovered.�

Abstract The purpose of this project was to conduct research in the use of high power lasers to induce the texturing of surfaces for aerospace applications. The project goal was to produce a black surface, created by the close placement of microscopic holes or lines which would trap incident light rays. The first step was to become familiarized with the laser and micromachining techniques. Micromachining is a manufacturing method that allows for the creation of microelectromechanical systems (MEMS). The Nd:YAG fiber laser was utilized to texture samples of aluminum and steel. A systematic approach to the project was developed in which the holes were consecutively brought closer to each other. The material left between the holes constituted a microstructure, which funneled light downwards via reflection towards the surface for absorption. Although a black surface was created, several factors needed to be optimized. A different approach was adopted in which lines were micromachined, meaning a laser was used to create microscopic features, in order to create a grid pattern. The remaining material resembled pillars that reflected incident light rays towards the surface for absorption. In addition, air and argon atmospheres were used during the machining process to help prevent the oxidation of the aluminum or steel sample. Several black surfaces were successfully created using this micromachining approach. Laser micromachining samples were characterized for light reflection and scattering. The process described allows for a fabrication process of black and white surfaces on a variety of materials that is of interest to the aerospace industry as well as the NIA and NASA.


Ami Patel

-Ami Patel

Ami Patel is currently a third-year Aerospace engineering major. Ami Patel knew that her future lay in aerospace ever since the 5th grade. Every year since then, Ami worked her way towards her dream. At U.Va, she has been involved with Alpha Omega Epsilon: Professional Engineering Sorority (President) and Sigma Gamma Tau: National Aerospace Honor Society (President). Ami is also involved with research at the Aerospace Research Lab for the Hy-V program, whose goal is to obtain ground and flight data for the further advancement of scramjet technology. She is also a founding member of the AIAA Conference Committee, which is preparing to host the annual Student Paper Conference at U.Va. next April. During her time here, she has even started up an athletic team, called HooRaas, which is a competitive Indian folk dance group. Last summer, Ami was chosen to intern at the National Institute of Aerospace through the Langley Aerospace Research Summer Scholar (LARSS) program, which is where she conducted the research presented in this paper. She was able to explore the basics of laser micromachining and study the effects of high power laser texturing of surfaces for aerospace applications.

The primary objective of this research project was to create low reflectance surfaces using laser texturing processes for aerospace applications. These low reflectance surfaces, called black surfaces, were created by texturing the surface of a metal at the microscopic level. The research outlined in this article has several applications for both NASA and the aerospace industry at large. The material created could be an efficient light absorber for thermal heat generation. This material processing method can also provide control of surface emissivity, meaning the amount of radiation escaping from the metal surface (Champion, Darrin, & Osiander, 2006).

parts per spacecraft and thus decreasing the cost of launches in payload (Champion, Darrin, & Osiander, 2006). However, new machining methods had to be invented in order to create MEMS, such as laser micromachining. The research outlined by this article explores such a method for the potential production of MEMS. Laser micromachining is an important tool for the miniaturization trend in technology. This method allows for the production of more complex shapes in accordance with less material damage than normal machining processes (Dahotre, & Harimkar, 2008). In general, laser material processing can create products in a non-intrusive manner with a precision that approaches the wavelength of the laser light (HelvaBackground jian, 1999). The 1990s marked the emergence of micro- There are several different laser micromachinelectromechanical systems (MEMS) in aerospace ing techniques. The technique used in the following applications. MEMS were the beginning of the ef- experiment is “direct writing� in which the laser beam fort to rescale aerospace macrosystems to microsys- is focused directly on the substrate surface and a pattems. These systems had numerous benefits to the tern is created by translating the surface with respect aerospace field including decreasing the number of to the stationary laser beam or vice versa (Dahotre & 21

Harimkar, 2008). Direct focusing of the laser beam is the easiest procedure, as it only requires lenses instead of chemicals and additional metals. For microdrilling processes, the focal length is chosen so that the focal diameter corresponds to the required hole diameter (McGeough, 2002). The minimum diameter of the laser beam is equivalent to the wavelength of the laser. There also exist different material removal mechanisms; ablation is used in the following experiment. Ablation occurs when the material is removed using thermal and/or non-thermal interactions. A non-thermal interaction is when the energy of the photons break the bonds, and a thermal interaction is when energy is converted to heat (Dahotre & Harimkar, 2008). Thermal ablation processes were used in the experiment to vaporize the surface and thus create a depression. The light of the laser beam is absorbed by the metal surface, which increases the temperature of the metal. The metal begins to melt then vaporize as the temperature increases. Once the laser beam is removed, the remaining metal solidifies and a depression is left in the surface. This process can be used either to drill holes or laser lines. The various attributes of the laser allow for the success of micromachining. The laser (light amplification by stimulated emission of radiation) is a unique energy source due to its coherence, monochromaticity, directionality and brightness (Nayak, 2007). The directionality of the laser allows for energy to be delivered to the substrate surface without contact, unlike traditional light sources, which disperses in all directions (Migliore, 1996). Brightness, coherence and low beam divergence all account for the tight focusing ability and high intensity of the laser (Helvajian, 1999). This focusing ability allowed for the ablation process described above to occur. Laser micromachining introduces many benefits to materials processing. The ability of using a contact-less method allows for materials to be processed without sticking to the surface, or more specifically the crystal planes. As stated before, structures can be resized to the micron level (10-6 m). A larger variety of materials can also be processed over large areas while maintaining high precision (Helvajian, 1999). High precision attributes to one of the many 22

applications of laser micromachining. The many benefits of laser micromachining allow for special applications of materials processing that were not possible before. The fabrication of very precise microholes and cuts in materials is now possible. There already exists a process for drilling microscopic holes for boundary layer suction on airplanes as developed by British Aerospace (McGeough, 2002). Also, dissimilar materials can be accurately fused together using the laser. Most importantly, microengineered devices and components can now be developed, which would have a serious impact for the aerospace industry. The development of microengineered components would allow for considerable weight reduction concerning space crafts. For future research, the micromachining of other materials besides metals, such as glass or ceramics, would allow for applications outside of the aerospace field (Helvajian, 1999). Research Approach Microdrilling Holes The main goal of this research was to create a black surface through the laser texturing of surfaces. As an incident light ray strikes the textured surface, the light will reflect downwards by bouncing between the peaks of the ablated microstructure. The surface will absorb the remaining energy of the light ray. Figure 1 portrays this light absorption process in greater detail.

Figure 1: Light Absorption Process

Initially, the light trapping microstructure was created by the close placement of tiny holes. The remaining material between the holes would constitute the reflective peaks of the microstructure, as shown in Figure 2. A systematic study was undertaken in which the holes were brought increasingly closer together and a color change was observed. However, the process of manufacturing this microstructure was time-consuming. It required several hours of intense labor working with the laser because every adjust-

lars that constituted the light-trapping microstructure. This approach was much more efficient, taking about half the time as the prior approach. Therefore, this approach was used for the remainder of the project. Hypothesized Microstructures The hypothesized microstructures for each of the surfaces created are presented below. Initially, the microstructure for a black surface was created by the

Figure 4: Light-trapping microstructure for microdrilled holes Figure 2: Close placement of microscopic holes

ment had to be made manually. The whole process was not efficient enough, so another approach was taken towards creating the microstructure. Micromachining Lines The new approach for creating the microstructure involved micromachining lines instead of microdrilling holes. As depicted in Figure 3, the holes would be ablated in a grid pattern, first horizontally then vertically. The remaining material resembled pil-

Figure 3: Micromachined

Figure 5: Microstructure for Black Surface

Figure 6: Microstructure for White Surface


close placement of holes, as shown in Figure 4. The approach of micromachining lines was adopted in order to optimize manufacturing factors. The hypothesized microstructure shown below resembles an array of square pillars. These pillars represent the peaks that funnel incident light rays towards the surface for absorption. During the course of this research, the development of white surfaces was also discovered and further explored. The microstructure for a white surface, as shown in Figure 6, represents the ablated surfaces as a square hole. Experimental Setup The experimental setup is shown in Figure

became popular in the 1960s (Paschotta, 2008). In this particular crystal, the neodymium atoms partially replace yttrium ions. The atoms in the crystal are excited by using a flash or arc lamp. This laser can be utilized in either pulse mode or continuous wave regime (Dahotre, 1998). In this research project, the Nd: YAG laser was used in pulse mode at a repetition rate of 30 kHz. The power of the laser was used in the range of 4.5 to 9 watts. A short focal length was chosen so that the holes could be micromachined very precisely. The focal length used in the experiment was a 45 mm Thorlab lens, as seen in Figure 9. The translation stage (Figure 9) was automated in the horizontal direction. The speed was set at Translation Stage

Figure 9: Experimental Setup Figure 7: Schematic of Laser Micromachining Experiment Setup

7. The same setup was used for both approaches described above. The light beam traveled from the laser and was directed by a mirror through a converging lens. The converging lens focused the laser beam to a tight circle on the substrate surface. The Nd: YAG laser is a solid-state laser where the active medium is a neodymium doped YAG (yttrium aluminum garnet Y3A15O12) Figure 8: Nd: YAG Fiber crystal. YAG is a synthetic laser crystal that 24

2.5 mm/s. A Newport Universal Motion Controller/ Driver was used to control the motorized translation stage in the horizontal direction. The stage was controlled manually in the vertical direction. A grinder was used to polish all unfinished surfaces used as samples. Surfaces tend to become less reflective with roughness. Thus, the laser beam may undergo two or more reflections off of local peaks and valleys (Migliore, 1996). This would affect the quality of the ablated lines or holes, and therefore create an inefficient light-trapping microstructure. A microscope was used to examine the microstructures created by laser micromachining. The microscope was equipped with a camera so photographs could be taken of the substrate surface. Typically, a

magnification of 500x was used to examine the surface. For this research project, samples of aluminum, silicon and stainless steel were used for the study. The photographs of the different microstructures are shown below. Also, air and argon environments were used to prevent the oxidation of the metal surfaces. Vents were used to control the flow of the gases. Figure 12: White Surface of Aluminum 500x magnification

create the black surfaces. White Surfaces

Figure 10: Microscopic holes on aluminum surface

During the research, it was discovered that white surfaces could also be created by texturing the surface with a laser. Although the study of white surfaces was not an original goal, the concept was further examined for the remainder of the project. White surfaces can be created by using a lower power setting and creating a shallower cut with the laser. White surfaces were successfully created on aluminum and silicon samples. The table below describes the properties used to create the white pattern in Figure 12 on an aluminum surface.

Results Microdrilled Holes A black surface was successfully created by the close placement of microscopic holes. As stated before, the method was too time-consuming, so another approach was used. Aluminum was the only metal substrate used for this process. The following table lists certain characteristics used to create the textured surface shown in Figure 10. SEM Imaging After ablating the aluminum samples, they were further analyzed using SEM (scanning electron microscope) imaging. As shown below, the microstructure on the white surface (see Figure 6) is a lot shallower than the black microstructure in Figure 14. Thus, the black surface has a darker color since

Figure 11: Black Surface 500x magnification

Micromachined Lines Black Surfaces Black surfaces were also created by the close placement of lines in a grid pattern. Table 3 exhibits the machining characteristics that were used to

Figure 13: Aluminum “White� Surface Laser Power: 5.4 Watts


there are deeper grooves for incident light rays to be trapped. The white surface has a more reflective surface due to a shallow microstructure and thus has a lighter color. The SEM images were crucial for obtaining a better understanding of the actual shape of the microstructures. They showed that the microstructure for the black surface does not really resemble a pattern of pillars, as described by Figure 14. Rather, it seems to represent a deeper version of the white surface microstructure and is more similar to Figure 6.

