2025 Journal for Undergraduate Research Opportunities

The Journal for Undergraduate Research Opportunities

University of Georgia

Center for Undergraduate Research Opportunities

University of Georgia undergraduate students present their research as part of the CURO Research Symposium on April 7-8, 2025. The Symposium featured 723 student presenters.

Letter from Honors Leadership

Dr. Margaret A. Amstutz, Dean, Jere W. Morehead Honors College

Dr. James N. Warnock, Associate Dean, Jere W. Morehead Honors College

Letter from the Editor

John Widener Norris, Editor-in-Chief

Editorial Board: Anna Grace Aiello, Sophia Ying Mei Beasley, Suchi Iyer, Ria Yesare, Hanif Zaman

About CURO

The Center for Undergraduate Research Opportunities

Life Sciences

Dynamics of Shrub Co-Invasion, Nutrient Cycling, and AM Fungal Abundance in a Southeastern U.S. Forest

Isabella Pellicano, Dr. Nina Wurzburger (mentor)

Public Service and Outreach

Analysis of the Opioid Crisis in Georgia: The Effect of COVID-19 and Urban-Rural Trends

Aarov Malhorta, James Byars (mentor)

Public and International Affairs

Brazil’s Humanitarian Visa Program for Haitian Migrants: A Critical Analysis of Policy Gaps, Regional Disparities, and Integration Challenges

Raquel Beatriz Caldas Laranjeira, Dr. Erin Little (mentor)

Social Sciences

The Moderating Role of Pubertal Timing on Perceived Parental Control and Internalizing Symptomology

Ashlyn Kingsley, Dr. Charles Geier (mentor), Dr. Assaf Oshri (mentor)

Technology, Engineering, and Mathematics

Development of a Novel Piezoelectric Model to Study Breast Cancer Bone Metastisis

Erika Landree, Dr. Cheryl Gomillion (co-author)

Special Thanks

Morehead Honors College Staff

Dear Reader,

The Center for Undergraduate Research Opportunities is pleased to present outstanding University of Georgia student work in this edition of JURO, the Journal for Undergraduate Research Opportunities. Its featured papers are a testament to the range and depth of research activity on campus.

For almost three decades, CURO has helped thousands of curious undergraduate students connect with the research mission of the institution and later carry their passion, energy and expertise into their chosen professional fields. Today, through the dedicated work of Andrea Silletti, CURO program coordinator, UGA students continue to engage with faculty across campus in critical research projects.

This publication would not be possible without the strong mentorship and support of UGA faculty and the talented students on the JURO editorial staff—thank you. We would also like to thank the faculty who assisted in reviewing these papers as well as the many donors who allow us to offer CURO activities to all UGA students.

We hope you enjoy witnessing the bright minds at work in these pages!

Sincerely,

Maragaret A. Amstutz Dean, Morehead Honors College

James Warnock Associate Dean, Morehead Honors College

Thank you to our faculty reviewers for the CURO Best Paper

Awards

Michael Adams, Distinguished Research Professor, Biochemistry & Molecular Biology, Franklin College of Arts & Sciences

Nicholas Allen, Baldwin Professor in Humanities and Director of the Wilson Center for Humanities & Arts

Mark Farmer, Professor of Cellular Biology, Franklin College of Arts & Sciences

Ted Furtis, Georgia Athletics Association Professor in Family & Consumer Sciences, College of Family & Consumer Sciences

Cheryl Gomillion, Associate Professor, College of Engineering

Susan B. Haire, Professor of Political Science & Head of the Department of Political Science, School of Public & International Affairs

Neil Lyall, Professor of Mathematics & Associate Dean, Franklin College of Arts & Sciences

Jean Martin-Williams, Meigs Distinguished Professor in the Hugh Hodgson School of Music, Franklin College of Arts & Sciences

Charlotte Mason, C. Herman & Mary Virginia Terry Professor of Business Administration, Terry College of Business

Susan Sanchez, Professor, Assistant Director of the Biomedical Health Sciences Institute, Chair of One Health

Andrea Silletti, CURO Program Coordinator, Morehead Honors College

James Warnock, Associate Dean, Morehead Honors College

Craig Weigert, Associate Department Head, Department of Physics, Franklin College of Arts & Sciences

Dear Reader,

It is my pleasure to present the fall 2025 edition of the Journal for Undergraduate Research Opportunities. University of Georgia faculty marked the papers submitted for consideration, after which the student editorial board selected manuscripts for publication. This competitive process underscores the merits of the authors whose work is contained in the following pages. My hope is that you will consider how each domain represented here offers meaningful contributions to our shared pursuit of truth. I encourage you to take this opportunity to read widely and to learn how various fields approach some of today’s pressing questions. This edition would not be possible without the work, talent, and tenacity of the 2025 JURO editorial team. We as a board are indebted to Dean Meg Amstutz, Dr. James Warnock, Ms. Andrea Silletti, and Ms. Stephanie Schupska for their invaluable support throughout this year-long publication process. We hope this edition not only conveys the extent of our university’s undergraduate research community but also serves as an impetus for students to consider what questions they might want to explore during their time at UGA.

Sincerely,

J. Widener Norris Editor-In-Chief

Anna Grace Aiello Chair of Design & Formatting

Sophia Beasley Chair of Communications

Suchi Iyer Chair of Public Outreach

Hanif Zaman Chair of Papers

Ria Yesare Design & Formatting Assistant

CURO

THE CENTER FOR UNDERGRADUATE RESEARCH OPPORTUNITIES

The Center for Undergraduate Research Opportunities (CURO) facilitates sustained, progressive, faculty-mentored undergraduate research during any year of undergraduate study at the University of Georgia, including students’ first semester on campus. Research can be conducted in any discipline, through any major, and with any GPA.

Through CURO, students can:

• Pursue a faculty-mentored research project and earn course credit hours that count towards degree completion

• Gain access to presentation and funding opportunities

• Form a mentoring relationship focused on conducting research and professional development

• Develop a deeper understanding of your chosen field by working closely with a faculty mentor

During the 2024 academic year, 2100 UGA undergraduates enrolled in “R-suffix” research-focused courses, and 723 presented at the annual CURO Symposium held in April.

FROM A MENTOR’S PERSPECTIVE

Professors who mentored this year’s authors reflect on the collaborative process of undergraduate research

Dr. Nina Wurzburger is an associate professor in the Odum School of Ecology. Her research focuses on plant-soil relationships, ecosystem ecology, and plant root symbioses. She served as a research mentor and Principal Investigator for this year’s author Isabella Pellicano.

What originally inspired you to pursue your field of research, and how do you share that passion with your students?

My favorite course as an undergraduate was “Trees and Forests” taught by Dr. Bledsoe. It was in this course that I first learned about the fungal-root symbiosis called mycorrhiza. Dr. Bledsoe ended her lectures with a list of unanswered questions about the role of mycorrhizal fungi in forests. I was so intrigued that I asked Dr. Bledsoe if I could conduct research in her lab. Thankfully, she said yes and I’ve been conducting research on forest ecosystems ever since. I try to emulate Dr. Bledsoe’s teaching style by conveying my enthusiasm and by highlighting the pressing questions that challenge ecologists.

What skills or qualities make a student researcher stand out?

I think there are two qualities that make a student researcher stand out: curiosity and determination. Intellectual curiosity helps a student generate new questions, which then motivates experiments and data analysis. Then, determination is necessary for remaining focused, overcoming obstacles, and ultimately, bringing a project to completion.

How do you help students connect their research experience to their academic or career goals?

I work with each student to tailor their project to their interests and broader goals. As a result, each student’s project has a unique emphasis on lab skills, experimentation, data analysis or literature synthesis.

FROM A MENTOR’S PERSPECTIVE

Professors who mentored this year’s authors reflect on the collaborative process of undergraduate research

Dr. Cheryl T. Gomillion is an associate professor in the College of Engineering. Her research focuses on tissue engineering and regenerative medicine. She served as a research mentor and Principal Investigator for this year’s author Erika Landree.

Can you share a memorable moment/accomplishment from mentoring undergraduate researchers?

There are so many that I can highlight. Since beginning my lab, I’ve mentored over 40 undergraduate researchers through CURO and other programs. It’s been exciting to see the simple things like their excitement when an experiment works or when something they’ve learned about in class is directly applicable to what they’re doing in lab. Even though it’s always bittersweet when students move on, I’ve been most excited when they reach the stage of leaving lab and moving on to the next thing. Many have gone to medical school and are doctors now. I’ve had others that went to graduate school and now hold master’s and doctorate degrees. I recently had a full circle moment where a student that began in my lab as an undergrad continued as a Ph.D. student in the lab and just finished her degree. She is now on to a postdoctoral fellow position and I’m excited to see what follows. Being able to see the evolution and maturity that students experience through mentored research experiences is what I find most rewarding.

LIFE SCIENCES

Class of 2026

Isabella Pellicano is a fourth-year student studying plant biology, ecology, and Spanish. She is interested in how global change influences plant-mycorrhizal interactions and soil biogeochemistry. Isabella has served as a nature program instructor, a student researcher with the Demarche and Wurzburger Labs, and an REU intern at the Kellogg Biological Station. After graduation, she hopes to teach English abroad before pursuing a PhD in ecology.

Isabella Pellicano

DYNAMICS OF SHRUB CO-INVASION, NUTRIENT CYCLING, AND AM FUNGAL ABUNDANCE IN A SOUTHEASTERN U.S. FOREST

ISABELLA PELLICANO DR. NINA WURZBURGER

Abstract

Invasive shrubs vary in their functional traits, such as nitrogen (N) fixation, and may differentially impact soil dynamics. Further, co-occurrence of multiple invasive shrubs may magnify or otherwise complexify changes to soil processes; however, this relationship is not thoroughly understood. In this study, I investigate how co-invasion of Autumn Olive (E. umbellata), an N-fixer, and Chinese Privet (L. sinense), a non-fixer, influence dissolved inorganic nitrogen content (DIN), extracellular enzyme activity associated with nitrogen and phosphorus availability (NAG, LAP, AP), and arbuscular mycorrhizal fungal (AMF) spore abundance. I quantified these variables by collecting soil from a Southeastern US forest where shrubs naturally occurred in mixed plots and single species plots. Total DIN and LAP were higher in mixed plots compared to E. umbellata-only plots, and AP was positively correlated with DIN across treatments. These results suggest that mixed plots are relatively N-rich compared to single species plots and may experience P-limitation. AMF spore abundance did not vary with treatment or sampling distance, however NAG decreased with increasing AMF spore abundance across treatments. The stated relationships, or lack thereof, between spore abundance and enzyme activity may be the result of drought stress. Further, the lack of expected trends between measured variables might be clarified by investigating the impact of invasive shrubs on soil pH. Future studies should take an experimental approach to investigate how E. umbellata and L. sinense influence soil pH, total N, and, most importantly, how these changes may influence ecosystem health and native plant success.

I. Introduction

Invasive plants are pervasive in the southeastern United States and may impact the diversity of native species, nutrient cycling, soil chemistry, and soil microbial communities (Miller et al.). Some invasive plant species have actinorhizal associations with N2-fixing bacteria and are known as N-fixing species. This trait likely improves their competitiveness and may influence other soil organisms, such as fungi, and neighboring plants by increasing the concentration of bioavailable nitrogen in the

soil (Kuebbing et al., “Two co-occurring invasive woody shrubs”; Scherer-Lorenzen et al.). Further, when invasive plant species co-occur, they may reciprocally or unidirectionally influence each other’s success, though the means through which this occurs is not well understood (Kuebbing et al., “Co-occurring nonnative woody shrubs”). Better understanding how multi-species co-occurrence impacts soil properties is essential as plant invader species rarely exist independently in nature and negative effects may be amplified by the presence of several species (Kuebbing et al., “Two co-oc-

curring invasive woody shrubs”).

Elaeagnus umbellata, commonly known as Autumn Olive, is a highly invasive, nitrogen-fixing shrub that grows particularly well in nutrient-poor, disturbed soils and may impact nutrient cycling and microbial communities (Malinich et al.; Goldstein et al.). It is unclear whether these soil-level impacts serve as a mechanism for invasion or are rather a side effect of invasion (Malinich et al.). Further, how other, non-fixing invasive shrubs benefit from, magnify, or otherwise respond to these shifts is unclear. Malinich and colleagues have suggested that the impact of E. umbellata on the surrounding microbial community depends on the community’s proximity to the shrub, which may characterize the impact of E. umbellata on neighboring co-invaders (Malinich et al.).

Another problematic invasive shrub found in the southeastern United States is Ligustrum sinense, commonly known as Chinese Privet, which is often found in wetlands, floodplains, and other open habitats with a history of land disturbance (Mitchell et al.). Unlike E. umbellata, L. sinense does not fix atmospheric nitrogen but may influence soil inorganic nitrogen levels through decomposition and stimulation of N-mineralization (Mitchell et al.). Further, Kuebbing and colleagues suggest that L. sinense has non-additive effects, underscoring the importance of studying this shrub in communities with other non-native plants (Kuebbing et al. “Two co-occurring invasive woody shrubs; Cash et al).

Both E. umbellata and L. sinense associate with arbuscular mycorrhizal fungi, or AMF. AMF form mutualistic interactions with host plants through which plants receive nutrients, especially phosphorus, via fungal root hyphae, and fungi receive plant carbon (Hawkins et al.). This association is particularly beneficial for plants under nutrient-limited scenarios. E. umbellata, an N-fixer, may increase soil nitro-

gen and thereby become phosphorus (P)-limited, as soil nitrogen is abundant but available P is insufficient. This P-limitation could lead to higher mycorrhizal dependence and increased AMF presence, but this relationship is not thoroughly understood (Shannon et al.). Investigating extracellular enzyme production could clarify this relationship. Fungi, among other microbes, produce a range of extracellular enzymes to break down macromolecules and increase nutrient availability for plant hosts (Bell et al.). Enzymes differ in the substances they degrade, and therefore in the nutrients they make available. It is largely accepted that extracellular enzyme activity is inversely related to nutrient availability, with microbes investing fewer resources in degrading already abundant nutrients (Sinsabaugh and Moorhead, “Resource allocation to extracellular enzyme production”). Therefore, quantifying activity of various enzymes in soil where invaders grow could provide insight regarding nutrient cycling effects.

The functional differences, overlapping niches, AMF associations, and varied proposed impacts of E. umbellata and L. sinense on neighboring plants, soil microbial communities, and nutrient cycling make these shrubs ideal candidates for investigating the impacts of plant co-invasion on ecosystem processes. To investigate relationships between E. umbellata, L. sinense, and soil traits, I conducted an observational study in a temperate forest in a southeastern US where the shrubs naturally coexisted and occurred independently, during which I collected data on soil inorganic nitrogen concentration, extracellular enzyme activity, and spore abundance.

In light of the above, I hypothesize that: 1) inorganic nitrogen concentration will be highest in E. umbellata only plots, followed by plots with both shrub species, then the L. sinense only plots; 2) N-degrading enzyme activity

(NAG, LAP) will be lowest in E. umbellata only plots, followed by the mixed plots, then the L. sinense only plots, and that P-acquiring enzyme (AP) will follow the opposite trend due to P-limitation; and 3) AMF spore abundance will be higher in mixed plots compared to single species plots.

Co-invasion, N-fixation, and fungal symbioses are important considerations in establishing a holistic knowledge of how invasive plant species impact soil biogeochemistry. Understanding these processes will help inform conservation efforts and promote native biodiversity. For instance, land managers could prioritize removing the invasive plant species with the most severe impact on soil microbial communities or nutrient cycling. Efficient practices like these can maximize benefits for native soil and plant communities.

II. Methods

A. Study Site & Soil Sampling

I collected soil samples in September 2024 from Whitehall Forest in Athens, Georgia with permission from the University of Georgia Warnell School of Forestry and Natural Resources. Whitehall Forest covers approximately 840 acres and includes managed, pine timber plantations as well as unmanaged areas where invasive shrubs dominate. I located an unmanaged site in the southwest portion of the property, east of the Middle Oconee River, with established populations of E. umbellata and L. sinense growing in close proximity to each other. Within this area of overlap, I established three spatial blocks where both species were present: block 1 was located in a slight depression southwest of a gravel road, block 2 was along the border of managed pine growth and unmanaged forest, and block 3 was in a depression sloping down to the northeast of the gravel road. All three blocks were located at around 190 meters elevation (Fig. 1).

In each block, I established three 2 m x 2 m treatment plots: one plot with E. umbellata only (EU), one with L. sinense only (LS), and one mixed plot with both shrubs present (BO), for a total of 9 plots across 3 blocks. Spatial blocks were separated by approximately 100 meters and plots within blocks were separated by 3 meters, when possible; however, at times I was constrained by the natural arrangement of shrubs across the landscape.

I used a trowel to collect soil at 0 cm to 10 cm depth, as drought conditions made using a soil probe challenging. In each plot, I collected soil from 3 radii, distanced 20 cm, 50 cm, and 70

2: Soil sampling schematic for a single plot. cm, from a central shrub (Fig. 2).

The central shrub was E. umbellata in EU and BO plots, and L. sinense in LS plots. Soils from the three distances were kept separate for analyses and tools were sterilized between samples. I sampled blocks 1 and 2 during a drought

Figure 1: Map displaying sampling locations in Whitehall Forest with spatial blocks shown in colored squares (Google Earth, earth.google.com/web/)

Figure

prior to Hurricane Helene, and sampled block 3 following the storm. I collected a total of 27 soil samples (3 blocks, 3 plots per block, 3 distances per plot).

B. Study Site & Soil Sampling

I adapted the Wurzburger Lab Protocol described in Tierney et al. to quantify inorganic nitrogen. Immediately following field collection, I returned to the lab and added 10 g of field-moist soil to bottles with 50 mL of 2M KCl. I shook samples vigorously for 15 seconds, then placed them in a shaker at 150 RPM for 3.5 hours. I allowed shaken samples to settle for 1 hour, then filtered the supernatant through Whatman #42 filter paper and froze extracts for ammonia and nitrate analyses. I expressed DIN data in ppm (mg N/kg soil). I simultaneously dried portions of soil for gravitational water content calculations and stored remaining soil at either 5°C for later spore extraction or -40°C for later enzyme assays.

C. Fluorometric Enzyme Assays

I used a protocol modified from Bell et al. by the Wurzburger Lab to quantify three microbial extracellular enzymes: leucine aminopeptidase (LAP) and β-N-acetylglucosaminidase (NAG), which influence nitrogen acquisition through degradation of peptides/amino acids and chitin, respectively, and acid phosphatase (AP), which influences phosphorus acquisition by hydrolyzing organic phosphorus to inorganic phosphates (Sinsabaugh et al., “Ecoenzymatic stoichiometry of microbial”). I adjusted pH of the sodium acetate buffer to 5, the pH typical of soils at Whitehall Forest. I expressed enzyme activity in μmol/g dry soil/hour.

D. Spore Extraction:

Wet Sieve & Decant Method

I modified the Chung Lab spore extraction protocol, based on Walker et al., in order to quantify spore abundance rather than morphotype diversity. I blended 50-100g of soil with

distilled water for 10 seconds, filtered through 500 μm, 53 μm, and 38 μm sieves, and collected the remaining soil particles (containing spores) from the two smaller sieves into 50 mL falcon tubes. After adding 25 mL of 480 g/L sucrose solution to each tube, samples were centrifuged for 3 minutes at 1500 RPM. I then decanted the supernatant through the 38 μm sieve and rinsed with distilled water. I collected the remaining material into a clean falcon tube and pipetted a 500 μL subsample into a petri dish for spore counting under a dissecting scope. I used lined paper to aid in counting and averaged two counts, one at low and the other at high magnification. I expressed spore abundance as spore counts/gram dry soil.

E. Statistical Analyses

I analyzed data using lme4, lmerTest, and emmeans R packages to run analysis of variance on linear mixed effects models on dissolved inorganic nitrogen, enzyme concentration, and spore abundance data, holding block as a random effect. I used linear models to investigate possible relationships between my three variables of interest. I used a significance level of p < 0.10 for all analyses.

III. Results

A. Dissolved Inorganic Nitrogen

Dissolved inorganic nitrogen concentrations were significantly different among treatments, but not among sampling distances. Total DIN differed among treatments (F2,16=6.2; p=0.0099, Fig. 2A) where it was significantly higher in mixed plots compared to E. umbellata-only plots. This difference is attributed to differences in ammonium concentration (F2,16=6.2; p=0.0101, Fig. 3B), as nitrate concentration did not differ by treatment. DIN was low overall across treatments with values ranging from 0.961 to 6.69 ppm in dry soil (Fig. 3A).

B. Enzyme Activity

Extracellular enzyme activity varied by treatment for LAP only, and only AP activity was related to DIN. Enzyme activities did not significantly vary among sampling distances. LAP activity was significantly different among treatments (F2,16 = 3.3; p= 0.097); it was higher in plots with both shrubs than in E. umbellata-only plots (Fig. 4). LAP and NAG were not correlated with DIN (Fig. 5B/C); however, AP activity showed a significant positive correlation with DIN across treatments (F1,25 = 5.7; p= 0.025; R2=0.15, Fig. 5A).

C. AMF Spore Abundance

Spore abundance (spores per gram dry soil) did not vary significantly by treatment nor by distance. However, in E. umbellata-only plots, spore abundances appear to decrease with increasing distance from the central E. umbellata shrub (Fig. 6). Across all treatments, NAG concentration decreased with increasing spore abundance (F1,20 = 5.0; p= 0.036; R2=0.16, Fig. 7C). Spore abundance showed no significant correlation with AP or LAP activities across treatments (Fig. 7A/B).

IV. Discussion

This study employs a biogeochemical perspective to investigate how plant invasions influence ecosystem scale processes. Understanding these complex relationships is integral to informing management actions and laying the groundwork for conceptualizing invasion mechanisms. My hypothesis that DIN would be highest in E. umbellata only plots was not supported. DIN was lowest in EU plots and highest in mixed plots; this is surprising as it is often expected that soil under N-fixing plants, like E. umbellata, will be high in inorganic N. This unexpected finding may suggest that the relative magnitude of inorganic nitrogen con-

Figure 3: (A) Total dissolved inorganic nitrogen (DIN) and (B) Ammonium across treatments and at three sampling distances. Treatments with different lowercase letters are significantly different (p<0.1).

Figure 4: LAP activity (ln(μmol/g/hr) across treatments at three sampling distances. Treatments with different lowercase letters above the boxplot are significantly different (p<0.1).

tribution from increased decomposition may outweigh the contribution from atmospheric N-fixation. In other words, plots with L. sinense present (BO and LS plots) may display higher DIN levels due to increased decomposition rate and N mineralization stimulated by labile, or easily degraded, L. sinense leaf litter (Mitchell et al.). Alternatively, nitrogen gains

from fixation may be retained within E. umbellata biomass and therefore have little detectable impact on local N availability (Brookshire et al.). This proposition is especially plausible given the drought conditions present during sampling and overall low soil DIN. With these limited nutrient conditions and possible drought stress, E. umbellata is unlikely to release excess N into the surrounding soil. These data, considered alongside the AP activity/ DIN correlation, suggest that mixed plots may experience a higher degree of P-limitation than single-species plots. Across treatments, AP activity increased with total DIN. This potentially indicates that soil microbes in the relatively N-rich, mixed-species plots are investing more heavily in P-acquisition than microbes in EU or LS plots are.

Previous studies have found lower LAP and NAG activity related to high inorganic N (Uwituze et al.; Shi et al.). Surprisingly, my results do not show any correlation between LAP/NAG activity and DIN. However, LAP and NAG activity may also be influenced by changes in pH, which I did not measure in this study. Higher soil pH can indirectly increase enzyme activity by providing suitable conditions for microbial activity (Cheng et al.). To my knowledge, no studies have directly investigated E. umbellata impacts on soil pH, but it is well established that L. sinense can increase soil pH (Cash et al.). Therefore, complex pH dynamics may be at play, which could influence extracellular enzyme activity. For instance, higher soil pH caused by L. sinense could explain my finding that LAP activity was significantly higher in mixed plots containing L. sinense than in E. umbellata-only plots. However, continued research is needed to clarify the role of these shrubs in influencing soil pH and subsequent soil effects.

My hypothesis that AMF spore abundance would be higher in mixed plots compared to

Figure 5: Extracellular enzyme activities (ln(μmol/g/hr)) against DIN (ppm dry soil) across treatments. Solid dark grey lines display significant slopes and dashed grey lines display non-significant slopes.

single species plots was not supported. While spore abundance did not significantly vary across treatments or distances, EU spore abundance appeared to increase at closer proximity to the central shrub, anecdotally corroborating the findings described in Malinich et al., 2017. If future studies utilize a larger sample size or spatial range, I speculate a clearer relationship between spore abundance and N-fixer proximity would likely be found and may have implications for facilitating co-invasion of other invasive plants (Kuebbing et al., “Two co-occurring invasive woody shrubs”).