Figure 14: Aluminum “Black� Surface Laser Power: 6.9 Watts

One reason for this change of shape could be that the material was not properly ablated, or removed, before the softened metal solidified.

turing on the color of the sample. The aerospace applications listed earlier can also be studied for future feasibility. Application Applications for the research conducted here are still developing, as a broader study of these reflective surfaces still needs to be completed. However, some hypothetical applications are described below and would require further study for the heat capacities of such microstructures. One problem concerning aerospace structures in space is heat dissipation from electronic parts on the craft. Extensive thermal control is needed to maintain the proper operating temperature range during missions (Champion, Darrin, & Osiander, 2006). Radiation heat transfer, the dominant form of heat transfer in space, is dependent on the properties of those materials exposed in space. If the reflective surfaces studied above are shown to be effective heat absorbers, they could be mounted on space crafts as passive thermal control systems. The control of surface emissivity could be an important factor for creating a thermal control system using these reflective surfaces.

References Champion, J.L. & Darrin, M.A.G. & Osiander, O. (Eds.). (2006). MEMS and microstructures in Conclusion aerospace applications. USA: Taylor & Francis This research study proved that the Nd: YAG Group fiber laser can successfully create black surfaces. The method used in this research is a cheaper alternative Dahotre, N. B. (Ed). (1998). Lasers in surface engimeans of producing black surfaces than is already neering (Vol. 1). Ohio: ASM International. used in the aerospace industry. The production of white surfaces was also discovered and further stud- Dahotre, N. B. & Harimkar, D. P. (2008). Laser fabriied. Controlling the laser power and surrounding encation and machining of materials. New York: vironment can create a gradient of color, from white Springer. to black surface. However, the laser focus position needs to be well controlled because it heavily impacts Department of the Army. (1991). Painting and Markthe color of the sample. ing of Army Aircraft. (Technical manual; TM The research conducted here can be further 55-1500-345-23). Washington, D.C.: U.S. Govstudied by turning the whole production process to ernment Printing Office automation, where there is no manual control at all. Also, a wider range of materials, such as ceramics or glass, can be studied to see the affects of surface tex26

El-Bandrawy, M. & Gupta, M. C. (2005). Femtosecond Laser Micromachining of Periodical Structures in Si <100>. Materials Research Society Symposium Proceedings, 850,173-178. Helvajian, H. (Ed.). (1999). Microengineering aerospace systems. USA: The Aerospace Corporation.

Acknowledgements Thanks to the National Institute of Aerospace and NASA Langley for their financial support, to Dr. Mool Gupta for guiding my research and to Dr. TehHwa Wong for helping me in the lab. Finally, thanks to Debbie Murray and Sarah Pauls of the LARSS program for allowing me this research opportunity.

McGeough, J. (Ed.). (2002). Micromachining of engineering materials. New York: Marcel Dekker, Inc. Migliore, L. (Ed.). (1996). Handbook of laser materials processing. New York: Marcel Dekker, Inc. Nayak, B. (2007). Ultrafast â&#x20AC;&#x201C; Laser â&#x20AC;&#x201C;Induced Surface Texturing and Crystallization of Semiconductors for Photovoltaic Devices (Doctoral dissertation, University of Virginia, 2007). Paschotta, R. (Ed.). (2008). Encyclopedia of laser physics and technology (Vol. A-M). USA: Wiley, John & Sons, Inc.


“When the mice are taken out of the hyperoxia condition and returned to normal oxygen level, a local tissue hypoxia will cause pathological growth of retinal superficial vessels stimulated through EphB4 and ephrinB2 signaling pathways. ” -Katelyn Mason

Abstract Retinopathy of prematurity (ROP) is a potentially blinding retinal disease that affects prematurely born infants. ROP is characterized by abnormal blood vessel growth in the retina. In order to investigate a treatment for this condition, the mechanisms behind blood vessel growth involving ephrinB2 reverse signaling in pathological retinal neovascularization must be understood. Litters of transgenic ephrinB2lacZ/+ and WT mice were subjected to the murine model of ROP. EphrinB2lacZ/+ and WT mice were housed in 75% O2 from P7 until P12. The neovascularization response was quantified through the analysis of the avascular tissue/total tissue ratio and the formation of preretinal neovascular tufts. Retinas harvested at P17 were whole-mounted and stained with lectin and analyzed to assess avascular tissue to total tissue ratio. Whole eyes harvested at P21 were sectioned, stained with hematoxylin and eosin, and the number of preretinal nuclei per section was measured. The avascular tissue/total tissue ratio was significantly increased in the ephrinB2lacZ/+ mice compared to the WT mice (0.26 ± 0.0063 vs. 0.14 ± 0.017, p < 0.005). Also, the number of preretinal vascular tufts per section was significantly decreased in ephrinB2lacZ/+ mice as compared to WT controls (22.9 ± 2.2 vs. 39.1 ± 3.7, p ≤ 0.001). The results from this study indicate that mice with decreased ephrinB2 reverse signaling exhibit an attenuated neovascularization response in the murine model of ROP. Thus, our results provide evidence for a pro-angiogenic role of ephrinB2 reverse signaling in postnatal pathological retinal neovascularization.


Katelyn Mason

Role of EphrinB2 Reverse Signaling in Pathological Retinal Neovascularization

Katelyn Mason is a third-year biomedical engineering student who expects to receive her B.S. in May 2011. She is from Winchester, Va. and went to John Handley High School. She has worked in the Peirce-Cottler laboratory for vascular and tissue systems bioengineering since January 2008. Alyssa Taylor, who began the research on retinal neovascularization in 2006, has been Katelyn’s graduate mentor on the research in retinal vasculature. She received the Jefferson Trust fund for perinatal research for this research project. She will be presenting this work at the Association for Research in Vision and Ophthalmology (ARVO) conference in May 2011. Katelyn is also a co-author with Alyssa Taylor on a presentation and an ASEE paper on the impact of team and advisor demographics on engineering capstone projects. She will continue with both of these research projects this summer in the Peirce-Cottler lab. Outside of the lab, Katelyn is involved in Delta Gamma sorority, Tau Beta Pi Engineering Honor Society, the Raven Society, and a global development project in South Africa through Jefferson Public Citizen.

Retinopathy of Prematurity (ROP) is a form of retinopathy that arises from premature babies that undergo a controlled increase in oxygen levels to maintain their blood levels. This induced tissue hypoxia causes blood vessels in the retina of the anterior of the eye to grow abnormally and remain fragile. These weak vessels leak blood into the eye, causing blindness and retinal detachment from the back of the eye. Annually, in the United States, there are about 15,000 cases of ROP in prematurely born babies and about 10% of those develop severe ROP that requires medical attention and potential blindness (National Eye Institute). This is an increasing problem as the number of infants with this condition continues to rise, despite several clinical treatments, due to the increasing number of infants that survive as a result of improved neonatal care (Phelps, 2004). Current treatment for ROP involves a laser treatment which burns away the abnormal blood vessels in the periphery of the eye. This treatment is crude and only partially successful. There is a need to investigate more successful treatment options for ROP by

understanding the mechanisms behind signaling molecules involved in blood vessel growth. The treatment for ROP can also be applied to several other retinal diseases that affect a large number of Americans, such as diabetic retinopathy, which is damage to the eye caused by long term diabetes. EphB4 receptor, which acts though forward signaling, and EphrinB2 ligand, which acts through reverse signaling, are key regulators in retina postnatal angiogenesis and microvascular remodeling. This receptor and ligand pair is primarily expressed by endothelial cells of developing arteries and veins (Zamora, 2005). As a result of the bidirectional signaling pathway and complicated expression patterns, the roles of EphB4 and EphrinB2 still remain unknown. The purpose of this work is to characterize the specific role of EphrinB2 reverse signaling in these processes. The murine model of ROP, which uses a mouse model to take advantage of genetic manipulations to reveal molecular pathways of retinal neovaculariztion, was used in this study. Mouse pups are 29

born with undeveloped vascularization in the retina, so they can be used to simulate prematurely born human babies with incomplete vascularization (Smith, 2004). Mouse pups are subjected to hyperoxia with 75% O2 for 5 days. During hyperoxia, blood vessels in the superficial retina stop growing to the center of the eye. When the mice are taken out of the hyperoxia condition and returned to normal oxygen levels (21% O2), a local tissue hypoxia occurs because the body is receiving significantly less oxygen than when they were in the chamber. This local tissue hypoxia causes an increased growth of blood vessels to supply enough oxygen to the eye. These blood vessels grow abnormally and cause blood to leak into the eye, and in some cases cause retinal detachment. We hypothesize that when returned to normal oxygen conditions, pathological growth of these superficial vessels is stimulated through EphB4 and ephrinB2 signaling pathways. Genetically mutated ephrinB2lacZ/+ mice were used to study the role of EphrinB2 reverse signaling. These transgenic ephrinB2lacZ/+ mice possess defective ephrinB2 reverse signaling with less than ¼ normal levels of ephrinB2 reverse signaling but undisrupted forward signaling through EphB4. The WT mice were used as control mice to compare against the genetically mutated mice.

Retinas harvested at P17 were whole-mounted and analyzed for the avascular/total tissue ratio. Whole eyes harvested at P21 were sectioned and analyzed for the formation of preretinal neovascular tufts. All procedures used were approved by the University of Virginia Animal Care and Use Committee.

Retinal Harvest and Fixation The two groups of mice at P17 and P21 were euthanized by overexposure to carbon dioxide. Both eyes were excised from the ephrinB2lacZ/+ and WT mice by applying pressure to the skull surrounding the eye cavity to force the eye upwards followed by clipping the optic nerve in the back of the eye. The eye was then stored in phosphate buffered saline (PBS) in a microcentrifuge tube. A small clipping of the end of the tail of mouse pup was taken and the DNA was analyzed to determine if the mouse was ephrinB2lacZ/+ or WT. The retinas harvested at P21 were fixed in paraformaldehyde (PFA) and sent to the University of Virginia Histology Core for sagittal cross sectioning of the eyes in order to view the neovascular tufts. The eyes are embedded in paraffin and cut into 10 representative sections each 5 microns thick. The retinas harvested at P17 were whole mounted on parafilm under a Zeiss Stemi 2000 dissecting microscope (Zeiss, Chester,VA). To begin, the eye was punctured with a 30G needle along the dark Materials and Methods equilibrium of the eye between the between the sclera lacZ/+ Transgenic ephrinB2 mice and the iris. A small incision was cut from the hole Male ephrinB2lacZ/+ were bred with CD-1 fe- in order to visualize after fixation. The eye was then males resulting in litter of approximately 50% wild fixed in 4% PFA for 30 minutes at 4°C. Following the type (WT) mice and 50% ephrinB2lacZ/+ mice. PCR fixation, the eye was cut along the equilibrium stemgenotyping was used to separate WT mice from ming from the initial hole. The cornea and lens were ephrinB2lacZ/+ as described previously (Davis, 2004). then carefully separated from the sclera and retina. These mice were obtained from Dr. Mark Henken- The sclera and choroids layers folded into a cup shape meyer from the University of Texas Southwestern. surrounding the retina. The retina was placed back in PFA for fixation for another 45 minutes at 4°C. The Exposure of Mice to Hyperoxia and Metrics anterior of the eye was removed from fixation and the lacZ/+ Litters of transgenic ephrinB2 and WT black choroid and scelera were pulled back from the mice were used in the murine model of ROP. Ephrin- retina using fine tipped tweezers. Paint brushes were B2lacZ/+ and WT mouse pups were housed in 75% O2 used instead of metal so not to damage the delicate, from P7 until P12. Two metrics were used to quan- metal sensitive retina. The retina was transported to tify the neovascularization response in the retina: a small amount of PBS on mounting slide. A straight the analysis of the avascular tissue/total tissue ratio blade razor was used to relieve tension along the sides and the formation of preretinal neovascular tufts. of the retinal envelope so the retina could lay flat. A 30

few more incisions with a razor were made and the retina was spread out in a butterfly shape. The remaining PBS was removed, allowing the retina to dry out and adhere to the slide. Immunohistochemistry The retinas harvested at P21 were stained with hematoxylin and eosin by the University of Virginia Histology Core in order to visualize the nuclei of the preretinal neovascular tufts. The whole mounted retinas harvested at P17 are stained with lectin to visualize endothelial cells of the microvasculature. The retinal tissue was co-stained with isolectin and smooth muscle (SM) alpha-actin with BS-I Isolectin B4 Biotin Conjugate and CY3-conjugated monoclonal anti-SM alpha-actin antibody (Sigma, Chicago, IL). Lectin is an endothelial cell marker and SM alpha-actin is a smooth muscle cell marker. Endothelial cells and smooth muscle cells line the blood vessels, so by staining for these, the vasculature can be visualized. Pap pen was applied in a thick circle surrounding the retina on the slide as a barrier to keep the liquid on the retina. The retinas were washed 4x 10 minutes with 0.1% PBS/Saponin and permeablized with Triton-X 100 for 1 hour. Blocking for nonspecific binding was completed with 5% Normal donkey serum (NDS) in 0.1% PBS/Saponin solution for 1 hour. Primary antibody solution with 1:100 BS-I Isolectin B4 Biotin Conjugate (Sigma-Aldrich, St. Louis, MO) and 5% NDS in antibody solution was applied to the retinas and they were incubated overnight at 4°C. Afterward, they were washed 8 times for 5 minutes per wash with 0.1% PBS/Saponin. The secondary antibody with 1:1200 Streptavidin AlexaFluor-647 (Sigma-Aldrich, St. Louis, MO), 1:200 CY3-conjugated monoclonat anti-SM alpha-actin, and 5% NDS in antibody solution was applied to the retinas for 2.5 hours at room temperature. The retinas were then washed 9 times for 10 minutes per wash with 0.1% PBS/Saponin. Cover slips were applied over the retinas using a mounting medium. Image Acquisition Confocal microscopy with a Nikon TE 2000E2 (Nikon, Melville, NY) microscope was used to obtain 10x images of superficial retinal networks for

retinal whole mounts. In order to visualize and analyze the entire retina, montages were made. Autopano Pro V2.0 (Kolor,Challes-les-Eaux France) was used to blend and render 8 to 12 10x images of a retina into a single montage. Image Analysis The images were quantified in a blinded manner. The 10x montage images of superficial retinal networks were then analyzed to assess avascular tissue/total tissue ratio. Using ImageJ (NIH, Bethesda, MD), the image montages were scaled by measuring a large vessel from the original 10x image in the pixel per micron ratio that was set up from the scale bar and translating that width to the whole retina montage. The total area of the retina was measured and recorded from the image montage. The avascular areas stemming from the center of the retina between arteriole/venule pairs were measured and added together. The total amount of avascular area was divided by the total area of the retina to obtain a ratio of avascular tissue to total tissue. The neovascular tufts penetrate the intraluminal membrane of retina, so sectioning the whole eye allows for greater 3D imaging of the vessels throughout the eye rather than just the superficial vessels in the retina. The preretinal tufts neovascular tufts are quantified by counting the nuclei of the neovascular tufts that extend into the vitreous. The average number of nuclei per sections is calculated using the mean number of nuclei in the 10 sections of the eye. Statistical Analysis For the statistical analysis of the data gained in both parts of this study, SigmaStat (Aspire Software International, Ashburn, VA) was used. The Student’s t-test was used to compare the two groups, ephrinB2lacZ/+ and WT mice. Statistical significance was asserted at p≤0.05. Results Avascular Tissue/Total Tissue Ratio The retinas harvested at P17 were whole mounted and stained with lectin to detect the presence of SM alpha-actin in the arteries, veins and capillaries in the retina. Images of these superficial 31

Fig. 1. Avascular Retinal Tissue for ephrinB2lacZ/+ and WT mice. WT mic eexhibit a greater degree of neovascularization compared with attenuated ephrinB2 reverse signaling (ephrinB2lacZ/+). These whole mounted retinas harvested at P17 and stained with alpha-actin are outlined in yellow to highlight the avascular area. The ephrinB2lacZ/+ mouse exhibited an avascular tissue/ total tissue ratio of 0.264 compared to the WT mouse ratio of 0.149.

vessels were taken at 10x and blended together in a single montage of each retina. The avascular areas stemming from the center of the retina was quantified along with the total area of the retina. Representative montage images of an ephrinB2lacZ/+ and WT mice with the avascular areas circled in yellow is shown in Figure 1. The images show that the avascular area in the ephrinB2lacZ/+ mouse is noticeably greater than in the WT mouse retina where the vascularization has reached further into the center of the retina. Using the Student’s t-test in SigmaStat, with an ephrinB2lacZ/+ mice n=4 and WT mice n=9, the avascular tissue/total tissue ratio was compared between the two groups. The results showed a significant increase in the ephrinB2lacZ/+ mice compared to the WT mice (0.26 ± 0.0063 vs. 0.14 ± 0.017, p < 0.005) (Fig. 2). Preretinal Neovascular Tufts The retinas harvested at P21 were sectioned and stained with hematoxylin and eosin to visualize nuclei. Ten sections per eye, each 5 microns thick, were imaged at 20x. The number of preretinal neovascular tufts were quantified and averaged by the 32

mean number of tuft nuclei counted for the 10 sections. Figure S1 shows a sectioned image of the eye in WT compared to ephrinB2lacZ/+ mice highlighted to show the nuclei of the preretinal tufts. SigmaStat analysis with Student’s t-test was used to compare the two mice groups. The number of preretinal vascular tufts per section was significantly decreased in ephrinB2lacZ/+ mice as compared to WT controls (22.9 ±

Fig. 2. Avascular Tissue/ Total Tissue Ratio for ephrinB2lacZ/+ and WT mice. At P17, eyes were harvested, whole mounted, and stained using alpha-actin after undergoing the standard murine model of ROP. Quantificaiton of avascular tissue/total tissue ratio shows a significant increase in avascular ratio of ephrinB2lacZ/+ mice compared to WT littermate controls. Data displayed=avascular tissue/total tissue ratio +SEM. *p<0.005

2.2 vs. 39.1 Âą 3.7, p â&#x2030;¤ 0.001). This information is displayed graphically in Figure S2. This is the same trend significantly decreased neovascularization response in the ephrinB2lacZ/+ mice versus the WT mice.

nal vascular tufts in the mutant ephrinB2lacZ/+ compared to the WT littermate control mice. This supports the hypothesis of the significance of ephrinB2 reverse signaling in neovascularization. The purpose of our study was to understand the effects of ephrinB2 reverse signaling in angiogenFig. S1. Mice with attenuated ephrinB2 lacZ/+ reverse signaling (ephrinB2 mice) esis and neovascularization. From the mice pups that exhibit decreased neovascularization as underwent the murine model of ROP, the microvascompared to WT littermate controls. cular remodeling was easy to see and quantify with Compared to WT control mice (left), H & E-stained whole-eye sections taken from lectin staining. This allowed the effects of ephrinB2 ephrinB2lacZ/+ mice exhibit decreased prereverse signaling to be compared between mice that retinal neovascular tuft formation (arhave the gene expression and mice that have the gene rows) (right). 5 micrometer thick wholeeye sections were obtained at P20, after 5 expression of ephrinB2 knocked out. Using two difdays of hyporoxia exposure followed by ferent metrics at two different time points allowed 8 days in room air. Conclusion for further analysis and increased confidence in the Qualitative analysis between ephrinB2lacZ/+ results that were found in the effects of ephrinB2 reand WT mice that underwent the murine model of verse signaling. ROP was used to further the understanding of the The results of this study which indicate a correlation between EphB4/ephrinB2 signaling and retinal neovascularization following the murine model of ROP are supported by previous works. During and after the ROP model with increased oxygen, there was a significant increase in the gene expressions for ephrinB2 and EphB4 respectively along with vascular endothelial growth factor (VEGF), which also promotes angiogenesis (Ehlken, 2009). Furthermore, ephrinB2 reverse signaling was shown to be active during angiogenesis by phosphorylation during the time when mice are developing circulation in their retina (Salvucci, 2009). Considering our data and Fig. S2. Comparison of preretinal neovascular tuft formation in WT versus ephrinB2lacZ/+ mice after exposure to murine model of the supporting literature, EphB4/ephrinB2 may be ROP. At P20, eyes were harvested, sectioned, and stained using therapeutic targets for Retinopathy of Prematurity H&E. Quantification of preretinal tuft formation indicates that along with other retinal neovascularization problems WT mice display a significantly greater amount of retinal neovascularization than ephrinB2lacZ/+ mice under the standard muto reduce angiogenesis. rine model of ROP. The number of preretinal nuclei per section The investigation into the bidirectional role (protruding past the ILM) was counted in a blinded analysis. Data of EphB4/ephrinB2 signaling pathway will be taken displayed=average number of preretinal nuclei per section + SEM. *p<0.001. further by injecting sEphB4 into ephrinB2lacZ/+ and WT mice that have undergone the standard murine specific roles of EphB4 and ephrinB2 signaling in model of ROP. This soluble monomeric protein, retinal hypoxia-induced neovascularization. Ephrin- sEphB4, is a form of EphB4 which blocks EphB4/ B2lacZ/+ mice exhibit a decreased neovascularization ephrinB2 signaling by binding and blocking the actiresponse compared to WT control mouse as indicated vation of ephrinB2 reverse signaling through EphB by two separate metrics: avascular tissue/total tissue receptors. This soluble protein has been shown to inratio and preretinal neovascular tufts. There was a hibit the angiogenic effects of growth factors such as statistically significant increase in avascular tissue/ VEGF (Kertesz, 2005) and thus reduce the extent of total tissue ratio and decrease in number of prereti- the angiogenic response. Injecting this soluble pro33

tein into the retina to target the EphB4/ephrinB2 proteins could serve as a pharmaceutical therapeutic approach to ROP as it would decrease the extent of neovascularization upon the return to normoxic conditions. The ephrinB2lacZ/+ mice are characterized to have defective ephrinB2 reverse signaling, but undisrupted EphB4 forward signaling. By injecting the sEphB4 protein, the neovascularization results will be able to be quantified without any EphB4/ ephrinB2 forward or reverse signaling, providing a larger picture on the role of EphB4/ephrinB2 signaling. This larger picture is necessary for the development of effective therapeutic interventions in retinal diseases such as ROP. This investigation into the effects of EphB4/ephrinB2 signaling in retinopathy of prematurity can be applied to similar diseases which affect a greater percentage of the population. Choroidal neovascularization (CNV) is age-related macular degeneration in which there is neovascularization in the corodial layer of the eye which can lead to deterioration of vision. The injection of sEphB4 has shown to reduce chorodial angiogenesis and overall CNV volume (He, 2005). Diabetic retinopathy is another disease that affects the eye in much the same way as ROP. This disease is caused by diabetes evoking neovascularization which can lead to hemorrhaging and the development of small scars. The ultimate result of this is vision loss. The effects of EphB4/ephrinB2 signaling in the retina can be applied to diabetic retinopathy in the same way as it can be applied to ROP. Thus, a treatment for ROP can be applied to diabetic retinopathy. The manipulation of ephrinB2 reverse signaling or both EphB4 forward signaling and ephrinB2 reverse signaling may result in therapeutic outcomes for treatment of various types of retinal diseases. With greater investigation, we hope to indicate how disrupting EphB4/ephrinB2 signaling could be a putative therapeutic target for the treatment of ROP.


References Davis C, Yokoyama N, Chumley M, Cowan C, Silvany R, Shay J, Baker L, Henkemeyer M. (2004). Bidirectional signaling mediated by ephrin-B2 and EphB2 controls urorectal development. Developmental Biology. 271: 272-290. Ehlken C, Martin G, Lange C, Gogaki E, Fiedler U, Schaffner F, Hansen L, Augustin H, Agostini H. (2009). Therapeutic Interference with EphrinB2 signaling inhibis oxygen-induced angioproliferative retinopathy. Acta Ophthalmologica. He S, Ding Y, Zhou J, Krasnoperov V, Zozulya S, Kumar SR, et al. Soluble EphB4 regulates choroidal endothelial cell function and inhibits lazor-induced choroidal neovascularizaion. Investigative ophthalmology and visual science. 2005;46(12):4772-9. Kertesz N, Krasnopero V, Reddy R, et al. (2005). The soluble extracellular domain of EphB4 (sEphB4) antagonizes EphB4-EphrinB2 interaction, modulates angiogenesis, and inhibits tumor growth. The American Society of Hematology. 107(6):2330-2338. National Eye Institude, National Institute of Health. <> Salvucci O, Maric D, Economopoulou M, Sakakibara S, Merlin S, Follenzi A, Tosato G. (2009). Eph rinB reverse signaling contributes to endothe lial and mural cell assembly into vascular structures. Blood Journal. 114:1707-1716. Phelps , DL. (2004). The Early treatment f retinopa thy of prematurity study . Pediatrics, 114(2). Smith LE, Wesolowski E, McLellen A, Kostyk SK, Dâ&#x20AC;&#x2122;Amato R, Sullivan R, et al. Oxygen-induced retinopathy in the mouse. Investigative ophthalmology and visual science. 1994; 35(1):101-11.