Unexpectedly, NAG activity was negatively correlated with spore abundance across treatments. This is in contrast with previous studies that found positive relationships between AM fungal spore density and extracellular enzyme activity (Sheng et al.; Bai et al.). While I did not initially hypothesize relationships between spores and enzymes, this correlation may offer insight into the complexity of AMF function in response to environmental conditions. Fungi may increase their spore production and simultaneously decrease extracellular enzyme activity in response to drought stress (Bogati and Walczak). While I expected to find positive correlations between spore abundance and extracellular enzyme activity, this drought stress hypothesis could explain why correlations were unexpectedly negative (NAG) or insignificant (AP and LAP). Further, drought stress

present during sampling could explain why spore counts were similar across treatments, as the influence of water limitation may have outweighed shrub-specific soil impacts.

A. Limitations

Spatial and temporal limitations likely impacted the results of this study. My single sampling event and small spatial scale may not have effectively captured nutrient and fungal dynamics under combined E. umbellata and L. sinense invasion. Further, assessing only extractable DIN may not have captured N dynamics as holistically as measuring N mineralization would have. Quantifying soil phosphorus directly would have also given more support to my proposed P-limitation mechanism. Assessing pH may have clarified the impact of L. sinense on nitrogen cycling. I did not quantify pH in this study and was therefore prevented from assessing how L. sinense may impact decomposition rates through changes in pH or vice versa. I intend to resample sites from this study to understand the potential role of pH. Climate and weather conditions may have affected my results. I sampled Blocks 1 and 2 during drought conditions and sampled Block 3 shortly after Hurricane Helene. These extreme moisture conditions may have increased dissolved available nutrients, or eliminated stress caused by drought, leading to a pulse of microbial activity and subsequent increases in extracellular enzyme activity and DIN (Borowik and Wyszkowska). Finally, I was unable to assess two influential processes: initial invasion mechanism and facilitation of additional invasive species. Determining the order of shrub establishment and shrub age was difficult; I collected data on shrub stem count as a proxy for shrub size, but the shrub diameter varied so greatly within and between species that it was not a useful metric of size nor age.

Figure 6: AMF spore abundance (number of spores/g dry soil) displayed across treatments at three sampling distances

Figure 7: Extracellular enzyme activity (ln(μmol/g/hr)) against spore abundance (number of spores/g dry soil) displayed across treatments at three sampling distances

V. Future Directions & Conclusion

Future studies should conduct experiments to directly assess the impact of N-fixing and non-fixing invasive shrubs on nutrient cycling and fungal communities. A greenhouse experiment to compare the magnitude of E. umbellata and L. sinense impacts on N using different

strategies (N-fixation and increased decomposition rate, respectively), would be informative. A common garden experiment with nutrient enrichment to artificially create N or P limitation could help clarify the relationships found between DIN and enzyme activity found in this study. Further, studies should aim to quantify how E. umbellata and L. sinense impact soil pH when grown separately and together, as pH shifts could clarify enzyme activity patterns. From a conservation perspective, it is imperative that future studies also directly quantify how plant co-invasion impacts native plant communities.

Understanding the soil-level impacts of co-invasion is essential to characterize how invader species differentially, or jointly, influence native plant success and ecosystem health. Investigating patterns in plant invader mechanisms and subsequent ecosystem changes is essential to predicting the long-term effects of invasion and justifying best management practices.

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PUBLIC AND INTERNATIONAL AFFAIRS

Raquel Beatriz Caldas Laranjeira is a Brazilian student at UGA majoring in International Affairs and Political Science. A Leadership Fellow through ILA, she is pursuing a Master’s in International Policy through the Double Dawgs program. She serves as a Lead Peer Learning Assistant, Honors Ambassador, and is active in International Student Life. After graduation, she plans to attend law school and pursue a career in diplomacy and global organizations.

Raquel Beatriz Caldas Laranjeira

Class of 2026

BRAZIL’S HUMANITARIAN VISA PROGRAM FOR HATIAN MIGRANTS: A CRITICAL ANALYSIS OF POLICY

GAPS, REGIONAL

DISPARITIES, AND INETEGRATION CHALLENGES

RAQUEL BEATRIZ CALDAS LARANJEIRA

DR. ERIN LITTLE

Abstract

Migration is a defining feature of the modern global landscape, reflecting the complex interplay between human agency, state sovereignty, and international legal frameworks. The ways in which migrants are classified—whether as refugees, asylum seekers, or economic migrants— carry profound implications for their access to safety, legal protections, and livelihoods. These categorizations are not neutral but serve as political tools that shape the rights and identities of migrants while reinforcing the control states exert over their borders. In Brazil, the humanitarian visa program for Haitian migrants, introduced in the aftermath of the devastating 2010 earthquake in Haiti, illustrates both the potential and limitations of migration policies in addressing large-scale displacement. While the program provided a legal pathway for entry, it fell short in addressing the systemic inequalities and structural barriers faced by migrants upon arrival. Brazil’s humanitarian visa program for Haitian migrants was an inadequate response to the multifaceted challenges of forced migration, as it failed to provide the necessary infrastructure, support systems, and integration policies to ensure migrants’ socioeconomic stability and dignity. Drawing on regional disparities in Brazil, legal frameworks, and migration data, this analysis demonstrates how the program left many Haitian migrants vulnerable to exploitation, poverty, and marginalization, exacerbating the inequities already present in Brazil’s underdeveloped regions. A comprehensive examination of Brazil’s historical, legal, and socioeconomic contexts underscores the need for a more equitable and inclusive migration strategy that addresses both immediate humanitarian needs and long-term integration challenges.

In the present global landscape, the way we classify migrants can profoundly affect both individuals and the States they seek to enter. Classification into categories like “refugees,” “illegal migrants,” or “asylum seekers” is a politically significant act that goes beyond mere technical labeling (Thomaz). These terms carry significant weight, often determining whether a person gains access to physical safety, legal protections, resources, and fundamental rights, shaping their identities and political agency. Categorization also serves as a political tool for states to assert control over human mobility, reinforcing their sovereignty by determining who is included or excluded from their territory. These labels are not able to reflect the true diversity of migrants’

experiences and motivations, as they are often based on arbitrary and contingent assumptions. Instead, they represent the state’s attempt to regulate and manage population movement (Thomaz). Ultimately, these categorizations reveal the power dynamics underlying migration policies, where the needs of individuals are often secondary to state interests and control.

International Legal Frameworks for Migration and Asylum

The 1951 Convention Relating to the Status of Refugees is a cornerstone of modern international refugee protection. The Convention is based on Article 14 of the 1948 Universal Declaration of Human Rights, which asserts that “Everyone has the right to seek and to enjoy in other countries asylum from persecution.” In 1950, the United Nations High Commissioner for Refugees (UNHCR) was established, and in 1951, the Geneva Convention relating to the Status of Refugees was adopted (Marino and Rampazzo). The Convention officially came into effect on April 22, 1954, and has only been amended once by the 1967 Protocol, which removed the initial geographic and temporal limitations. This change made the Convention applicable to refugee situations arising anywhere in the world and at any time, but only for states that are parties to the Protocol. Article 1 of the 1951 Convention explicitly defines a “refugee” as an individual who:

Owing to well-founded fear of being persecuted for reasons of race, religion, nationality, membership of a particular social group or political opinion, is outside the country of his nationality and is unable or, owing to such fear, is unwilling to avail himself of the protection of that country; or who, not having a nationality and being outside the country of his former habitual residence as a result of such events, is unable or, owing to such fear, is unwilling to return to it.

The Geneva Convention laid the foundation for international refugee law, a subset of human rights law (Souza).

The International Covenant on Civil and Political Rights (ICCPR), adopted on December 16, 1966, establishes key rights in Article 12 regarding freedom of movement. Section 1 guarantees that “everyone lawfully within the territory of a State shall, within that territory, have the right to liberty of movement and freedom to choose his residence.” Section 2 states that everyone has the right to freely depart from any country, including their own. According to Article 12, section 3, restrictions on these rights are permitted only when “necessary to protect national security, public order...public health or morals, or the rights and freedoms of others,” and they must align with other rights in the ICCPR.

In 1969, another important human rights treaty was signed, The American Convention on Human Rights, or the Pact of San Jose, Costa Rica. This Convention establishes a comprehensive set of civil and political rights for individuals in the Organization of American States (OAS) member states, aiming to protect and promote human dignity across the region. Article 22, section 7 states that “Every person has the right to seek and be granted asylum in a foreign territory, in accordance with the legislation of the state and international conventions, in the event he is being pursued for political offenses or related common crimes.” The Convention in Article 22 affirms that everyone

lawfully within a State Party has the right to move freely within it, reside there according to the law, and leave any country, including their own. According to Article 22, these rights can only be restricted “to the extent necessary in a democratic society” for reasons like national security or public health (“Basic DocumentsAmerican Convention”). That same article further asserts that “No one can be expelled from the territory of the state of which he is a national” and prohibits deportation to a country where one’s “right to life or personal freedom is in danger” and bans the “collective expulsion of aliens (“Basic Documents - American Convention”).

The 1984 Cartagena Declaration on Refugees is one of the most significant legal, political, and strategic frameworks for refugee protection in Latin America and the Caribbean. It marked a milestone in regional cooperation, strengthening a tradition of solidarity and asylum for refugees. The Cartagena Process, a unique model of regional collaboration and shared responsibility, was established following this Declaration, with Latin American countries adopting 10-year plans, such as the Mexico Plan of Action and the Brazil Plan of Action, to address displacement challenges. The Declaration emphasized the need to “enlarge the concept of a refugee” to reflect regional realities, drawing on precedents like the OAU Convention and the Inter-American Commission on Human Rights. It recommended a definition that includes individuals “whose lives, safety or freedom have been threatened by generalized violence, foreign aggression, internal conflicts, massive violation of human rights or other circumstances which have seriously disturbed public order” (“Cartagena Declaration on Ref-

ugees” 34) Additionally, the Declaration reinforced the “peaceful, non-political and exclusively humanitarian nature of grant of asylum” and highlighted the critical principle of non-refoulement, including the “prohibition of rejection at the frontier,” which it recognized as a core element of refugee protection and a “rule of jus cogens” in international law.

The concepts of “refugee” and “asylum seeker” differ in scope and application, though both aim to protect individuals fleeing unsafe conditions. Refugee status, as defined by the 1951 UN Refugee Convention and its 1967 Protocol, applies to individuals outside their home country who have a well-founded fear of persecution due to their race, religion, nationality, social group, or political opinion (Kingston). It is an internationally recognized status that provides safety in other countries. In contrast, asylum is a broader legal and political mechanism that allows a country to offer protection to individuals within its borders, often for political reasons (Marino and Rampazzo). While refugee status is about specific criteria for being displaced, asylum rules can differ regionally and might include political, territorial, diplomatic, and military issues (Marino and Rampazzo). Additionally, refugee status needs an evident fear of persecution, while asylum status often requires proving actual persecution (Marino and Rampazzo).

The International Rescue Committee (IRC) classifications of “refugees” and “asylum seekers” reflect these distinctions within the framework of international law but follow a narrower interpretation. According to the IRC, a refugee is someone forced to flee their home due to war, violence, or persecution, often with little or no warning (International Rescue

Committee). An asylum seeker, on the other hand, is an individual seeking international protection whose refugee status has not yet been legally determined. To apply for asylum, these individuals must enter the country they want to seek protection and file their claim. The IRC points out that asylum helps foreigners who cannot return home due to persecution without legitimate reasons (International Rescue Committee).

International law and the IRC focus primarily on persecution as grounds for protection; however, the Cartagena Declaration (1984) takes a broader view of these grounds, expanding them to address forced displacement in the Global South. The Declaration widens the definition of refugees to include people fleeing widespread violence, internal conflict, serious human rights abuses, and other major disruptions to public order (“Cartagena Declaration on Refugees”). This broader view covers complicated displacement situations that international law may not fully address. Unlike the IRC definitions that match the 1951 UN Refugee Convention closely, the Cartagena Declaration offers a framework that is more appropriate for the different reasons for displacement found in Latin America and other areas suffering from systemic violence and rights violations.