Zamora DO, Davies MH, Planck SR, Resenbaum JT, Powers MR. Soluble forms of EphrinB2 and EphB4 reduce retinal neovaxcularization ina model of proliferative retinopathy. Investigative ophthalmology & visual science. 2005:46(6):2175-82.

Acknowledgments Alyssa Taylor is Katelyn Masonâ&#x20AC;&#x2122;s graduate student mentor who has worked in the Peirce-Cottler Laboratory since 2004. She will be defending her PhD in May 2010 and will be joining the department of Bioengineering at the University of Washington as a teaching faculty member. Alyssa has played an integral role in teaching Katelyn all of the techniques of the research as well as allowed her to gain an understanding of the research and the context. Dr. Shayn Peirce-Cottler is the head of the vascular and tissue systems bioengineering lab where this project was conducted. She oversees the entire research project and is a mentor for both Alyssa Taylor and Katelyn Mason. Dr. Paul Yates in the Ophthalmology Department at the University of Virginia has been a collaborator on the research project.


“New information put forth suggests that certain existing sunscreens have many side effects and don’t effectively block our bodies from two types of UV light. ” -Haarthi Sadasivam

Abstract Sun tanning is a “sport” that has been embedded into popular culture. Sunscreens have been used to fuel our obsession with tanning but what exactly are the health consequences of such sunscreens? In my paper I discuss the negative impacts of different types of sunscreens on human health and the environment. Some types of sunscreens, which involve chemical manufacturing, do not effectively block our skin from harmful rays and have a variety of visible health risks. Although these risks are not “visible” in the second, more abundant, type of sunscreens, which contain “natural” nanoparticles, they have the potential to be harmful. As a relatively undiscovered field and a research hot spot, there are many things we do not know about nanotechnology- and nanoparticle- containing sunscreens. These types of sunscreens could have harmful health effects that we have not become aware of yet and have been proven to be very detrimental to the environment, where fears of biotoxicity and bioaccumulation persist. These problems with chemically produced sunscreens and nano-particle sunscreens call for more research and regulation.


Haarthi Sadasivam

The Shady Side of Sunscreen

Haarthi is a first-year at the University of Virginia with an intended major in computer engineering and a minor in engineering business. She was first introduced to the budding field of nanotechnology in the fall semester of 2009 where she learned about its benefits and the potential it has for our future development. After an interesting class about the real world effects of nanotechnology, Haarthi became very interested with their effect on sunscreens. As a result, she chose to investigate the critical role of nanotechnology in this field and the numerous effects it has on the environment and our health.

Sunscreens have been used for many years to absorb or deflect damaging ultraviolet rays emitted from our sun and are an essential part of our daily lives. However, new information put forth suggests that certain existing sunscreens have many side effects and don’t effectively block our bodies from two types of UV light. Other sunscreens with nanoparticles that do manage to effectively shield our skin from all UV light have been found to have numerous environmental consequences. This leads to an important question: Should we use the sunscreen with better UV protection even if it may harm the environment and cause health problems? There are many different types of sunscreens, currently all of which contain different substances with different functions. Sunscreens containing Zinc oxide and Titanium dioxide are considered “physical blockers” since they reflect and scatter UV rays. Although some ingredients of Zinc oxide play absorbing roles, for the most part the particles sit atop the skin and provide a barrier against the UV rays, deflecting them away from the skin and preventing any harm. Other sunscreens, including those containing Oxybenzone, Octinoxate, and Octocrylene, called “chemical blockers,” absorb the UV rays into the skin’s top layer. These sunscreens aim to either deflect the UV rays or absorb their energy and convert it into something less harmful (Environmental Working Group, 2007). Researchers, however, have found many problems with chemically produced sunscreens. Some of the most common substances used in these sunscreens like Benzophenone and Avebenzone are powerful free radical generators (highly active chemical reactors) that are activated when UV light energy strikes them. In the case of Benzophenone, its double bonds break and produce two free radical sites that then react with other molecules to produce damage to fats, proteins, and DNA in cells. This free-radical generation increases their cellular damage and in certain cases has been known to produce the most common form of skin cancer (Pickart, 2008). In addition to this problem, large amounts of applied sunscreens have been found to enter the bloodstream though the skin. Scientists have found that about 35% of sunscreen particles like Avebenzone penetrate the skin and enter through to the

blood stream (Mayo Clinic Staff, 2009).This combined with their “estrogenic activity” has been linked to many health problems, such as gender blending, from chemical interference with normal sexual development. These characteristics of Avebenzone and many other chemical sunscreens allows them to be easily absorbed through the epidermis, and as a result, they have been linked to increases in cancers, birth defects in children, and reproductive damages in men (Pickart, 2010). These sunscreens are also said to lead to more toxins on average in each of these categories: birth defects and reproductive harm (40% more), neurotoxins (20% more), endocrine system disruptors (70% more), and immune system damaging chemicals (70% more). (Environmental Working Group, 2010) Along with their damaging properties, recent studies have shown that chemically produced sunscreens are not 100% effective at combating both UVA and UVB rays. Some forms dissolve in the skin within 30 minutes and leave the skin open to all forms of Ultraviolet rays, while others are effective at combating only UVA rays, which are responsible for causing aging effects and skin cancer (Library of Congress, 2010). They don’t have the capability of attacking UVB rays, which can cause sunburns, cataracts, and immune system damage. In order to rectify this situation many industrial companies have begun to combine many different chemical particles to offer a broader range of protection, hoping that different chemicals will protect a person from both UVA and UVB radiation. This, however, increases the amount of exposure to different chemicals and results in unexpected interactions between these chemicals leading to unexpected consequences. Taking a step back from these chemical sunscreens leads us to natural sunscreens containing Zinc oxide and Titanium dioxide particles. Both Zinc oxide and Titanium dioxide are natural minerals that provide a thin film coating of fine particles over the skin, ensuring a protective physical barrier over the skin. Zinc oxide can screen both UVA and UVB light (the only one to do so) and Titanium dioxide has the ability to protect our skin from UVB light. In addition, these minerals are FDA approved and are stable elements that will not degrade in the sun. These chemical-free products also start working immediate37

ly upon application and are found to be less irritating to the skin. Why would we use chemically produced sunscreens if these naturally made sunscreens are much more beneficial and work efficiently? In this case, it often has to do with the aesthetic appearance of these sunscreens. When ZnO and TiO2 are used, they scatter visible light, causing a whiteish opaque appearance in the places where they are applied. This appearance turns off consumers from buying these natural products and has thereby led many researchers to explore ways to reduce the white coloration and make the product more transparent. Researchers have found that there is a relationship between particle size and the degree of scattering. As the particle size approaches the nano-scale, thus reaching opacity, UltraViolet waves pass through the particles instead of reflecting off the particles. The optimum particle size, calculated using the refractive index of TiO2 and ZnO and the wavelength of visible light (500-800nm), is now in the nano-scale of about 1 to 100 nanometers (Bird, 2008). To meet these requirements, the natural elements are modified to reduce the intensity of the UVA and UVB portions of the electromagnetic spectrum and minimize interactions with the visible portions, which causes the substances to become transparent on the skin surface. The change in the optical properties of the two minerals, described below, is achieved solely by the modification of the particle size and not by any chemical alterations (Lowe, 1996). In industries, TiO2 and ZnO are purified using different techniques such as high-temperature oxidation and crystallization, and go through processes such as the “chloride process” and the “sulfide processes” to be fully purified. Once the pure material is obtained, various techniques such as crystal size control precipitation and high temperature heat treatment are used to get to the optimal particle size. Once optimal size (now in the nano-scale) is reached, a coating is added to the TiO2 and ZnO to improve chemical stability while reducing their dispersible characteristics. If agglomeration (the bunching up of particles) occurs, the material still reflects the visible light range, leading to a white appearance. Otherwise, nano TiO2 and ZnO become very effective at 38

blocking harmful UV rays and do not leave an unsightly residue. (Nicholas, 2010) One of the main problems that manufacturers face is nanoparticle ability to generate free radicals. Both Titanium dioxide and Zinc oxide are photoactive: they have the ability to absorb UV light in the presence of moisture thus converting water molecules into active hydroxyl free radicals. This chemical reactivity could erode the components that hold the sunscreen together. This could cause skin cancer and skin damage if free radicals penetrate the surface and enter skin cells. Researchers, however, have found ways to suppress this activity in sunscreen products by altering the structures of nanomaterials. By changing the crystalline structures of titanium dioxide and by adding material, they have managed to reduce photocatalytic activity substantially. For example, a sunscreen company, Oxonica, incorporated a small amount of manganese into titanium dioxide nanoparticles, which allowed the absorbed UV energy to be spread out, eliminating the formation of free radicals. This made the sunscreen safer and allowed it to last longer in the sunlight (Oxanica, 2009). While nano-particle-containing sunscreen products have overcome the problem of skin penetration, chemical sunscreens have yet to find a solution. Nanoparticles do not, for the most part, penetrate through the thickness of the outer stratum corneum, the outermost layer in the skin, into the tissues below. Penetration into just the stratum corneum presents good evidence that nanoparticles do not fully penetrate the skin and reach living cells below. The fact that nanoparticles do not reach past the outer layer of the skin suggests that they do not get into the blood stream where they move around throughout the body penetrating vital organs. In an experiment, researcher Lademann could not detect any absorption of nano-TiO2 particles past the epidermis after repeated application on the hands of volunteers over a period of seven days. Using an electron microscope, he determined that only the upper stratum corneum and hair follicles showed evidence of particle penetration. In another experiment, he tested the penetration of ZnO particles though the skin and monitored the location of the particles and the amount of zinc penetration through the epider-

mis. The results he found suggested that the total amount absorbed was found to be less than .03% of the applied amount. The study suggested that this amount of epidermal penetration into the dermis is negligible (Cross, 2007). Other experiments done throughout the years on layers of healthy human skin and pig skin (which is very similar to human skin) suggest that sunscreen nanoparticles generally do not penetrate past the layers of the stratum corneum and into the dermis. Although in some experiments zinc oxide was said to have penetration rates of <.03%, this was considered minimal. Some accumulation of the particles in the follicles was also deemed safe as long as there was no movement into deeper tissues. In all of these experiments done on healthy skin there was no strong evidence that the particles reached the blood stream or â&#x20AC;&#x153;vital tissuesâ&#x20AC;? (Environmental Working Group, 2007). Compared to chemically manufactured sunscreens, natural sunscreens, with products such as nano Titanium dioxide and nano Zinc oxide, are generally healthier for humans. They are better at shielding consumers from both types of harmful Ultraviolet rays and are gentler on the skin surface. Many studies show that they sit on the surface of the skin and block UV radiation for several hours after application. These sunscreens give a clear product that looks good and also works well. With all these positive benefits, one has to wonder why nanoparticle based sunscreens remain such a controversial topic. Many of these controversies come from the present lack of information and the uncertainty that surrounds these nanoparticles. Although many studies show that skin penetration is negligible, it has been pointed out that the experiments have only been conducted on healthy skin cells. Other studies show that nano-particle skin penetration depends only on the thickness and durability of the skin. This suggests that the thinner and undeveloped skin coverings are open to higher levels of nano-particle penetrations, which means that they could reach the bloodstream and cause damage to the body. (Environmental Working Group, 2007) For example, in cases of psoriasis, a skin condition where the skin does not have a protective bar-