Brazil’s Migration Policies in Their Historical Context

Brazil’s approach to migration has evolved significantly over the years, shaped by historical, political, and humanitarian considerations. From the late 19th century, when migration policies were designed to attract labor for the growing coffee economy, to contemporary

efforts aimed at addressing complex global crises, Brazil’s migration framework reflects both the nation’s aspirations and the challenges it continues to face. Historically, Brazil implemented laws and policies to attract and support migrants, reflecting its economic priorities and the influence of domestic stakeholders like coffee producers. As time went on, these laws adjusted to fit international agreements, such as the 1951 Refugee Convention, while also dealing with regional issues through agreements like the Cartagena Declaration.

In recent years, Brazil has faced the challenge of managing non-traditional forms of displacement, including those driven by environmental disasters, as seen with the influx of Haitian migrants following the 2010 earthquake. This period marked a shift in Brazil’s migration policy, moving beyond traditional refugee definitions to adopt innovative mechanisms, such as humanitarian visas. However, the vague and incomplete classification of “humanitarian immigrant,” which lacks clear legal protections and leaves many in a precarious legal and social position, has sparked debates about its effectiveness. The following sections delve into Brazil’s historical and legal migration frameworks, the humanitarian visa mechanism, and the broader implications for Haitian migrants, illustrating the complexities and contradictions within the country’s migration policies.

After the Proclamation of the Republic on November 15, 1889, Brazil’s new government prioritized migration as a key foreign policy focus. Diplomatic efforts abroad were supported by domestic laws aimed at attracting immigrants, heavily influenced by coffee producers’ interests. A notable example is Decree No. 528, issued on June 28, 1890, by the provisional

ever, the National Committee on Refugees (CONARE), responsible for asylum decisions, denied the refugee claims of Haitians, arguing they did not meet the international definition, despite their pleas to escape a crisis-stricken Haiti (Thomaz). Instead, CONARE referred these cases to the National Immigration Council (CNIG), which issued the humanitarian visas, acknowledging the dire situation in Haiti but avoiding a refugee status designation (Miura). Brazil’s verdict on Haitian asylum claims was based on extreme poverty and environmental causes following the 2010 earthquake. CNIG’s Resolution 97, passed in 2012, granted humanitarian visas to Haitian migrants due to deteriorating living conditions. The visas, valid for 5 years and renewable every 2 years, required migrants to prove regular working status to remain in Brazil. This policy highlighted the ambiguity of the humanitarian immigrant category, where migrants were seen as disaster victims but were expected to work to extend their stay (Thomaz). While UNHCR praised Brazil’s humanitarian approach, local NGOs like Conectas argued that Haiti’s conditions warranted refugee status under Brazilian law, as the severity of the humanitarian crisis aligned with Brazil’s legal standards for widespread human rights violations (Thomaz).

Challenges Faced by Haitian Migrants

Despite Brazil’s willingness to accommodate Haitian migrants, the outdated immigration laws posed substantial challenges, especially as migration surged after the visa quota was lifted in 2012. The 1980s-era legislation failed to account for environmental migrants, leaving many Haitians without proper documentation, which hindered their access to basic rights

such as housing, bank accounts, and formal employment. Consequently, many Haitian migrants were pushed into informal labor markets, where they often faced exploitation and discrimination (Miura). The situation exposed critical gaps in Brazil’s legal framework, highlighting the need for migration policies that could adapt to contemporary humanitarian crises, particularly for migrants displaced by environmental disasters and other emerging causes of forced movement.

Brazilian authorities systematically denied Haitian claims for refugee status, despite the country’s broad legal definition of refugees, which includes individuals fleeing grave and widespread human rights violations (Silva; Macêdo) The central reason for these denials was that Haitians failed to meet the criteria of a well-founded fear of persecution as defined by the 1951 UN Refugee Convention and Brazil’s Refugee Statute (Kingston). Although the 2010 earthquake and Haiti’s severe hardships were acknowledged as devastating, they were not considered grounds for refugee status since natural disasters do not constitute persecution under international law. Besides, the term “environmental refugee,” which defines people displaced because of environmental catastrophes, is not defined either by international or Brazilian legislation (Souza). Instead, the Brazilian government invited Haitians to seek humanitarian visas, which provided another alternative for legal stay.

The humanitarian visa program introduced in Brazil in 2012 provided Haitians with a more accessible pathway to legal status, balancing ease of entry with significant limitations in rights. Designed with lower requirements, the program allowed applicants to qualify by

presenting a valid passport, proof of residency in Haiti, and a clean criminal record, all for a relatively modest cost of $200 (Jubilut et al.; Macêdo). Initially, the program was restricted to a maximum of 1,200 visas annually and only at specific locations; later, the eligibility was expanded to allow Haitians to apply at any Brazilian consulate. However, bureaucratic hurdles—for example, those involved in obtaining the necessary documents—led to large backlogs and delays for those trying to immigrate through legal channels (Jubilut et al). Many were stopped at Brazilian airports for lack of proper documentation (Silva). The visa provided an avenue to permanent residence through the National Immigration Council but did not contain refugee protections, such as non-refoulement, thus rendering the holder subject to deportation (Jubilut et al.). Because the program was based on administrative resolutions rather than formal legislation, the humanitarian visa framework was legally precarious and subject to political change, echoing the dilemma of balancing expedited migration with solid protections.

Initially, the Brazilian government perceived the Haitian migration as a temporary phenomenon, a transit flow toward French Guiana. This miscalculation led to a delayed and inadequate response, as the government was slow to recognize the scale and complexity of the situation. Brazil’s migration policies and legislation, largely unchanged since the 1980s, were ill-equipped to handle the unique challenges posed by the Haitian migration. The existing legal framework, focused primarily on traditional notions of refugee status based on persecution, did not adequately address the complexities of forced migration driven by

environmental disasters and socioeconomic vulnerabilities. The response from government agencies, including the Federal Police, was hampered by bureaucracy, limited resources, and a lack of specialized training in handling the specific needs of Haitian migrants (Miura). Visa application processing was slow, and the capacity to provide essential services like housing, healthcare, and language training was inadequate.

The humanitarian visa program was specifically designed for Haitians affected by the earthquake, excluding other nationalities facing similar hardships. The program also deliberately avoided granting refugee status to Haitians, a decision that sparked criticism from international organizations and human rights advocates. This reluctance to recognize Haitians as refugees stemmed in part from economic considerations, as the government prioritized attracting skilled labor and managing the economic impact of migration over fully adhering to international human rights standards (Silva; Kingston). The complex interplay of factors driving Haitian migration, including socioeconomic vulnerabilities, political instability, and environmental degradation, could not be fully addressed by a visa program alone (Kingston). Although the visa program granted legal residency, it did not adequately address the challenges of integrating Haitian migrants into Brazilian society. The responsibility for providing essential services like housing, language training, and job placement often fell on civil society organizations and NGOs, which struggled to keep pace with increased demand (Kingston). The lack of robust government support created difficulties for Haitians in accessing public services, securing stable

employment, and overcoming language and cultural barriers (Thomaz).

For many Haitians fleeing to Brazil, the journey itself was marked by hardship and danger, especially for those dependent on smugglers, known as coyotes, to enter the country illegally. Many Haitians were misled by smugglers who provided unreliable information about wages and opportunities in Brazil, leading to inflated expectations that resulted in disillusionment and hardship upon arrival. The lack of access to humanitarian visas made many turn to those taking advantage of their urgent need for profit. Typical routes for Haitian migrants began with flying to South American countries such as Peru, Ecuador, or Bolivia, which had relatively more lenient entry requirements (Silva; Yates). Migrants were then forced to undergo long bus or boat rides and dangerous walks on foot through rough terrain to reach Brazil’s Amazonian border (Miura). Key entry points included the towns of Assis Brasil and Brasiléia in the state of Acre and Tabatinga in the state of Amazonas. These journeys, which cost thousands of dollars, were filled with risks such as theft, violence, and bribery (Silva; Miura). Upon reaching the border, Haitian migrants still had to face additional obstacles, including corruption and overcrowding, which often placed them on a waitlist before they could finally enter Brazil (Miura). Such a perilous journey made Haitians physically and emotionally drained before they could even begin their lives in Brazil. The conditions upon arrival were dire. Overwhelmed by the influx of migrants, local infrastructure was inadequate to provide proper shelter and support. Migrants were often housed in overcrowded and unsanitary make-

shift camps, such as an abandoned football arena in Brasiléia, which lacked basic necessities like bathrooms, running water, and proper sanitation (Silva; Miura). These conditions posed serious health risks, further exacerbating migrants’ vulnerability. Language barriers, as most Haitians speak Creole, and the cultural differences in these remote border regions added another layer of difficulty, leaving many isolated and struggling to communicate with locals (Miura).

Haitian migrants in Brazil faced significant economic challenges that hindered their ability to achieve financial stability and improve their living conditions. Often confined to low-paying jobs, they were frequently exploited, earning lower wages than their Brazilian counterparts for the same work (Silva; Yates). This exploitation stemmed from their vulnerable position as newly arrived immigrants, often desperate for employment. While the 2014 World Cup and the 2016 Olympics opened up initial job opportunities, the following economic slowdown meant that work became progressively harder to find for Haitians as well (Yates). They experienced increasing competition from Brazilian workers and other migrant groups, who further reduced their chances. This economic vulnerability was further exacerbated by the higher cost of living in major Brazilian urban areas, where insufficient wages made it difficult to afford basic necessities such as housing, food, and transportation, leaving little or no chance of sending any money back to their families in Haiti (Kingston). Moreover, linguistic barriers significantly curtailed chances of better-paying jobs, while the lengthy and complex process necessary to authenticate Haitian diplomas and professional qualifications thwarted many

from reaping the benefits of their skills and education in enhancing their economic situations (Souza). This combination of factors left many Haitian migrants trapped in a cycle of economic precarity and marginalization.

Despite the many challenges they encountered, Haitian migrants often used Brazil’s border towns only as temporary transit points rather than long-term destinations. Driven by the need for economic opportunities and better living conditions, many continued their journey to larger urban centers. São Paulo has become a major magnet, offering job opportunities and highly structured support networks (Kingston; Macêdo) Other urban areas, such as Rio de Janeiro, Curitiba, and Porto Alegre in both the South and Southeast also attracted migrants (Silva). However, the greater promise these cities provided, the more hindrances they presented: high cost of living, high competition for jobs, and the problem of finding adequate housing.

Addressing Regional Disparities and Policy Shortcomings

To investigate the socioeconomic and policy-related challenges surrounding Haitian migration to Brazil, this study employs a mixed-methods approach that integrates data visualization, regional analysis, and migration mapping. By utilizing tools like the Atlas Brasil website and data from the Brazilian Institute of Geography and Statistics (IBGE), this research highlights regional disparities in development and their impact on migration dynamics. Additionally, maps illustrating Haitian migration routes and internal movements provide critical insights into the logistical and humanitarian challenges faced by migrants and host regions.

By merging these data tools with an examination of laws and historical details, the study seeks to cover the structural inequalities and policy shortcomings that influenced Haitian migration to Brazil.

Using the Atlas Brasil website, which allows users to explore and compare various development indices across Brazilian locations, I created Map 1 highlighting the Municipal Human Development Index (IDHM) for all Brazilian states in 2014. This map uses data derived from the National Household Sample Survey (PNAD), conducted by the Brazilian Institute of Geography and Statistics (IBGE). PNAD is a vital survey that investigates the socioeconomic characteristics of Brazilian society. Its primary objective is to produce information essential to the study and understanding of Brazil’s socioeconomic development (IBGE, “Volume Brasil | IBGE”).

By gathering data on aspects such as education, income, health, and living conditions, PNAD provides critical insights into regional disparities across Brazil. For this map, the PNAD data contributed to the calculation of the IDHM for Brazilian states in 2014. The IDHM combines indicators of longevity, education, and income, allowing for a comprehensive overview of human development across the country. Notably, 2014 was a pivotal year for Haitian immigration, as Brazil received the highest number of Haitian immigrants, rising from just 595 in 2010 to nearly 30,000 in 2014 (Sousa). This influx underscores the importance of understanding regional disparities, as the IDHM data contextualizes the challenges faced by both migrants and the regions receiving them.