rier, titanium dioxide particles have been found in living cells (Environmental Working Group, 2007). A recent study, which examined nanoparticle fluorsphere penetration by tape stripping debris from the skin openings to the follicles, suggested that significant nanoparticle penetration occurred in the case of psoriasis (Nohynek, 2007). This alarming data suggests that nano-particles could also have a higher chance of entering sunburned skin and should not be applied to open wounds or in places where there is a break in hair follicles. In addition small children and the elderly, who are less likely to have strong, healthy, well developed skin, should also take care when using nano-particle containing sunscreens because of skin penetration problems. Even if the usage of nanoparticle sunscreens was restricted only to those with healthy skin, health risks still remain. Studies have shown that accidental oral exposures to nano ZnO and nano TiO2 in pools and beaches could have more direct damages than skin penetrations. Oral exposures, which could easily occur every time water is swallowed in the pool, introduce nanoparticles into our body where they could be harmful. When oral exposure tests with reasonable doses were performed on animals, researchers found altered enzyme levels and found that the nano TiO2 passed out of the intestines and provoked damages to organs. This evidence suggests that the small sizes of nano Zinc oxide and nano Titanium dioxide allows them to be easily transported throughout the body. This enables them to be accumulated in cellular organs where they could easily penetrate vital organelles and tissues. (Cross, 2007) Health problems associated with nanoparticles do not end with the oral exposures. Powdered mineral sunscreens or sprays that contain nano ZnO and TiO2 could be inhaled through the nose or the mouth. The inhalation of these nano-sized particles can provoke inflammatory responses because of the high surface area and high reactivity of these small particles. Studies on nano-titanium particles show that when nanoparticles are inhaled, they can reach the brain, provoke oxidative stress, and possibly reduce the flow of air through the lungs. One form of TiO2 used called anatase has been deemed a carcinogen and has repeatedly shown higher penetrating and 39

reactive rates in lungs, in some cases even leading to lung cancer (Cross, 2007). To stop these harmful effects, the use of powdered or spray sunscreens should be censored. Another controversial aspect of nanoparticle containing sunscreens is their impact on the environment. In the environment, small separate instances have the potential to accumulate in substantial amounts with enormous consequences. Bioaccumulation and biotoxicty are some of the biggest fears in this field. Through research and various studies, nano ZnO and TiO2 have been found to have serious health implications on many species. Nano ZnO and nano TiO2 create numerous problems in the lower levels of the food chain. For example, a study has shown that Zooplankton and filter feeding invertebrates that make up the basis of the aquatic food chain have the capability of absorbing these nanoparticles. These aquatic organisms usually feed on particles such as bacteria, viruses, and other organic molecules, but with selective filtering they could ingest nanoparticles because of their small size. This could result in harmful effects for them and create problems for other species that consume these organisms. In this case, the biotoxicity of these organisms would pass on to their consumers and would multiply at each food level in the food chain, resulting in problems for organisms at the higher end of the food chain (Borm, 2006). Another example of the trickling effect can be seen with the nano substanceseffect on bacteria. Nano TiO2 and ZnO have been found to harm certain microbes that perform vital roles in the environment such as removing ammonia from water waste treatment systems, cleaning up toxic wastes, reducing phosphorus in lakes, and creating carbon dioxide in the soil (ArticlesBase, 2006). The nanoparticles most often used in sunscreens, which usually end up in sewage treatment plants after being washed off in showers, have the potential to eliminate microbes and bacteria that treat waste water and play vital roles in ecosystems. In an experiment performed in the University of Toledo, researchers added different amounts of TiO2 to water containing bacteria (which were grown in the lab and were stained with a green fluorescent gel). The researchers soon observed significant dam40

age to the bacteria’s cell walls after adding 10 to 100 milligrams per liter of the nanosubstance. The damage, which was indicated by the change in the cell membrane color from a green fluorescent color to a red glow, occurred surprisingly fast. In this case, the cell wall damage in the bacteria indicated that the bacteria lost its previous functionality (Cimitile, 2009). In another study, researchers determined exactly how these nanoparticles act as biocidal (a chemical substance capable of killing living organisms in a selective way). When put in with sunscreen nanoparticles, researchers found that the bacterium acquired dark splotches and was destroyed. The nanoparticles had an electrical charge opposite to that of the bacteria, which caused them to be attracted towards each other. As a result when the particles and the bacteria came in contact, the nanoparticle’s sharp edge penetrated the thick outer shells because of the attraction to bacteria in different locations. Additionally, since nanoparticles are bases, they softened the exteriors of the bacteria and chemically damaged them by stealing electrons. Titanium oxide nanoparticles are able to destroy a bacterium in about five minutes and produce hydroxyl radicals in pure water (Kalaugher, 2002). A recent release by the Environmental Health News stated that beneficial soil bacteria cannot tolerate silver, copper, and zinc oxide nanoparticles. This damage to the bacteria occurs in very small levels of exposure. In their experiment, they determined that the level of exposure that would create problems was equivalent to two drops of nanoparticles in an Olympic sized swimming pool (Cimitile, 2009). This data suggests shocking implications about the future impact of these beneficial microbes in the soil. In some cases bacteria are harmful to human, plant, and animal health; therefore eliminating nanoparticles’ antimicrobial properties would be very beneficial. But there are also “good bacteria” in the environment which play a vital role in the human health and the ecosystem. If these beneficial bacteria were eliminated by the use of nano titanium dioxide and zinc oxide then this would cause a trickling effect that could pose numerous problems both for humans and our environment.

Other cases of sunscreen nanoparticle damage includes their effects on zebra fish, rats, and daphnia. A researcher named Furgueson exposed zebrafish embryos to Zinc oxide nanoparticles in a laboratory. His findings indicated that some of the zebrafish died while others were left with dramatic mutations causing serious malformation in their eyes and tails. Some of the embryos even suffered heart failure (Shetler, 2009). Using an optical microscopy technique, scientists characterized the nanometer sizes of individual nanoparticles. Using the brightness and the color of Zinc oxide nanoparticles the scientists were able to trace the nanoparticles as they were transported through chorion pore canals into the embryo. These chorion pore canals exist to provide a protective covering for the embryo and to separate it from the outside environment, but since the ZnO nanoparticles were very small, they were able to penetrate through to the embryo by passively diffusing through the protective covering. In the case of zebrafish, researchers found that once these nanoparticles traveled through the chorian canals, they have to ability to affect the phenotypes of certain embryos causing certain mutations (Berger, 2007). These nanoparticle toxicity tests, which were also performed on hairless mice, showed similar results. Their skin, which is less than half as thick as human skin, has greater permeability that allows for greater absorption of nanoparticles. In a study when TiO2 nanoparticles were added to the drinking water of lab mice, the mice began showing genetic damage by the fifth day (TechWire, 2009). The TiO2 nanoparticles in the water seeped through the skin’s surface and reached the living cells leading to double strand DNA breakage and oxidative stress in the mice (Bird, 2009). This nanoparticle absorption through the skin, suggests that nanoparticles could enter through the skin of many animals (that do not have the same protective coverings as humans) and cause damage. This puts the lives of numerous animals and plants at risk. It is important for us to establish what concentrations of these nanoparticles have the potential for significant damage. In most cases, as the nanoparticle concentration increases, the amount of the impact on organisms increases. What concentrations

of the nanoparticles found in “natural” sunscreens actually cause these impacts? In certain cases (like with the bacteria and the filter feeders) research has shown that small concentrations of the nanoparticles (similar to those that exist right now) can actually cause inhibitory/harmful effects. In the case of the mice and the zebra fish the experimental concentrations that researchers have used are unrealistic as of now. However, the continual disposal of sunscreens and the increasing use of nano ZnO and TiO2 in many manufactured products will only serve to increase the concentrations of these nanoparticles in the environment. As the concentrations increase drastically over the next few years, mutations and deaths could increase significantly and affect many different species. The impacts of nano ZnO and TiO2 on zooplankton, bacteria, zebra-fish and the other organisms mentioned above raise many concerns that nanoparticle usage could bioaccumulate and mimic the role of DDT in our environment. In some of these cases, even when the nano-oxides do not seem to have much impact on a particular organism, they have far reaching consequences because the organisms that they affect play vital roles in the ecosystem. Like DDT, nanoparticle toxicity could increase exponentially within each level of the food chain resulting in the significant problems for the environment. The environmental and health problems associated with nanoparticle-based sunscreens call for regulation. Since many aspects of nanotechnology research cannot be fully conducted on a large scale, it is hard to fully understand the negative aspects of sunscreens. In addition, the regulatory process is far behind the speed at which new sunscreens are being produced and entering the market. As of now, regulatory programs are basing their standards on mass concentrations. Some regulating agencies have also been predicting toxicity of nanoparticles based on studies with “conventional” particles that have similar chemical structure. This poses a problem because the role of nanoparticles is greatly dependent on size and differs greatly as the particles get to a nano-scale. This can be seen with Zinc oxide and Titanium dioxide nanoparticles. In their “macro” state these particles are found in nature and do not have any harmful effects on humans 41

or the environment. Once they get down to the nanoscale, however, they gain penetration abilities and act very differently. Their high surface area-to-mass ratio allows for greater exposure, and allows the nanoparticles to be more potent at far lower concentrations (Florini, 2009). Another problem with the regulatory process is that no specific agency handles sunscreens. Different agencies like the FDA, EPA, the TSA look at different aspects of their effects. The FDA has, in the past, looked at the effects of nano Zinc and TiO2 on food products and determined that food intake does not have any correlation with health effects. Neither the FDA nor the EPA have looked at the effects of these particles on aquatic ecosystems (Florini, 2010). In the past, environmental regulation on toxic particles has started only when significant problems have risen, but with the case of nanoparticles this would not work. The particles would be too wide spread for us to be able to control their impacts on the environment. All this information about sunscreens and regulations puts us in a difficult spot. Sunscreens are an essential part of our lives but possibly could do more harm than good. Chemical sunscreens do not adequately protect our bodies from damaging UV light and have been found to have many detrimental health effects. Although “Natural sunscreens” are not harmful and are very capable of protecting us against UV light, consumers shun them because they are not aesthetically pleasing. Natural sunscreens with nanoparticles are pleasing and do manage to block the skin from UV light, but have many harmful effects. Although there is little skin penetration in healthy skin, if these nanoparticles manage to find a way into the body, through sprays or damages in the skin, they could have many health consequences. In addition to these health consequences, they also have numerous environmental consequences that could drastically change our ecosystem. So what should we do to fix this problem? Would it be wise to continue using nanoparticle sunscreens despite the environmental risks? Or would it be better to give up sunscreens altogether and suffer the risks of skin cancer? To rectify this situation, there should be a major increase in sunscreen and nano-material risk re42

search. The US government, along with private firms and governments of other countries, should spend more time and money to address the health and environmental implications of the different types of sunscreens and needs to focus on finding ways to eliminate the negative aspects of nano-material sunscreens. So far, federal regulatory agencies have not fulfilled their role in identifying, managing, and avoiding the downsides of sunscreens. Raising consumer awareness about the problems with nanoparticle containing sunscreens would help put pressure on sunscreen companies and would initiate research and regulation in this field. A balance must be struck between regulation and research in order to avoid potentially detrimental effects of using sunscreens. References Bird, Katie. “Natural Nano-sunscreens - A Contradiction?.” 23 04 2009. Web. 14 Feb 2010. < com/Products-Markets/Natural-nano-sunscreens-a-contradiction+optimal+particle+siz e+%2B+nano+zinc+oxide+%2B+sunscreens &cd=1&hl=en&ct=clnk&gl=us>. Bird, Katie. “Exposure to nano titanium dioxide could up cancer risk, says study.” NutraIngredients. Web. 14 Feb 2010. <http://www.>. Cross, Sheree. Human Skin Penetration of Sunscreen. “Nanoparticles: In-vitro Assessment of a Novel Micronized Zinc Oxide Formul a t i o n . ” 2 0 0 7 . h t t p : / / w w w. n a n o a r c h ive . org/762/1/000098701.pdf “EW’s 2009 Sunscreen Investigation.” Environmental Working Group, Web. 14 Feb 2010. <1http:// ais>. “EWG’s 2009 Sunscreen Investigation.” Nanotech-

nology and Sunscreens. Web. 14 Feb 2010. <>.