Map 1 uses a gradient color scheme to represent the IDHM, with lighter shades (green/ light grey) indicating lower development levels, typical in the North and Northeast Regions, which struggle with limited resources and infrastructure. Medium shades (grey) represent moderate development, while darker shades (dark blue) indicate higher IDHM values, found in more developed states in the South and Southeast Regions with better socioeconomic conditions. This color coding highlights Brazil’s stark regional disparities, underscoring the challenges faced by low-IDHM states like Acre in managing migratory inflows compared to more developed regions like São Paulo. The IDHM map reveals striking regional disparities in development, which contextualize the challenges faced by Haitian immigrants. The North Region, particularly the state of Acre, served as the main entry point for Hai-

tians arriving through countries such as Ecuador, Peru, and Bolivia. Cities like Brasileia, in Acre, bore the brunt of this influx. However, Acre and much of the North Region have some of the lowest IDHM values in Brazil, reflecting limited infrastructure, education, and income opportunities. This lack of resources significantly hindered the region’s ability to integrate and support immigrants effectively. This crisis was further complicated by legal and policy challenges. Many Haitians sought refugee status, but their applications were often denied for not meeting the criteria set by the National Committee for Refugees (Conare) (Thomaz). Instead, the National Immigration Commission (CNIg) issued them residency on humanitarian grounds (Thomaz,). In 2013, Brazil removed its limit of 1,200 visas for Haitian migrants and broadened humanitarian visa issuance to Brazilian embassies in places

Map 1 - Regional Disparities in Brazil: Municipal Human Development Index (IDHM) by State (2014)

like the Dominican Republic, Ecuador, and Peru (Miura). This policy aimed to decrease illegal migration and relieve border pressure, yet it also put more responsibility on Brazil to provide basic living conditions for Haitians arriving legally.

Acre bore the brunt of these challenges, with its capital, Rio Branco, and nearby municipalities like Brasileia experiencing a humanitarian crisis. Overwhelmed, the state government eventually shut down overcrowded shelters and began relocating migrants to other cities, including São Paulo, located over 3,000 km away (Miura). This relocation brought new issues, as São Paulo’s municipal government was not ready for the sudden influx of thousands of migrants. Many Haitians arrived urgently needing food and shelter, putting pressure on groups like Missão Paz, a Catholic NGO that offers legal help, housing, and mental health support (Miura).

Even though many Haitian immigrants started in Acre, many moved to the South and Southeast Regions, attracted by higher IDHM values and better job opportunities. These areas, especially São Paulo and Rio Grande do Sul, had better infrastructure to help them settle in. Between 2011 and 2013, Haitian participation in Brazil’s formal labor market grew significantly, with a 406% increase from 2011 to 2012 and another 254% from 2012 to 2013 (Sousa). However, structural challenges, such as labor exploitation and marginalization, persisted. Many Haitians faced poverty, precarious housing, and limited access to essential services such as healthcare, education and, legal documentation, reflecting systemic shortcomings in immigrant integration policies.

The economic downturn of 2015 compounded these challenges. Job losses following Brazil’s unfinished World Cup projects and broader economic instability forced many Haitians to leave Brazil for countries like the United States, Mexico, and Chile. The Obama administration’s work visa program provided an additional incentive for migration out of Brazil (Sousa). Ultimately, while Brazil’s humanitarian visa rules initially helped with legal migration, the country’s lack of resources and immigrant support systems showed how unprepared Brazil was to handle large numbers of migrants.

In addition, I collected Map 2, the Haitian Migration Routes (2010–2019) map, from the website Migration for Development and

Map 2 - Haitian Migrant Routes (2010-2019)

Equality (MIDEQ), as featured in the article “Migration Data Gaps and the Challenge of Understanding Haitian Mobility in the Global South” by Mário Fidalgo (2020). This map visualizes the routes Haitian migrants took to reach South America, particularly Brazil, from their home country, highlighting key transit countries such as Ecuador, Peru, and Bolivia, as well as cities that played a significant role in their journey. It identifies important entry points into Brazil, including Tabatinga (Amazonas) and Brasileia (Acre), regions that were overwhelmed due to their limited infrastructure and resource capabilities. The map also illustrates the regional movement of Haitian migrants within South America, emphasizing how Brazilian border states like Acre, with their IDHM, bore the initial burden of migration. The reliance on poorly prepared entry locations led to humanitarian crises because local infrastructures were ill-equipped to handle the needs of migrants. The IDHM map further underlines the socioeconomic challenges that these regions are experiencing, further magnifying their inability to house large populations of migrants sustainably.

Haitians would frequently move to South and Southeast Regions, into states like São Paulo and Curitiba, where they could find better IDHM values and more opportunities. They hardly settled in the Northeast Region since it was one of the regions with already-low IDHM rates, similar to those in the Northern states, which could not offer good resources or opportunities for them.

Together, these maps emphasize the need for coordinated regional migration policies to better manage transit flows and address the development disparities that exacerbate migra-

tion-related challenges. They also reveal significant humanitarian gaps in the reception and integration of migrants, particularly in regions with low IDHM, demonstrating the importance of creating inclusive policies to ensure migrants’ access to basic rights and opportunities.

An analysis of the maps reveals how regional disparities in development influenced Brazil’s response to the Haitian migration crisis. The IDHM values highlight why some areas, like the Southeast, became popular for immigrants, while others, like Acre, struggled significantly. Strengthening immigrant integration policies is essential for addressing the challenges of mass migration and ensuring a more humane response to future crises.

Regional Disparities and Their Impact on Haitian Migrants

Tables 1, 2 and 3 were constructed using data from Atlas Brasil, which aggregates socioeconomic and demographic indicators for Brazil. The dataset combines information from the Brazilian Census (Censo) for the years 2000 and 2010, conducted every 10 years by the Brazilian Institute of Geography and Statistics (Instituto Brasileiro de Geografia e Estatística – IBGE), providing detailed insights into the population and social conditions. For the years 2012 to 2020, data from the National Household Sample Survey (Pesquisa Nacional por Amostra de Domicílios – PNAD) was used. PNAD, also conducted by IBGE, is an annual survey that complements census data by tracking trends in education, income, health, and other key socioeconomic factors. The provided tables detail key socioeconomic indicators for six Brazilian regions—Acre (AC), Amazo-

nas (AM), Bahia (BA), Distrito Federal (DF), Paraná (PR), and São Paulo (SP)—from 2000 to 2020.

Between 2000 and 2020, socioeconomic indicators across Brazilian regions revealed both significant progress and persistent disparities, reflecting the uneven development of the country. While states like Acre (AC), Amazonas (AM), and Bahia (BA) showed improvement, they continued to lag behind wealthier regions such as the Distrito Federal (DF), São Paulo (SP), and Paraná (PR), highlighting the entrenched inequalities between Brazil’s North, Northeast, and more developed South and Southeast.

One of the most revealing indicators of Brazil’s regional disparities is the IDHM, which shows the uneven pace of progress across the country. By 2014, Acre had made significant improvements but still lagged behind wealthier regions like São Paulo and the Distrito Federal. These differences highlight how regions in the North and Northeast struggled to keep pace with the South and Southeast, where development levels remained consistently higher.

Healthcare improvements were evident in the reduction of infant mortality rates, though disparities persisted across regions. By 2014, Acre and Bahia showed marked declines but still had significantly higher rates than São Paulo and Paraná, reflecting the uneven quality of healthcare services across regions. Illiteracy rates also declined steadily between 2000 and 2020, with regions like Acre and Bahia showing notable progress. However, these less-developed states continued to face higher levels of illiteracy compared to wealthier states like São Paulo and Paraná, emphasizing persistent educational inequalities.

Table 1 (above)

Table 2 (above)

Table 3

Economic disparities remained stark throughout this period. By 2014, the Distrito Federal had solidified its position as the wealthiest region, with significantly higher per capita income than Acre and Amazonas. This economic gap underscores the limited opportunities available in underdeveloped regions, further complicating the challenges faced by Haitian migrants who settled there.

While Brazil made notable progress in reducing illiteracy, improving health outcomes, and raising incomes from 2000 to 2020, regional disparities remained deeply entrenched. The year 2014 exemplifies the dual realities of progress and inequality, emphasizing the challenges faced by states like Acre and Amazonas in catching up with wealthier regions. Addressing these imbalances requires sustained and coordinated efforts to improve education, economic opportunities, and healthcare access in the country’s most vulnerable areas.

The differences in economic and social conditions across regions in Brazil show important details about the movement of Haitian migrants and shed light on why the humanitarian visa program did not meet their needs well. Migrants arriving in northern states, such as Acre and Amazonas, encountered serious problems because of the area’s weak infrastructure, low IDHM scores, and high rates of illiteracy and infant deaths. For example, Acre’s IDHM score in 2014 was just 0.715, much lower than São Paulo’s 0.825, indicating great lacks in public services and job chances. As a primary entry point for Haitian migrants, Acre was ill-equipped to provide adequate support, resulting in humanitarian crises in cities like Brasiléia and Rio Branco. Food shortages, overcrowded shelters, and overburdened pub-

lic health systems created harsh conditions for both migrants and the local population. The lack of opportunities in these regions drove Haitian migrants to relocate to more developed states, such as São Paulo and Paraná, in the Southeast and South, where higher IDHM values promised better prospects. This internal migration aligns with the socioeconomic patterns revealed in the data. São Paulo, for example, had a per capita income of R$1,093.25 in 2014, significantly higher than Acre’s R$514.59, a difference that made the state an attractive destination for migrants seeking economic stability. However, even in wealthier regions, Haitian migrants faced significant challenges. Despite the humanitarian visa granting them legal status, many struggled with informal employment, labor exploitation, and poverty. The absence of robust integration policies meant that legal entry alone did not translate into meaningful access to rights, services, or stable livelihoods.

The humanitarian visa program, while initially designed to provide legal pathways for Haitian migrants and reduce dependence on human smugglers, proved inadequate in addressing the broader structural issues migrants faced upon arrival. The data on illiteracy rates, per capita income, and IDHM highlight how Brazil’s underdeveloped regions, which served as key entry points, lacked the capacity to integrate migrants effectively. Additionally, the large gaps between these underdeveloped regions and wealthier states led many Haitians to move south for better chances. This situation showed that the program failed to ensure equitable support or coordination across regions, placing disproportionate pressure on underdeveloped entry points and leaving many

migrants without adequate assistance. Even migrants who got to live in better IDHM states like São Paulo faced many socioeconomic challenges. The labor market in these regions, while offering more formal job opportunities, often subjected migrants to precarious working conditions. Many Haitians found low-paying jobs that took advantage of them with almost no legal protections, showing issues in Brazil’s labor rights system. Additionally, access to housing, education, and healthcare remained limited, further marginalizing migrants despite their legal status. The stark contrast between their expectations of better living conditions and the realities of exclusion and vulnerability reveals the shortcomings of the humanitarian visa as a comprehensive solution.

Lessons from Brazil’s Humanitarian Visa Program

In essence, while the humanitarian visa addressed one aspect of the migration challenge—providing legal entry—it failed to consider the socioeconomic realities Haitians faced upon arrival. The data on regional disparities in development, coupled with the lived experiences of migrants, illustrate how these legal protections did little to mitigate the structural barriers to integration. The program’s limitations are evident in the continued struggles of Haitian migrants to secure stable employment, access essential services, and escape cycles of poverty. As such, the humanitarian visa was not an ultimate solution but rather a partial measure that left many migrants vulnerable to the same systemic inequalities faced by Brazil’s most underdeveloped regions. A more comprehensive approach, combining legal protections with targeted investments

in infrastructure, social services, and migrant integration programs, would have been necessary to ensure a humane and effective response to the Haitian migration crisis.

Brazil’s humanitarian visa program for Haitian migrants provided legal entry but failed to address the deeper challenges migrants faced. Without strong support systems, infrastructure, or integration policies, many Haitians struggled with poverty, exploitation, and limited access to basic services. Regional disparities, especially in underdeveloped areas like Acre, worsened these issues, pushing migrants to wealthier states where challenges still remained. The program, while a step forward, was not enough to meet their needs. This case serves as an example for other countries and regions, showing that truly integrating migrants requires not just legal protections but also comprehensive social and economic support systems. While such efforts demand significant investment, they must be addressed collectively by states, international organizations, and nonprofits together to ensure effective and humane migration policies worldwide.

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Branco Pereira, Alexandre. “Refuge in Brazil: An Ethnographic Approach.” Antípoda. Revista de Antropología Y Arqueología, no. 43, Apr. 2021, pp. 197–214, www.scielo.org.co/pdf/antpo/n43/1900-5407-antpo-43-197.pdf, https:// doi.org/10.7440/antipoda43.2021.09.

“Cartagena Declaration on Refugees.” UNHCR US, 1984, www.unhcr.org/us/media/cartagena-declaration-refugees-adopted-colloquium-international-protection-refugees-central.

Delgado-Wise, Raúl. “A Critical Overview of Migration and Development: The Latin American Challenge.” Annual Review of Sociology, vol. 40, no. 1, 30 July 2014, pp. 643–663, https://doi.org/10.1146/annurev-soc-071811-145459.

Dragomir, Cristina-Ioana. “Protecting Climate Refugees Requires a Legal Definition.” Al Jazeera, 2 Mar. 2024, www.aljazeera.com/ opinions/2024/3/2/protecting-climate-refugees-requires-a-legal-definition.

Fidalgo, Mário. “Migration Data Gaps and the Challenge of Understanding Haitian Mobility in the Global South.” MIDEQ - Migration for Diversity and Equality, 2020, www.mideq. org/en/inequalities/inequalities-resources/migration-data-gaps-and-challenge-understanding-haitian-mobility-global-south/. Accessed 15 Sept. 2024.

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International Rescue Committee. “Migrants, Asylum Seekers, Refugees and Immigrants: What’s the Difference?” International Rescue Committee (IRC), 17 June 2024, www.rescue. org/article/migrants-asylum-seekers-refugees-and-immigrants-whats-difference.

Jubilut, Liliana Lyra, et al. “Humanitarian Visas: Building on Brazil’s Experience - Forced Migration Review.” Forced Migration Review, 27 Aug. 2024, www.fmreview.org/jubilut-andrade-madureira/.

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PUBLIC SERVICE AND OUTREACH

Aarov Malhotra is a third-year Foundation Fellow and Stamps Scholar. He serves as Georgia Rep. Spencer Frye’s Chief of Staff and has completed internships with the Colorado Attorney General’s Office and the Centers for Medicare and Medicaid Services. On campus, he runs the Athens Prison Tutorial and represents the Honors College and School of Public and International Affairs as an ambassador. Eventually, Aarov hopes to become a consumer protection lawyer.

Aarov Malhotra Class of 2027

ANALYSIS OF THE OPIOID CRISIS

IN GEORGIA: THE EFFECT OF COVID-19 AND URBAN-RURAL TRENDS

AAROV MALHOTRA JAMES BYARS

Abstract

How has the opioid crisis affected Georgia’s different counties over time? While existing studies have found that rural status is not correlated with opioid overdoses or opioid overdose deaths in Georgia, the five counties with the highest rates of fatal opioid overdose are all rural. Additionally, although rates of prescription opioid use have declined nationwide since 2012, opioid deaths have increased during the same period. Given this, it is critical to understand the relationship between geography and opioid outcomes to determine how best to address the opioid crisis in different parts of Georgia. This study compared county-level opioid overdose death rates between the years 2010 and 2023 and county-level prescription opioid dispensing rates between the years 2019 and 2023 through line charts and choropleth maps. Fulton County and Madison County were utilized as case studies to compare the evolution of the opioid crisis in Georgia’s urban and rural counties. This study found that the COVID-19 pandemic accelerated opioid-related overdose death rates in Georgia’s urban and rural counties through increased use of illicit opioid substances. This research demonstrates the importance of policy solutions tailored to the needs of both urban and rural areas impacted deeply by the opioid crisis.

Introduction to the Opioid Crisis

Opioids are a class of drugs that activate the brain’s opioid receptors. These receptors block pain signals sent from the body, leading to the use of opioids for pain management. Humans have used opioids and their derivates for thousands of years, cultivating sources like the opium-producing poppy plant. Opioids derived from natural sources are termed “natural opioids” or “opiates” and include morphine and codeine (Johns Hopkins Medicine).

In the 1990s, the pharmaceutical company Purdue Pharma developed a potent, “semi-synthetic” opioid called OxyContin. Such semi-synthetic opioids are made by processing opiates within a laboratory (National Institute of Drug Abuse). After FDA approval, OxyCon-

tin prescription rates increased significantly. Between 1999 and 2010, prescription opioid sales quadrupled (Congressional Research Service). Over the same period, opioid-related overdose deaths more than doubled, indicating the first wave of the opioid crisis marked by the rise in semi-synthetic opioids (Congressional Research Service). In 2010, most of the sourcing for heroin, an illegal semi-synthetic opioid, shifted from South America to Mexico. This change reduced the costs of sourcing heroin and rapidly reduced its price within the United States’ illicit market, causing the rate of heroin-related deaths to increase by nearly 500% by 2016 (Congressional Research Service). The rise in heroin deaths between 2010 and 2016 marked the second wave of the opioid crisis. In 2016, “synthetic” opioids synthesized en-

tirely in a lab became the leading category of opioids related to opioid deaths (Congressional Research Service). The most well-known illicit opioid is fentanyl, a drug 50 times more potent than heroin (Texas Health and Human Services). Although fentanyl has FDA-approved medical uses, its illegal analogues have led to a doubling of drug overdose deaths between 2015 and 2022 (Spencer et al. 1). The surge of synthetic opioid use, led largely by fentanyl, has been deemed the third and current wave of the opioid crisis (Congressional Research Service).

Opioid use disorder is defined by a compulsive and addictive use of opioid substances despite internal desires to stop use (Johns Hopkins Medicine). While withdrawal from opioid use disorder is usually not fatal, death can result when in combination with pre-existing conditions or complications (Mosel). Those who quit opioids “cold turkey,” or without any gradual decline of use, often experience vomiting, diarrhea, insomnia, muscle and bone pain, and high blood pressure. Thus, it is crucial that those addicted to opioids seek professional medical treatment. Naloxone, an opioid antagonist, rapidly works against overdoses by reversing the effects of other opioids (National Institute on Drug Abuse). The most common treatment for opioid addiction is Methadone, an opioid agonist that assists with addiction recovery (National Institute on Drug Abuse). Methadone binds to the same opioid receptors that more addictive drugs target and elicits a weaker response. By mimicking stronger drugs, methadone reduces cravings, and its limited euphoric effects ensure it does not further addict patients.

Literature

The first and second waves of the opioid crisis affected counties differently depending on economic factors. In Georgia, uninsurance rates were inversely associated with opioid

overdose deaths between 1999 and 2015 (Valentini et al.). The relationship indicates that prescription drugs obtained through insurance coverage drove mortality in past phases of the opioid crisis in Georgia. Nationally, while overdose-related mortality increased by 6% between 2019 and 2020, heroin-related mortalities decreased by 6.5%, and prescription-related overdoses decreased by 5% (Ciccarone). Such trends indicate that illicit substances (such as fentanyl) have replaced prescription opioids as the primary drivers of the opioid crisis.

The effects of the third wave of the opioid crisis culminated in a nationwide increase in mortality during the COVID-19 pandemic. Between March 2020 and October 2022, roughly 60,000 additional people died of opioid-related causes than regression models fitted with pre-pandemic overdose deaths data had predicted (Laing et al.). US Life Expectancy decreased for the first time in nearly a century in 2020 and 2021, when Americans experienced the peak of isolation while quarantined from the effects of COVID-19 (Centers for Disease Control and Prevention). It has been determined that the worsening opioid crisis, exacerbated by the effects of the COVID-19 pandemic, was a significant contributing factor to the drop in life expectancy (Graham). The most frequent age at which opioid use begins is sixteen; consequently, the loss of many younger individuals to the opioid crisis had a profound impact on life expectancy, as their deaths represented a significant number of years lost (Austic et al.). The estimated economic burden of opioid use disorder and fatal opioid overdoses in 2017, including costs of healthcare, crime, lost productivity, lost years of life, and worsened quality of life, was $1.02 trillion US dollars (Florence et al.).

As the opioid crisis has magnified, rural areas have been left ill-equipped to handle a rise

in opioid use disorder, fatal overdoses, and non-fatal overdoses. Rural areas of Colorado, North Carolina, Washington, Ohio, and Pennsylvania implemented overdose-abatement policy initiatives such as first responder opioid training, arrest alternatives for opioid offenses, and drug courts at significantly lower rates than metropolitan areas of such states (Swann et al.). In 2020, median driving times to opioid use disorder treatment centers were higher and more variable in rural areas than in urban areas (Kiang et al.). Since most opioid treatment requires daily interventions, barriers such as driving time could be concerning for rural residents. Although over 25% of Georgia’s 2019 population was located within a 15-minute drive of a methadone clinic, 0% of the population of the four counties with the highest opioid overdose death rates were within such a distance, and all five counties with the highest opioid overdose death rates were all designated as rural (Anwar et al.). Despite such evidence that rural areas have been harder hit by the opioid crisis, rurality was not found to be significantly correlated with rates of opioid mortality in Georgia, even after adjusting for demographic and socioeconomic factors (Valentini et al., Roth et al.). Thus, in this comparative study, we hope to address the notable gap in the literature by assessing how opioid prescription rates and overdose deaths progressed between the second and third waves of the opioid crisis, from 2010 until after the pandemic, in Georgia’s rural and urban counties. Understanding the evolution of the opioid crisis into the present will help inform policy initiatives that effectively curb the impact of the opioid crisis across the entire state of Georgia.

Methods

In this study, we performed an analysis of county-level opioid death rates from 2010 to 2023 and county-level opioid dispensing rates

from 2019 to 2023 to identify the impact that prescription opioids had on the progression of opioid-related mortality during and after the COVID-19 pandemic in Georgia. We created visual representations of our findings using choropleth maps and line charts to demonstrate geographic and time series trends within opioid mortality and dispensing. The visual representations were separated into two interactive dashboard frames, with one relating to opioid-related mortality and the other relating to prescription opioid dispensing. The dual frames allowed for a direct comparison between the distribution of legal, prescribed opioid drugs with opioid-related overdose deaths in Georgia. Furthermore, two counties designated as urban (Fulton County) and rural (Madison County) as per the Georgia Department of Revenue were chosen to represent case studies on how the opioid crisis has impacted these counties in Georgia.

Dispensing rate data, defined as the county-level number of retail opioid prescriptions dispensed per 100 people, was obtained from the Centers for Disease Control and Prevention. Data from other states was removed, Georgia counties were placed in alphabetical order, and missing county-year units were identified using Microsoft Excel. When county-level data was not identifiable, null values were inserted to make up for such discrepancies.

Estimates of the number of fatal overdoses in each county during each year between 2010 and 2023 were obtained from the Georgia Department of Public Health’s Online Analytical Statistical Information System. To ensure that the fatal opioid overdose data would be visually comparable across counties on choropleths, county-level opioid overdose death numerical counts were divided by county population estimates, obtained from The Carl Vinson Institute of Government and Census data. Such calcula-

tions led to the production of opioid overdose rates used in our study.

Using Microsoft Power BI, we created two interactive dashboard frames related to opioid overdose death rates and opioid dispensing rates. The overdose frame included a state-level choropleth map of Georgia. Differences between county-level opioid overdose death rates in 2023 and 2010 were marked by a purple-green color gradient, with purple marking an increase and green marking a decrease. The overdose frame included a line chart of opioid overdose counts (measured as the number of opioid overdose deaths per county) aggregated from each county in Georgia, excluding those with null variables. Vertical markers signified important events and dates for the progression of the crisis. The marker labeled “Fentanyl” was placed in 2014, which is when rates of illicit fentanyl use began to rise in the United States (Drug Enforcement Agency). The marker labeled “Emergency” was placed in 2017, which was when President Donald Trump declared the opioid crisis a public health emergency and directed public focus and funds to address the crisis (The White House). Finally,

the marker labeled “Pandemic” was placed at the end of 2019 through the end of 2021. The dispensing frame first included a choropleth map of Georgia. Differences between county-level opioid dispensing rates in 2023 and 2010 were similarly marked by a purple-green color gradient, with purple marking an increase and green marking a decrease. The dispensing frame also included a line chart of the dispensing rate, as measured by the number of opioid prescriptions dispensed per 100 people, aggregated from across Georgia between 2019 and 2023.

Specific functions within Microsoft BI allow for an interactive map. For example, clicking on a specific county within the choropleth or selecting a specific county from the dropdown results in a line chart reflecting opioid overdose death counts for the specified county. This function was used to create a mortality frame and a dispensing frame for the two case study counties, Fulton County and Madison County.