Noyenhak, . “Grey Goo on the Skin? Nanotechnology, Cosmetic and Sunscreen Safety.” Critical Reviews in Toxicity. Web. 14 Feb 2010. < abs/10.1080/10408440601177780

Florini, Karen. “Nanotechnology: Getting it Right the First Time.” Organic Consumer Association, Web. 14 Feb 2010. <http://www.virlab. Oxonica. “Leaders in Nanotechnology” 2009. http:// Lecture_13_Materials/Nano%20Hazards/Getphp ting%20It%20Right%20the%20First%20 Time_%20Developing%20Nanotechnolo- Pickart, Loren. “Chemical Sunscreen Health Diagy%20wh...pdf>. ster.” Web. 14 Feb 2010. <>. “How Bacteria Influence Our Environment?.” 18 06 2008. Web. 14 Feb 2010. <http://www.articles- Pickart, Loren. “Toxic Estrogenic Chemical” Skin Biology. Web. 14 Feb 2010. ence-our-environment-454539.html>. <>. “How does sunscreen Work?.” Library of Congress. 12 09 2009. Web. 14 Feb 2010. <http://www.loc. “The Potential Risks of Nanomaterials: a Regov/rr/scitech/mysteries/sunscreen.html>. view Carried Out For ECETOC.” Particle and Fibre Technology. Web. 14 Feb 2010. Kalaughe, Liz. “Nanoparticles Destroy Bacteria.” < NanoTech Web. Web. 14 Feb 2010. <http:// content/3/1/11#IDA21HRC >. Shelter, Gordon. “Fish Kill: Nanosilver Mutates Lowe, Nicholas. “Sunscreens: development, evaluaFish Embryos.” Environmental Health Ortion, and regulatory aspects, Volume 15.” 1996. ganization, Web. 14 Feb 2010. <19http:// w w w. s c i e n t i f i c a m e r i c a n . c o m / a r t i c l e . “Nanoparticles from Sunscreens Damage Microbes.” cfm?id=nanotechnology-silver-nanoparticlesEnvironmental Health Organization, Web. 14 fish-malformation>. 20 http://www.nanowerk. Feb 2010. <http://www.environmentalhealthcom/spotlight/spotid=3045.php>. “Sunscreens: Answers to Your Burning Questions.” Mayo Clinic, Web. 14 Feb 2010. <http://www. “Nanotechnology & Sunscreens.” The Power of>. formation. Environmental Working Group, Web. 14 Feb 2010. < Acknowledgements metics/report/sunscreen09/investigation/Nano- I would like to thank Professor John Bean technology-Sunscreens>. for his inspiration and his commitment to helping me through the research process. I would also like to thank “Nanoparticles Used in Common Household Items my friends and family for their continual support. Cause Genetic Damage in Mice.” Nano Tech Wire. Web. 14 Feb 2010. <>. 43

Efficacy of Adipose-Derived Stromal Cell Homing and Role in Vascular Remodeling of Inflamed Tissue “Adipose derived stem cells are a promising alternative to using bone marrow stem cells to aid vascular remodeling in the clinic.”

Abstract Human adipose derived stromal cells (hASCs) have shown promise in aiding and maintaining angiogenesis in ischemic models and are capable of rescuing an ischemic hindlimb from autoamputation. Hypoxia preconditioning has been suggested as a possible pretreatment to increase the efficacy of hASCs. The goal of this study was to compare the effects of hASCs cultured in normoxia and hypoxia and bone marrow stromal cells (BMSCs) on angiogenesis. Treatment groups received injections of a proinflammatory agent, compound 48/80, prior to injection by one of the cell types or a vehicle control. Mesenteric windows were harvested at three time points and analyzed for vascular density, cell count, and pericyte morphology. Results supported previous findings indicating that hASCs and BMSCs have similar functionality and result in similar bimodal trends of vascular density 10, 30, and 60 days after treatment. They also suggest that hASCs are capable of releasing higher amounts of proangiogenic factors at day 10, which supports previous findings. The second part of this study sought to develop an in vivo model in which to explore different binding mechanisms believed to be involved in hASC homing to inflamed tissue. The mesenteric microvasculature model explored does not allow for flow of hASCs; this is believed to be because of their vascular binding capabilities. We continue to regard hASCs as a potential and accessible stem cell source for promoting angiogenesis, although their homing mechanisms remain unknown.


Carolyn Mulvey

-Carolyn Mulvey

Carolyn Mulvey is a third-year biomedical engineering student from Westfield, N.J. She attended high school at Miss Porter’s School in Farmington, Conn., where she developed her interests in the sciences. At the University of Virginia, she applied to the biomedical engineering department. She joined Dr. Shayn Peirce-Cottler’s lab in March, 2009 where she worked alongside a Ph.D. student at the time, Dr. Peter Amos. She was given responsibility and co-ownership of the work from the day she stepped into the lab and learned many useful techniques and skills from her mentor, Peter. Since his graduation in January, Carolyn has taken full responsibility of the work as it is being wrapped up for publication. She will move forward with successive studies this summer and while completing her capstone and thesis the following year. She hopes to attend medical school following graduation in 2011. Outside of her course work, she is a member of Alpha Phi sorority and she is an engineering school tour guide. She also tutors through the Center for Diversity and she is a member of Tau Beta Pi Engineering Honor Society. She has also spent time volunteering at Martha Jefferson Hospital and for the American Heart Association heart walk.

Stem cell therapy shows promise in treating atherosclerotic disease by promoting angiogenesis. Bone marrow stromal cells (BMSCs) have been used in the clinic, but are obtained in small quantities and through invasive procedures. Human adipose derived stromal cells (hASCs) have been shown to rescue an ischemic hindlimb from autoamputation by secreting angiogenic growth factors and antiapoptotic factors. This discovery sparked greater interest in the use of hASCs in the clinic, as they are accessible in large amounts through minimally invasive procedures. It was also discovered that the use of hypoxic hASC conditioned media increased endothelial cell survival, as compared to normoxic hASC conditioned media (Rehman, 2004). hASCs have been shown to enhance angiogenesis in inflamed tissue. Recent research supports two hypotheses about the role of hASCs in angiogenesis; the first suggests paracrine signaling through the release of proangiogenic factors as the mechanism and the second suggests that hASCs act as pericytes and physically support vessels. Injec-

tion of hASCs into tissue stimulated with compound 48/80, an inflammatory agent, revealed bimodal responses in both cell population and vascular density. Tissues injected with hASCs showed significantly increased cell entrapment and vascular density as compared to tissues injected with human lung fibroblasts, which served as the control. Changes in vascular density were positively correlated with a transient decrease then recovery of the hASC cell population (Amos, Stem Cells, 2008). The initial response supported earlier studies indicating the ability of ASCs to release proangiogenic factors (Miranville, 2004), which results in an enhanced angiogenic response to inflammation. The later response supports the hypothesis that ASCs can adopt a pericyte morphology and play a role in vessel maintenance by physically stabilizing vessels (Benjamin, 1998). Encouraging results indicating the role of hASCs in vascular remodeling led researchers to explore factors that might enhance the entrapment and signaling mechanisms of hASCs in vivo. Greater understanding of homing mechanisms could sug45

gest ways in which scientists could manipulate cells to enhance entrapment prior to therapeutic use. Static adhesion and laminar flow assays were used to assess different binding mechanisms hypothesized to contribute to ASC homing in vivo. Proteins used in the assay were selected based on their roles in cell adhesion, particularly in the endothelium, and in the extracellular matrix in the cells’ native adipose tissue. Results showed that hASCs were able to bind to vascular cell adhesion molecule-1 (VCAM1) and type I collagen during the laminar adhesion assay and to VCAM-1, ICAM-1, type I collagen, and fibronectin in the static adhesion assay. hASCs cultured in hypoxic conditions prior to testing showed increased hASC-binding affinity to VCAM-1 and ICAM-1 (Amos, Annals of Plastic Surgery, 2008) The current study compares the level of angiogenesis in inflamed tissue stimulated by hASCs normal culture conditions (NCC hASCs), HCC hASCs, and BMSCs in inflamed tissue. In the second study, we sought to develop an in vivo model for intra-arterial cell injection to investigate the mechanisms believed to be involved in ASC homing and adhesion.

were washed with buffer 24–48 hours after plating to remove unattached cells, and then resupplied with fresh medium. Plating and expansion medium consisted of Dulbecco’s modified Eagle’s medium (DMEM)/F12a with 10% fetal bovine seruma (FBS) and 1% antibiotic-antimycotica. Cultures were maintained at 37°C with 5% CO2 and media was chanaged three times per week. Human bone marrow stromal cells were obb tained . Briefly, this population was harvested from the posterior iliac crest (25 ml per site) of a 24-yearold Hispanic male (donor no. 1640) and diluted in PBS with heparin (125 U/ml of bone marrow). Samples were then separated using centrifugation and a Ficoll® gradient into mononuclear populations. Mononuclear cells were passaged once and frozen immediately before being shipped. Cells were grown to confluence after the initial plating (passage 0; p = 0), typically within 10–14 days. Adherent cells were released with either 0.5% trypsin-EDTA or Accutase cell detachment mediumc and replated at 2,000 cells/cm2 (p = 1). Cell cultures were passaged every 7–8 days until analysis. All cells used for injection studies were between passage 2 and 4, corresponding to approximately 11 or fewer Materials and Methods total population doublings. One day prior to injecIsolation, Culture, and Labeling of hASCs and tion, cells were labeled with the fluorescent marker BMSCs 1,1-dioetadeeyl-3,3,3’,3’-tetramethylindocarboeya Subcutaneous adipose tissue was obtained nine perchlorate (DiI, 5μM) according to the manufrom a 38-year-old female patient undergoing an facturer’s instructionsd . Cells were rinsed, trypsinelective liposuction procedure in the Department of ized, counted, and resuspended in sterile phosphate Plastic Surgery, University of Virginia, for in vivo buffered solution (PBS) for injection. Furthermore, experiments. The University of Virginia’s Human plated hASCs were labeled with DiI and maintained Investigation Committee approved tissue harvest in culture to confirm that DiI fluorescence did not diprotocols. Adipose tissue came from intraoperative minish visibly over time or with cell division. suction lipectomy. Cells were isolated from adipose tissue us- Hypoxic Preconditioning of hASCs ing methods previously described (Katz, 2005, Zuk, hASCs undergoing hypoxic precondition2001). Briefly, harvested tissue was washed several ing were placed in a Modular Incubator Chambere times and enzymatically dissociated (Katz, 2005, and perfused with 5% CO2, 95% N2 for 20 minutes Katz 2002). Dissociated tissue was filtered to remove to purge the chamber of oxygen. The chamber was debris, and the resulting cell suspension was cen- then sealed and incubated at 37°C for 48 hours. This trifuged. Pelleted stromal cells were recovered and method maintains the cellular microenvironment at washed several times. Contaminating erythrocytes less than 14.9 mm Hg oxygen (less than 2% O2), and were lysed with an osmotic buffer, and the stromal was previously shown to induce hypoxia-inducible cells were plated onto tissue culture plastic. Cultures factor-1alpha (HIF-1alpha) expression in rat pulmo46