Results

The results of this study took the form of choropleths, line charts, and combined-graph-

1: Statewide Changes in Opioid-Related Mortality Between 2010 and 2023

Figure

ic dashboard frames. Opioid overdose death rates in most of Georgia’s counties were notably higher in 2023 than in 2010 (Figure 1). Additionally, the progression of opioid-related death counts coincided with the event markers placed on the mortality frame line chart. In 2014, as illicit fentanyl use was on the rise, Georgia saw its first major increase in opioid-related deaths (Figure 1). The state saw slight decreases in opioid overdose death

matched statewide trends, with a higher death rate in 2023 than in 2010, a slight decrease in the opioid overdose death count in 2017, and a major surge during the COVID-19 pandemic (Figure 3). Madison County has a population of 32,191 (United States Census Bureau). Opioid overdose death rates were higher in 2023 than in 2010, just as they were in Fulton County and the state aggregate (Figure 4). Opioid overdose deaths also increased dramatically in

counts after 2017 when President Trump declared the opioid crisis a public health emergency (Figure 1). Georgia experienced a major surge in opioid mortality during the pandemic, from 2019 to 2021 (Figure 1). At the same time, however, the prescription opioid dispensing rate fell in nearly every Georgia county during the pandemic (Figure 2). No dispensing rates were identified for Echols County, and little dispensing rate data was available for Glascock, Taliaferro, and Long Counties.

Opioid overdose trends were relatively consistent between rural and urban counties. Fulton County has a population of 1,079,105 and is highly urban (United States Census Bureau). Opioid-related mortality in Fulton County

Madison County during the COVID-19 pandemic.

Trends in prescription opioid dispensing rates between 2019 and 2023 were highly consistent between Madison and Fulton counties, with dispensing rates falling steeply (Figure 5, Figure 6). In both urban and rural areas, rates of opioid overdose were higher in 2023 than in 2010, and opioid-related death counts increased sharply during the COVID-19 pandemic.

Discussion

This study aimed to assess differences in the progression of the opioid crisis between Georgia’s urban and rural counties by measuring

Figure 2: Statewide Changes in Prescription Opioid Dispensing Between 2019 and 2023

the trajectory of opioid-related mortality between 2010 and 2023 and investigating drivers of mortality trends during and beyond the COVID-19 pandemic.

Analysis revealed multiple parallels between opioid-related mortality and prescription opioid dispensing in rural and urban counties. Since the dispensing of prescription opioids fell while opioid-related mortality surged in both Fulton and Madison Counties during the pandemic, increases in mortality were likely due to increases in the use of illicit opioids such as fentanyl. However, opioid-related data exhibited much more variation in rural counties than in urban counties. Madison County’s mortality trajectory, for example, adhered to statewide trends less than Fulton County did, for this reason (Figure 4). Furthermore, the only two counties to experience increases in opioid dispensing during the pandemic were

Figure 3: Changes in Fulton County’s Opioid-Related Mortality Between 2010 and 2023

Figure 4: Changes in Madison County’s Opioid-Related Mortality Between 2010 and 2023

Pulaski County and Towns County, both rural counties with populations under 15,000 (United States Census Bureau). Smaller population sizes may lead to results indicative of random variance rather than evidence-based trends. Even so, increases in opioid-related mortality and decreases in prescription opioid dispensing were broadly consistent across the state of Georgia, indicating that illicit opioids drove surges in opioid-related mortality in both rural

and urban areas throughout the pandemic. Increased use of illicit opioids has significant implications for certain demographic groups affected by the opioid crisis. Of the 1,893 Illinois residents who died from opioid overdoses in 2016, white populations were overrepresented in those whose deaths involved prescription opioids, whereas Hispanic and African American populations were overrepresented among those whose deaths involved illicit opioids

Figure 5: Changesin Fulton County’s Prescription Opioid Dispensing Between 2019 and 2023

Figure 6: Changes in Madison County’s Prescription Opioid Dispensing Between 2019 and 2023

(Abbasi et al.). This data indicates that Hispanic and African American populations are growing more susceptible to the opioid crisis as prescription opioids give way to fentanyl and illicit substances as the most significant drivers of opioid-related mortality.

Moving forward, it is imperative to restrict access to fentanyl and increase access to opioid addiction services. Whether through law enforcement-related initiatives or public awareness campaigns, policymakers should make a concerted effort to reduce the use of fentanyl and prevent future mortality. While the supply of fentanyl must be reduced, there also exists demand for the substance. Making addiction treatment and overdose-reversal drugs such as naloxone more widespread, especially in underserved and rural areas with low treatment center density, will support those already addicted to fentanyl and illicit opioids. For example, Ohio and Virginia have leveraged partnerships with Federally Qualified Health Centers (FQHCs), care centers that are more widespread than methadone clinics, to expand addiction treatment options in rural areas (Recovery Research Institute, Manz et al. 1). Alternatively, increasing the number of residents covered by health insurance through Medicaid expansion and public insurance may allow for further addiction treatment in urban areas that have sufficient treatment center density.

Opioid overdoses are an important sign of community health and wellbeing, often categorized as “deaths of despair” (Beseran et al.). Between 1999 and 2015, unemployment rates were directly associated with opioid overdose deaths in Georgia (Valentini et al.). The relationship between higher unemployment and higher overdose deaths reinforces the “deaths of despair” term, as those experiencing feelings of hopelessness and isolation are more susceptible to fatal opioid overdose (Beseran et al.). The increase in opioid-related mortality

that this study demonstrated occurred across rural and urban Georgia counties because the COVID-19 pandemic provides further evidence that those in despair are more susceptible to negative opioid outcomes. Because the roots of the opioid crisis dig deep into a population’s community health and economic stability, solutions to the opioid crisis will necessitate diverse action. Addressing mental health issues, ensuring an adequate supply of housing, encouraging economic development, and supporting social revitalization will support all of Georgia in reducing the impact that the opioid crisis has on its residents.

In conclusion, the progression of the opioid crisis in Georgia mirrors national trends, such that opioid-related overdose deaths have surged in the wake of the COVID-19 pandemic while the dispensing of prescription opioids has declined. These trends, consistent between rural and urban counties, suggest a noteworthy rise in the use of illicit opioids such as fentanyl, despite difficulties in measuring the use of illegal substances.

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SOCIAL SCIENCES

Ashlyn Kingsley

Class of 2027

Ashlyn Kingsley is a third-year Health Promotion major at the University of Georgia. She is involved in the Pre-Physician Assistant Association, Undergraduate Pediatric Society, and Medicine in Literature book group. Her interests include refugee and immigrant mental health, psychosocial resilience in underserved youth, and rural engagement and healthcare access. She aims to blend clinical practice with public health advocacy to serve communities in need.

THE MODELING OF PUBERTAL TIMING ON PERCEIVED PARENTAL CONTROL AND INTERNALIZING SYMPTOMOLOGY

ASHLYN KINGLSEY

DRS. CHARLES GEIER & ASSAF OSHRI, MORRIGHAN WINGATE

Abstract

Parent-child relationships have been consistently shown to affect the risk for psychopathological symptomology in adolescence. However, adolescents undergo significant psychological and biological changes during puberty, which can moderate the effect of parenting on youth mental health. Previous literature found pubertal timing influenced the strength of the association between parent-child relationship quality and cigarette use, particularly among late-maturing girls. Further, early maturing adolescents with insecure perceived parental relationships exhibited higher levels of anxiety and depressive symptoms. Based on these results, pubertal timing can exacerbate the association between challenging parent-child relationships and youth physical and mental outcomes. Yet, there has been little research on how the influence of parent-child relationships on internalizing behaviors is altered by pubertal timing. To close this gap, data from a sample of 142 adolescents and their parents was used to investigate the hypothesis that the parent-child relationship is associated with the development of internalizing behaviors in youth and that this association is moderated by pubertal timing. Moderation analysis using multiple regression was utilized to evaluate the data from the Child’s Report of Parenting Behavior Index (CRPBI), Youth Self Report (YSR), and Peterson’s Pubertal Development Scale (PDS) to explore the interplay among these factors. The study found that among females with earlier pubertal maturation, there was a stronger relationship between psychological control and both anxious/ depressive symptoms and withdrawal symptoms. This effect was not observed in males. Findings from this research hold implications for health professionals understanding the complex interactions influencing adolescent development and physical and mental health outcomes.

Pubertal Timing

Puberty marks an important transition in adolescent development with its timing playing a crucial role in shaping an individual’s psychological well-being. The timing of pubertal onset, when compared to same-sex, same-age peers, has been found as a significant determinant in the relationship between pubertal maturation and risk for psychopathology in adolescence. The effects of pubertal timing tend to differ between biological sexes. The early timing hypothesis states that early maturing

adolescents may face greater difficulties due to being less prepared for the rapid changes associated with puberty (Shelton and van den Bree 2). This mismatch between physical maturity and emotional readiness can increase vulnerability to internalizing symptoms, such as anxiety and depression, explaining why pubertal timing is a critical factor in adolescent mental health outcomes.

Perceived Parental Control

Types of parental control play a role in the adolescent development of internalizing sympto-

mology. Adolescents tend to experience fewer internalizing symptoms when their parents demonstrate warmth, establish clear boundaries, and grant them autonomy (Gorostiaga et al. 7). Conversely, adolescents are more prone to anxiety, depression, and suicidal thoughts when their parents exert excessive control or resort to harsh disciplinary measures (8). Although the effects are not dramatic, they are notable and should be taken into consideration when creating programs to promote adolescents’ well-being by building positive parenting practices.

Internalizing Symptomology

Internalizing psychopathology refers to a broad category of mental health problems, associated with anxiety and depression. Research shows that internalizing problems are significantly increasing during adolescence (Kanwar 3). Moreover, pubertal timing has been associated with various internalizing problems, such as depression and anxiety, which significantly impact adolescents’ quality of life (2). Early maturation in girls has been linked to depression, while late maturation in boys has been associated with depressive symptoms (3). The contextual amplification model provides a framework for understanding the interplay between early puberty and problem behaviors, emphasizing the role of social contexts in shaping developmental outcomes (Ge et al. 27-29). This perspective acknowledges that the effects of early pubertal timing are shaped by various social and psychological factors, such as family dynamics, peer relationships, and environmental stressors. These factors can either exacerbate or mitigate the challenges associated with early maturation, thereby influencing adolescents’ adjustment and well-being. However, conflicting findings also exist, with some studies reporting no significant relationship between pubertal timing and internalizing problems. For example, while Western studies had substantial evidence

indicating that the timing of puberty predicts problem behaviors in adolescents, Asian studies were inconclusive. Studies have found that early puberty is linked to depression in boys (Strong et al. 354). This connection has also been observed among girls (Chen et al. 89). Earlier puberty has been linked to mental health problems (Fujimara et al. 795), while other studies have not found a connection between pubertal timing and internalizing problems (Chiang et al. 44; Lee et al. 318). Taken together, the research on the effect of pubertal timing on adolescent risk for psychopathology is inconsistent.

Proposed Study

Addressing this gap, this study examines how the perceived parent-child relationship influences the development of internalizing behaviors and how this association is moderated by pubertal timing. We hypothesize higher perceived parental psychological control will correlate with increased somatic, anxious/depressive, and withdrawal symptomology. We further hypothesize that these relationships will be moderated by early pubertal maturation. By understanding how parent-child relationships interact with pubertal timing to influence internalizing behaviors, we can better support adolescents’ mental health and well-being during this critical developmental period.

Methods

Utilizing data from the Child’s Report of Parenting Behavior Index (CRPBI), Youth Self Report (YSR), and Peterson’s Pubertal Development Scale (PDS), we employed moderation analysis using multiple regression to explore these dynamics.

Procedure

Two waves of data were gathered from a sample of 142 adolescents from rural communities in the Southeastern United States with an

average participant age of 12.89 years (SD = 0.85). The participants were recruited through the distribution of flyers in local communities, social media advertisements on various platforms, and referrals from previous participants. Fluency in English and the absence of significant developmental delays were prerequisites for participation. Informed consent and assent were received from the caregivers and adolescents, and both parties were notified that the researchers were mandatory reporters of suspected child abuse and neglect. Approval for all study protocols was derived from the University of Georgia Institutional Review Board . Baseline data were collected from January 2018 to March 2020, while wave 2 data were collected from May 2020 to October 2020. Trained researchers conducted home visits to collect baseline data, while online surveys were utilized during wave 2. Among the participants, 52% were females. The majority of the sample identified as European American (78.8%), followed by African American (11.5%), Latino (3.8%), Asian/Pacific Islander (1%), and other (4.8%).