nary endothelial cells (Palmer, 1998). Additionally, the presence of a hypoxia was verified in a previous study [9]. Animal Studies Experiments were performed using sterile techniques according to the guidelines of the University of Virginia Animal Care and Use Committee. Two studies of 45 and 39 male nude ratsf weighing 100-225 g were divided into six study groups receiving intraperitoneal (i.p.) injections: (a) hASC injection (1 x 106 cells), (b) hASC injection (1 x 106 cells) and 48/80 stimulation, (c) BMSC injection (1 x 106 cells), (d) BMSC injection (1 x 106 cells) and 48/80 stimulation, (e) hypoxic hASC injection (1 x 106 cells), (f) hypoxic hASC injection (1 x 106 cells) and 48/80 stimulation (g) vehicle control (sterile PBS), and (h) vehicle control and 48/80 stimulation. Stimuation of Microvascular Remodeling with Compound 48/80 and Cell Injection Compound 48/80g (condensation product of N-methyl-p-methoxyphenylethylamine with formaldehyde) is a pharmacological agent known to induce mast cell degranulation. Intraperitoneal injection of compound 48/80 into the rat mesentery stimulates microvascular growth and remodeling in the mesenteric vasculature (Nehls, 1992, Norrby 1986). This small-animal model is a well-established assay for studying angiogenesis (Anderson, 2004, Bocci, 1999). Compound 48/80 was injected into the peritoneum (1 ml/100-g animal weight) in 0.9% sterile NaCl on the first five consecutive study days. Two doses of each concentration (100, 200, and 300 μg/ml) were administered per day separated by 8 hours on the first three days. On day 4, one dose of 400 μg/ml was administered, followed by i.p. injection of 1 ml of sterile PBS containing cells or vehicle with 253⁄8-G, 0.5-inch needles. On day 5, rats in these study groups received a single dose of 500 μg/ml. Harvesting of Mesenteric Tissue Rats were anesthetized with intramuscular injections of ketamine (80 mg/kg body weight [bw]), atropine (0.08 mg/kg bw), and xylazine (8 mg/kg bw). Ten mesenteric windows were harvested from each

animal 10, 30, or 60 days after cell injection. Tissues were whole mounted on gelatin-coated slides. Immunohistochemistry To determine whether injected hASCs expressed markers consistent with a perivascular cell phenotype, tissues were immunostained for an array of markers known to be expressed by smooth muscle cells and pericytes: NG2, smooth muscle alpha-actin (SMA), and PDGF-beta receptor. Tissues were washed in PBS + 0.1% saponin three times for 10 minutes and immunolabeled with lectin from Bandeiraea simplicifolia (BSI-lectin) or Alexa Fluor 647conjugate (1:100; Molecular Probes), and/or antibody to SMA using purified FITC-conjugated clone 1A4 mouse monoclonal anti- SMA (1:500;)g, diluted in PBS buffer containing 0.1% saponin and 2%bovine albuminh at pH 7.4 (incubation for 1 hour at room temperature). Tissues were also stained with perivascular cell markers (Gerhardt, 2003), including antibodies to the following: NG2i (1:150, rabbit polyclonal) and PDGF-beta receptorj (1:100, rabbit polyclonal) AlexaFluor-488 conjugated secondary antibodies were applied for 1 hour at room temperature: NG2 and PDGF-beta receptor goat anti-rabbit IgG. Image Acquisition and Data Analysis Mesenteric tissues were examined with a Nikon Eclipse TE2000-E microscope equipped with confocal accessories (Nikon D-Eclipse C1) using x20 Nikon water/oil immersion and x60 Nikon oil immersion objectivesk. Images were digitized and analyzed using ImageJ ver. 1.37 softwarel. The number of DiIpositive cells per tissue area and total microvessel length were quantified. Pericytes are often defined by their histological and anatomical position in close physical contact with microvascular endothelial cells and morphological shape, which is typically elongated along the ablumenal surface of the microvascular endothelium. Injected hASCs were examined for pericyte-like morphology, defined here as cells whose processes extend along. vessels in a manner that conforms to the curvature of the vessel and whose cell bodies are no more than 5 μm from the abluminal surface of the endothe47

lium. Analysts were blinded to the treatment group during analysis. The “pericyte-like” cell behavior is distinct from smooth muscle cell morphology, which is characterized by wrapping of the smooth muscle cell around the abluminal endothelial surface in a direction perpendicular to the vessel axis and parallel to adjacent smooth muscle cells. Three fields of view (FOVs) were selected from each mesenteric window based on three criteria. First, images were taken of FOVs containing the region of highest vessel density with DiI cells. If there were not three different FOVs containing both cells and vasculature, next, areas of visible cells were imaged. Lastly, regions of the highest vessel density without cells were imaged. Statistical Analysis Results are presented in the form of mean ± standard error. Comparisons for data were made using the statistical analysis tools provided by SigmaPlot 5.0m. Data were tested for normality and analyzed using Analysis of Variance followed by a multiple comparisons test.

In Vivo Rolling Adhesion Assay Nude ratsf were used to develop the injection model and in an attempt to quantify cell rolling and adhesion. Rats received injections of HCC hASCs, NCC hASCs, and BMSCs (200,000 cell/ml PBS). Cell suspensions were injected at a physiological flow rate of 5 ml/min by a Harvard Apparatus PHD2000 syringe pumpn. As a positive control, 10μm G1000 Aqueous Fluorescent Polymer Microsphereso were injected at 200,000 microspheres/ml of both Ringer’s solution and PBS. Injections were also attempted at 0.645 and 0.4 ml/min. Flow through the vasculature in the mesenteric windows was imaged and recorded using a Nikon Eclipse 80i intravital microscope with a 10x objectivek, equipped with an MTI CCD72 camerap during injection.

Results Vascular Density and Cell Counts PBS vehicle groups did not satisfy the first two criteria of image acquisition, as described above, so images were acquired only of the areas of highest vessel density, resulting in higher vascular densities than expected (data not shown). In tissueswithout Mesenteric Artery Cannulation and Inflamma- treatment of 48/80, BMSCs exhibited lower cell retory Response using IL1-beta tention at day 10 than both populations of hASCs (p An in vivo model was developed to investigate < 0.005) (Figure 1). The population decreased by day mechanisms of hASC intravascular homing. Rats 30 and proliferated by day 60. Vascular density folwere anesthetized with intramuscular injections as lowed an inverse trend, with the highest density at day described above. The mesentery was exteriorized and 10 and significantly higher density at day 30 over the the superior mesenteric artery was exposed from the hASC populations (p < 0.05 as compared with HCC fat pad. The artery was ligated at two points between hASCs and p < 0.005 as compared to NCC hASCs) the jujeunal and ileo-colic artery branches and at (Figure 2). An increase in pericyte morphology was side vessels located between the upstream and down- not seen over time (Figure 3). NCC hASCs exhibited stream ligations. The artery was cannulated using bimodal trends in cell population and vascular denpolyethylene tubing (0.59 mm inner diameter) and sity. Cells proliferated by day 60, amounting to the secured with suture between the two ligations. The greatest population at the final time point. Prolifdownstream ligation was removed prior to injection. eration also corresponded to an increase in percent An inflammatory response was induced in of cells displaying pericyte morphology. HCC hASCs mesenteries exteriorized and superfused with 20 ng/ survived as the highest cell population at day 10 with ml of IL-1beta in PBS for a period of 1 hour prior significance (p < 0.005). The population decreased by to cannulation. Upregulation of VCAM-1 and ICAM- day 30 but was still significantly higher than NCC 1 was verified by intra arterial injection of VCAM-1 hASCs and BMSCs (p < 0.005), and continued to deand ICAM-1 antibodies. Images were acquired using crease slightly and was the lowest population at day the Nikon confocal microscope described above. 60. The decreasing HCC hASC cell population was inverse to the vascular density trend. Vascular den48

Figure 1: Cell Retention and Proliferation with no Compound 48/80 Stimulation Over Time. HCC hASCs had the highest cell population at day 10, and both populations of hASCs exhibited significant increase over BMSCs at day 10. NCC hASCs and BMSCs exhibited bimodal trends while HCC hASCs showed a slight decrease in population size from day 30 to 60 (#, p<0.05, *, p<0.005).

Figure 4: Cell Retention and Proliferation with Compound 48/80 Stimulation Over Time. NCC hASCs exhibited a bimodal trend, decreasing by day 30 and proliferating by day 60. HCC hASCs and BMSCs had significantly lower population retention by day 10. HCC hASCs displayed steady growth over the next two time points, while BMSCs greatly proliferated and the cell population peaked at day 30 (#, p<0.05, *, p<0.005).

Figure 2: Mesenteric Vascular Length Density with No Compound 48/80 Stimulation Over Time. BMSCs show significant increase in vascular length density at day 30 but decrease at day 60 relative to both HCC and NCC hASCâ&#x20AC;&#x2122;s (#, p<0.05, *, p<0.005). HCC hASCs exhibit growth over the course of all time points, and NCC hASCs display a bimodal trend.

Figure 5: Mesenteric Vascular Length Density with Compound 48/80 Stimulation Over Time. Cell treatments display similar trends with the Compound 48/80 inflammatory stimulus. HCC hASCs and BMSCs show vascular regression between days 10 and 30 and growth between days 30 and 60. NCC hASCs resulted in a lower vascular density and day 10 and growth over the remainder of the time course.

Figure 3: Cells Displaying Pericyte-Like Morphology. BMSCs exhibited a steady decrease in pericyte morphologies, while hASC populations increased in observed pericyte morphologies. These trends paralleled overall trends in vascular density (#, p<0.05;*,p<0.005).

Figure 6: Upregulation of VCAM with IL1-beta Stimulus. Il1-beta was dripped onto exposed mesenteric windows for a period of one hour. This resulted in an inflammatory response and upregulation of VCAM-1 as compared to control windows.


ure 7 depicts HCC hASCs having adopted a pericyte morphology. In Vivo Rolling Model The IL1-beta inflammatory stimulus was verified with injection of VCAM-1 antibody. As seen in Figure 8, VCAM-1 was noticeably upregulated over the one-hour inflammatory stimulus. Upon development of the surgical model, 3035 nude rats were used in attempts to collect rolling and adhesion data. In many of the surgeries, momentary flow of fluorescent cells was observed, but only two surgeries generated sufficient observation times for data collection. Five surgeries failed due to improper surgical techniques and nicking of surrounding vessels, which resulted in bleeding out. The majority of the remaining surgeries failed because injections leaked from the vessels. Very few of these Figure 7: HCC hASCs expressing pericyte morphologies. At day 60, HCC hASCs are seen associating within 5 micrometers and leakages occurred at the site of cannulation (1-3). spanning vessels. These images also show high vascular densities Failure occurred either after observing brief flow 60 days after stimulus with 48/80 and cell treatment. through vessels or in mesenteries in which no flow was observed. In the animals in which no flow was sity at day 10 was the lowest, and it increased over observed, it was assumed that vessels were shut the following two time points. This corresponds with shutting down during exposure time and tissue hana steady increase in observed pericyte morphology. dling during the surgery and that this might have Treatment with compound 48/80 resulted in been contributing to the bursting of vessels. In those significantly different trends. NCC hASCs exhibited in which we saw initial flow before leakage, several the highest population at day 10 with significance (p adjustments were made to the protocol in an attempt < 0.005) and again followed a bimodal trend, decreasing by day 30 and proliferating by day 60 (Figure 4). Vascular density exhibited a steady increase across all time points, which corresponded with a steady increase in observed pericyte morphology (Figures 5 and 6). The population of HCC hASCs increased across all time points, beginning from the lowest population and ending with the highest. Vascular density displayed a bimodal trend, which paralleled the percent of cells displaying pericyte morphology. HCC hASCs displaying pericyte morphology are shown in Figure 7. BMSCs exhibited significant proliferation from day 10 to 30, and had a significantly greater population than hASCs (p < 0.005). This was Figure 8: Upregulation of VCAM with IL1-beta Stimulus. Il1-beta was dripped onto exposed mesenteric windows for a period of one followed by a decrease in population by day 60. Vashour. This resulted in an inflammatory response and upregulation cular density was inversely related to the changes in of VCAM-1 as compared to control windows. cell population, which decreased by day 30 and increased from day 30 to 60. This trend paralleled the percent of cells displaying pericyte morphology. Fig50

to determine the point of failure. The infusion rate was lowered from 5 ml/ min to 0.645 ml/min; which was the flow rate used in laminar adhesion assays, and further to 0.4 ml/ min [8]. Cell suspensions in PBS resulted in leakages at all three injections rates. Choice of an injection solution was also examined. 10μm G1000 Aqueous Fluorescent Polymer Microspheres were used as a positive control when comparing the effects of PBS to Ringer’s solution, a solution with physiological ion concentrations, predicted to maintain homeostasis within the vessels. The injected microspheres emit green light, but both red and green-emitting microspheres were imaged using corresponding filters in order to confirm comparable imaging of cells and microspheres. When 3.75 ml of a 200,000 microspheres/ ml Ringer’s solution was injected through the arterial cannula, vessels maintained barrier function and no leakage was observed as microspheres were seen flowing through the microvasculature. The solution was then switched to a 200,000 microspheres/ml solution in PBS, which caused immediate leakage. hASCs suspended at 200,000 cell/ml in Ringer’s solution were injected into vasculature in which flow was observed. Upon injection at 0.4 ml/min, flow stopped in all vessels. Flow was still not seen when the infusion rate was increased to 0.645 ml/min. A more dilute suspension of hASCs (100,000 cells/ml) was injected into another animal, yielding the same results. Harvesting of mesenteric windows from a successful surgery, in which > 1ml of the BMSCs suspended in PBS were injected prior to observing leakage, revealed cells that spanned the inner diameters of vessels, as seen in Figure 9. Some cells even appeared to be simultaneously directed into two branches and became stuck.

Figure 9: BMSC Blockages Post Intra Arterial Injection. After injection in PBS, BMSCs are attributed to stopping flow by causing blockages in the microvasculature. Some cells appear to be stuck at the intersection of two branches, and all cells expand the entire vessel diameter.

cell treatment groups. NCC hASCs closely followed predicted trends seen in the previous study of vascular remodeling with and without stimulation with 48/80 (Rehman, 2004). We believe that the decrease in vascular density seen between days 10 and 30 without the inflammatory stimulus is due to the changing morphology of hASCs. It is believed that the initial mechanism of vascular remodeling is by proangiogenic secretions. By day 30, the cells change from a role of paracrine signaling cells and begin adopting a pericyte morphology, and they appear to play a smaller role in vascular remodeling during this change. The cell population also declined over this period, before indicating proliferation by day 60, during which an increase in pericyte morphology was seen. This data supports the theory of the second mechanism of the Discussion hASC role in vascular remodeling, which is that they Vascular Density and Cell Counts Imaging of vehicle control groups signifi- stabilize vascular and potentiate growth by taking cantly differed from cell treatment groups because on a pericyte morphology. HCC hASCs resulted in a of the FOV selection criteria; this resulted in data lower initial cell population after stimulus with comthat was skewed high for the vehicle control. Thus we pound 48/80. Cells were not introduced to 48/80 after concluded that the cell treatment groups could not treatment with hypoxia in order to explore the efbe directly compared to the vehicle group. Data col- fects; an adverse reaction to compound 48/80 may be lected in this experiment was used only to compare responsible for why HCC hASCs do not populate well 51

by day 10. Cells that did populate exhibited proliferation over the next two times points and exhibited a high percentage of pericyte morphologies by day 60; they exhibited the highest growth rate and percentage of pericyte morphologies. This corresponded with increased vascular density, which was suggested by predicted mechanisms. The population of BMSCs exhibited a trend that was not predicted, when the population decreased between days 30 and 60, suggested that they may not survive as well as hASCs over time. Overall, all three cell populations resulted in comparable vascular growth and increase in pericyte morphologies, suggesting equivalent efficacy as an angiogenic therapy. This supports previous studies comparing BMSC and hASC functionality. Studies have shown comparable morphologies and effects on vascular remodeling in ischemic tissue between BMSCs and hASC populations. hASCs have been found to incorporate into the vasculature and increase vascular endothelial growth factor secretions, angiogenesis, and cutaneous blood flow in an ischemic hindlimb model. Studies have concluded that hASCs are a viable source of neovascular progenitor cells, similar to BMSCs in clinical use (Miranville, 2004, Planat-Benard, 2004). Our results, which were generated by directly comparing cell populations with angiogenesis, further indicate comparable functionality and therapeutic benefits of BMSCs and hASCs. Without the inflammatory stimulus, BMSCs appear to exhibit a short-term effect on vascular density, which increased by day 30 and decreased by day 60. The decrease in vascular density corresponded with a decrease in percent of cells showing pericyte morphologies and was likely because the cells did not play a role in long-term vascular stabilization and maintenance. This is contrasted to the BMSC response with 48/80, in which the cell population increased by day 60 but a decrease in vascular density corresponded to a decrease in pericyte morphologies. This suggests that the pericyte morphology is integral to vascular maintenance. The overall decrease in vascular density and pericyte morphologies is contrasted to increases in both of the hASC populations. These results suggest that different cell populations exhibit different long term effects in tissues with and 52

without the 48/80 stimulus, and the BMSCs are less effective in tissues not exhibiting the long term effects post-inflammation, while hASCs support long term vascular remodeling in both cases. Different cell populations used in the experiment may have caused variations in the trends seen in hASCs as compared to previous findings, such as the lack of bimodal vascular density caused by NCC hASCs with 48/80 stimulus. The previous experiment used hASCs harvested from both liposuction procedures as well as paniculectomies. Data from in vitro rolling assays indicated that the two hASC populations have different functionalities, and this is a plausible explanation for variance seen here. In Vivo Rolling Model The majority of cannulation surgeries failed due to leakage during injection of cells. We explored three hypotheses for this phenomenon; the first was the bursting due to high pressure caused by the infusion flow rate, the second was the loss of vascular patency and the third was small vessel blockages caused by cell size or adhesion. Lowering the infusion flow rate to 0.645 ml/min and 0.4 ml/min did not reduce the incidence of cell injection leakages. Injection of microspheres at these flow rates did not result in leakages, suggesting that the pressure caused by infusion rate was not causative. We have concluded that the use of Ringer’s solution is preferable over PBS for intra-arterial injections. Injection of microspheres in Ringer’s allowed vessels to maintain patency, while injection in PBS caused immediate loss of patency. We suspect that a contributing factor to the improved homeostasis of Ringer’s solution over PBS is due to the presence of bicarbonate buffering. However, using Ringer’s for cell injections still resulted in leakages, indicating another contributing factor. Successful injection of microspheres led us to conclude that cell size was not causing blockages. The microspheres used were 10 μm in diameter and essentially rigid. hASCs in suspension are 11-15 μm in diameter and can flex and deform to fit through small vessels. Furthermore, decreasing the concentration of injected cells did not improve blockages. Conclusions drawn about injection rate, solution, cell size, and concentration, as well as results depicting

cells stuck in the vasculature, have led us to believe References that the greatest difference between the successful 1. Rehman J, Traktuev D, Li J et al. (2004). Secretion injection of microspheres and the leakages caused of angiogenic and antiapoptotic factors by by cell injections is due to their adhesive capabilities. human adipose stromal cells. Circulation, 109, We conclude that the binding capabilities of hASCs r52–r58. are too high to allow for travel through the microvasculature in the rat mesentery model and that they 2. Amos, P, Shang, Hulan, Bailey, A et al. (2008). The are adhering and causing blockages, thereby arrestrole of human adipose-derived stromal cells in ing flow. inflammatory microvascular remodeling and evidence of a perivascular phenotype. Stem Conclusion Cells. 26, 2682-2690. Vascular density results after stimulation with 48/80 indicated that hASCs exhibit similar efficacy 3. Miranville A, Heeschen C, Sengenes C et al. (2004). to BMSCs in vascular remodeling of ischemic tissue. Improvement of postnatal neovascularization HCC hASCs may exhibit higher secretion of angioby human adipose tissue-derived stem cells. genic factors at day 10, as compared to NCC hASCs, Circulation, 110, 349 355. contributing to a more dense vascular network; particularly, HCC hASCs had a much higher vascular 4. Benjamin LE, Hemo I, Keshet E. (1998). A plasdensity per cell at day 10, after stimulation with ticity window for blood vessel remodeling is compound 48/80. This supports previous findings defined by pericyte coverage of the preformed indicating a possible therapeutic benefit of hypoxic endothelial network and is regulated by PDGFculture conditions, which should be further explored. B and VEGF. Development, 125, 1591–1598. The initial low HCC hASC retention may have been caused by a negative reaction to 48/80 stimulus; the 5. Katz AJ, Tholpady A, Tholpady S et al. (2005). cells should be tested for effects of exposure. These Cell surface and transcriptional characterizaresults show that hASCs should continue to be tested tion of human adipose-derived adherent strofor use in the clinic, as they show promise of benefits mal (hADAS) cells. Stem Cells, 23, 412- 423. similar to BMSCs. hASCs would provide a less inva- 6. Zuk PA, Zhu M, Mizuno H et al. Multilineage sive, safer, and more accessible and abundant supply cells from human adipose tissue: Implicaof proangiogenic stromal cells for clinical use, potentions for cell based therapies. Tissue Eng tially available for the treatment of vascular diseases 2001;7:211–227. including atherosclerosis and peripheral arterial disease. 7. Katz, AJ. (2002). Mesenchymal Cell Culture: Adi Use of Ringer’s solution rather than PBS pose Tissue. In: Atala A, Lanza RP, eds. Methfor cell suspension proved to be a potential solution ods of Tissue Engineering. Burlington, MA: to the loss of barrier function during intra-arterial Academic Press, 277–286. injection. We believe that cellular adhesion is also to blame for the arrest of flow in this microvascular 8. Palmer LA, Semenza GL, Stoler MH, et al. (1998). model. Future work could involve development of a Hypoxia induces type II NOS gene expression surgical model for in vivo studies in a larger network. in pulmonary artery endothelial cells via HIFRolling and adhesion assays might be possible in the 1. Am J Physiol, 274, L212–219. femoral artery in a mouse model, which can also be visualized on an intravital microscope. 9. Amos, P, Bailey, A, Shang, Hulan, et al. (2008). Functional binding of human adipose-derived stromal cells: effects of extraction method and hypoxia treatment. Annals of Plastic S urgery, 53

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h. Fisher Scientific, Hampton, NH, i. Chemicon Int., Billerica, MA, j. Santa Cruz Biotechnology Inc., Santa Cruz, California, k. Nikon, Melville, NY, l. National Institutes of Health, Bethesda, MD, m. Systat Software, Inc., Chicago, http://www.systat. com/ n. Harvard Apparatus, Holliston, MA, http://www. o. Thermo Fisher Scientific, Waltham, MA, http:// p. Dage-MTI, Michigan City, IN,

Experimental Supplies Acknowledgements a. Invitrogen, Carlsbad, CA, http://www.invitrogen. This work was funded by the NIH grant com 1R21HL091312-01 (Peirce-Cottler). The author would like to thank Dr. Peter Amos for his leadership and guidb. STEMCELL Technologies, Inc., Vancouver, BC, ance throughout both experiments and Dr. Shayn PeirceCanada, Cottler for her mentorship. She would also like to thank Dr. Ji Song for her help with the intra arterial studies, c. Innovative Cell Technologies, San Diego, http:// Bryan Thorne for help with imaging the intra arterial studies, and Dr. Richard Price for supplying the microspheres used in the intra arterial studies. d. Molecular Probes, Eugene, OR, 54

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Acknowledgements Faculty Advisory Board

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The Spectra

Volume I, Issue 1 Spring 2010

Ian Czekala Ashley Keller

Ami Patel Katelyn Mason Haarthi Sadasivam Carolyn Mulvey


Collaborative Engineering of the Atacama Large Millimeter Array (ALMA) Expression and Purification of Inclusion Membrane Protein A (IncA) from Chlamydia trachomatis using Dual-Detergent System and Secondary Structural Determination using Circular Dichroism Spectroscopy High Power Laser Texturing of Surfaces for Aerospace Applications Role of EphrinB2 Reverse Signaling in Pathological Retinal Neovascularization The Shady Side of Sunscreen Efficacy of Adipose-Derived Stromal Cell Homing and Role in Vascular Remodeling of Inflamed Tissue

The Spectra Research Journal