Measures

Child’s Report of Parenting Behavior Index (CRPBI)

The Child’s Report of Parenting Behavior Index (CRBI) was utilized in this study. The CRBI is a 30-item self-report measure designed to assess the parent’s child-rearing level of control as perceived and reported by children (S. Schludermann and E. Schludermann). Responses were scored on a 3-point scale in which 1 point corresponds to “not like” and 3 points corresponds to “a lot like.” The total scores are summed to categorize the parent into 3 control levels: acceptance, psychological control, and firm control. Acceptance involves behaviors such as emotional support and equalitarian treatment. Psychological control utilizes behaviors for covert control over the child’s behavior . Firm control measures the parent’s ability to establish and maintain

healthy boundaries for the child.

Peterson’s Pubertal Development Scale (PDS)

The Peterson’s Pubertal Development Scale (PDS) was utilized in this study. The PDS is a 5-item self-report measure designed to assess physical development in boys and girls rated on a 4-point scale where 1 indicates no development and 4 indicates completed development (Earls et al.). Girls self-reported their changes in five areas: growth spurts in height, body hair (underarm and pubic hair), skin changes, breast growth, and menarche. Boys self-reported their changes in five areas: growth spurt in height, pubic and underarm hair, skin changes, deepening of voice, and facial hair growth. The total scores are averaged to determine the individual’s overall pubertal stage.

Youth Self-Report (YSR)

The Youth Self-Report (YSR) was utilized in this study. The YSR is a 112-item child self-report measure designed to assess behavioral competency and behavioral problems on a 3-point scale (0-not true, 1-somewhat true, 2-very true) (Achenbach). The YSR provides scores for DSM-oriented scales, including anxiety, somatic, and withdrawn behaviors. The self-report measure also assesses “Total Competency,” which measures competency in activities, social functioning, and academic performance.

Statistical Analyses

All statistical analyses were conducted using R Statistical Software (R Core Team). Descriptive statistics were calculated using the psych R package to assess normality and means for each variable used in the analysis (Revelle). Correlation tests were then run between the predictor variable (perceived parental psychological control) and outcome variables (internalizing symptoms). Spearman’s correlations were run due to the non-normality of the data. Thus, Spearman’s rank correlation ρ was used to calculate the association between variables,

while pairwise complete observations were used to account for missing data. The appropriate variables were then centered by subtracting the mean from each data point. Multiple linear regression was used to build and assess each moderation model for statistical significance. The age and race of participants were considered as covariates in each model. Covariates, main effects, and interaction effects were then compared using the Stargazer R package (Hlavac). After testing for the significance of effects, overall model significance and variation explained by the proposed model were evaluated. All significant interactions were then visualized through simple slope plots included in the interactions R package (Long).

Results

The study found no significant correlation between perceived parental psychological control and somatic symptoms. Additionally, early maturation did not significantly moderate the relationship between psychological control and somatic symptoms. While no significant correlation was found between perceived parental psychological control and anxious/depressive symptoms, a significant interaction (β = 0.15 **, SE = 0.07, p < 0.05) was observed for females, indicating a strengthened relationship between psychological control and symptoms among females with earlier pubertal maturation. Pubertal maturation did not moderate the relationship between psychological control and anxious/depressive symptoms in males. Additionally, a significant interaction (β = 0.10 *, SE 0.06, p < 0.10) was observed between psychological control and withdrawal symptoms for females, suggesting a strengthened relationship between these variables among females with earlier pubertal maturation. Pubertal maturation did not moderate the relationship between psychological control and withdrawal symptoms in males.

Figure 1: Simple Slope Model of Youth Anxiety/Depression Symptoms and Perceived Parental Psychological Control Across Female Pubertal Timings

Note: The The simple slope shows associations between youth self-report anxiety and depression symptoms and youth perceived parental psychological control by levels of pubertal development (±1 SD from the grand mean). Variables on the x and y axis are centered.

Figure 2. Simple Slope Model of Youth Withdrawal Symptoms and Perceived Parental Psychological Control Across Female Pubertal Timings

Note: Simple slope showing the associations between youth self-report withdrawal symptoms and youth perceived parental psychological control by levels of pubertal development (± 1 SD from the grand mean). Variables on the x and y axis are centered.

Discussion

Affective psychopathology increases during adolescence, a developmental period in which youth undergo significant psychological and biological changes due to puberty. The provided graphs plot significant interaction terms. Figure 1 shows the association between self-report anxiety and depression symptoms and parental psychological control as moderated

by pubertal maturation timing for females. Those showing early maturation (higher pubertal development scores) tend to report more anxiety and depression symptoms, as well as more perceived parental psychological control. This association can be explained through the contextual amplification hypothesis, which suggests that familial contexts play an important role in moderating the effects of early pubertal maturation on adolescent developmental outcomes (Ge et al. 29-30). These results indicate that early maturation may have intensified the negative consequences of perceived parental control, leading to increased internalizing symptomology. However, no significant correlation was found between perceived parental psychological control and withdrawal symptoms in girls. Figure 2 shows the linkages between self-report withdrawal symptoms and parental psychological control as moderated by maturation timing for females. Those showing early maturation (higher pubertal development scores) tend to report more withdrawal symptoms, as well as more perceived parental psychological control. This further supports the idea that pubertal timing moderates adolescents’ psychosocial responses to parental practices, especially among early maturing girls who appear most vulnerable to adverse familial contexts. These findings provide valuable insights into the nuanced interplay between perceived parental psychological control, pubertal timing, and internalizing symptomologies among adolescents, highlighting the importance of considering gender-specific dynamics in understanding developmental outcomes.

The implications of these findings are significant for health professionals understanding the complex interactions influencing adolescent development and physical and mental health outcomes. Health professionals should be made aware of these risk factors, as they can consider how physical health, mental health,

and the perceived climate of formative relationships are impacted by each other through a biopsychosocial approach. In addition, the study highlights the significant role of pubertal timing, specifically earlier pubertal maturation, in the relationship between perceived parental psychological control and anxious/ depressive symptoms and withdrawal symptoms in females. These findings call for unique prevention and intervention efforts aimed at mitigating the adverse effects of parental psychological control when considering the specific challenges faced by early maturing females.

Works Cited

Achenbach, Thomas M. Manual for the Youth Self-Report and 1991 Profile. University of Vermont, Department of Psychiatry, 1991.

Chen, Jie, et al. “The Influence of Pubertal Timing and Stressful Life Events on Depression and Delinquency among Chinese Adolescents.” Psych J, vol. 4, no. 2, 2015, pp. 88-97, Medline, doi:10.1002/pchj.83.

Chiang, Huey-Ling, et al. “The Association between Pubertal Development and Emotional/Behavioral Problems, Substance Use, and Suicidality among Adolescents.” Taiwanese Journal of Psychiatry, vol. 24, 2010.

Earls, Felton J., et al. Project on Huma Development in Chicago Neighborhoods (PHDCN): Master File, Wave 2, 1997–2000. Harvard Medical School, 2002. Distributed by Inter-university Consortium for Political and Social Research.

Fujimura, Yuko, et al. “The Relationship between Quality of Life and Pubertal Timing in Adolescence: The Toyama Birth Cohort Study, Japan.” J Adolesc Health, vol. 65, no. 6, 2019, pp. 790-98, Medline, doi:10.1016/j. jadohealth.2019.07.004.

Ge, Xiaojia, et al. “A Contextual Amplifica-

tion Hypothesis: Pubertal Timing and Girls’ Emotional and Behavioral Problems.” Understanding girls’ problem behavior: How girls’ delinquency develops in the context of maturity and health, co-occurring problems, and relationships, 2011, pp. 11-29, doi:10.1002/9780470977453.ch1.

Gorostiaga, Arantxa, et al. “Parenting Styles and Internalizing Symptoms in Adolescence: A Systematic Literature Review.” Int J Environ Res Public Health, vol. 16, no. 17, 2019, Medline, doi:10.3390/ijerph16173192.

Hlavac, Marek. stargazer: Well-Formatted Regression and Summary Statistics Tables. R package version 5.2.3, 2022, https:// CRAN.R-project.org/package=stargazer.

Kanwar, Palak. “Role of Pubertal Timing and Perceived Parental Attachment in Internalizing Problem Behaviours among Adolescents.” Psychol Rep, 2024, p. 332941241226684, Publisher, doi:10.1177/00332941241226684.

Lee, Chih-Ting T. et al. “Longitudinal Effects of Self-Report Pubertal Timing and Menarcheal Age on Adolescent Psychological and Behavioral Outcomes in Female Youths from Northern Taiwan.” Pediatr Neonatol, vol. 58, no. 4, 2017, pp. 313-20, Medline, doi:10.1016/j. pedneo.2016.04.004.

Long, Jacob A. interactions: Comprehensive, User-Friendly Toolkit for Probing Interactions. R package version 1.1.0, 2019, https:// cran.r-project.org/package=interactions.

R Core Team. R: A Language and Environment for Statistical Computing. R Foundation for Statistical Computing, 2023, https://www.R-project.org/.

Revelle, William. psych: Procedures for Psychological, Psychometric, and Personality Research. Northwestern University, R package version 2.3.6, 2023, https://CRAN.R-project. org/package=psych.

Schludermann, S, and E Schludermann. Questionnaire for Children and Youth (CRPBI-30). Unpublished manuscript, University of Manitoba, 1988.

Shelton, Katherine H., and Marianne B. van den Bree. “The Moderating Effects of Pubertal Timing on the Longitudinal Associations between Parent-Child Relationship Quality and Adolescent Substance Use.” J Res Adolesc, vol. 20, no. 4, 2010, pp. 1044-64, Medline, doi:10.1111/j.1532-7795.2010.00643.x.

Strong, Carol et al. “Childhood Adversity, Timing of Puberty and Adolescent Depressive Symptoms: A Longitudinal Study in Taiwan.” Child Psychiatry Hum Dev, vol. 47, no. 3, 2016, pp. 347-57, Medline, doi:10.1007/ s10578-015-0570-y.

TECHNOLOGY,

ENGINEERING, AND MATHEMATICS

Erika Landree

Class of 2026

Erika Landree is a Biological Engineering student in the Gomillion Lab, where she studies 3D printed breast cancer bone metastasis models. Her research experiences also include an internship at Poly-Med, Inc., studying bioresorbable medical devices and participation in an NSF REU program, assessing the effects of space-related magnetic fields on neuronal networks with Dr. Kunze. After graduation, she plans to pursue a Ph.D. in biomedical engineering.

DEVELOPMENT OF A NOVEL PIEZOELECTRIC MODEL TO STUDY BREAST CANCER BONE METASTISIS

ERIKA LANDREE DR. CHERYL GOMILLION

Abstract

Bone has been identified as the most prevalent site for breast cancer metastasis; however, the cause is not fully understood. In vivo, bone tissue experiences mechanical loading and creates electrical signaling as a result of piezoelectric properties. Evidence suggests that breast cancer cells undergo increased proliferation and migration in the presence of electrical fields caused by the electrical signaling of bone cells. Three-dimensional (3D) in vitro models have been widely investigated in an attempt to better understand the underlying mechanisms for breast cancer metastasis; however, current models fail to take into account the piezoelectric effect on breast cancer behavior. This work aims to develop a piezoelectric 3D in vitro breast cancer bone metastasis model to more accurately observe the interactions between breast cancer cells and the bone microenvironment. This model consists of a 3D printed scaffold made of polycaprolactone (PCL), demineralized bone matrix (DBM), and polyvinylidene fluoride (PVDF). Preliminary results show the PVDF-loaded scaffold provides a piezoelectric effect within the model when subjected to mechanical stimulation. A transwell migration assay was used to compare the migratory behavior of low-metastatic (MCF-7) and high-metastatic (MDA-MB-231) breast cancer cells. Recent results indicate that the addition of a piezoelectric component into the model recapitulates in vivo metastatic behaviors. This work indicates that the addition of a piezoelectric component can increase the physiological similarity of in vitro modeling systems. With a more physiologically accurate modeling system, novel cancer treatments and breast cancer behavior can be investigated.

Introduction

Breast cancer is currently the most common cancer diagnosis in the United States and remains a serious public health concern (“Cancer Statistics”). The disease disproportionately affects women, with a lifetime risk of 13.1%. Although the overall 5-year survival rate of female breast cancer is 91.2%, this number sharply drops to 31.9% once metastasis occurs (“Cancer Stat Facts: Female Breast Cancer”). Metastasis is classified by the spread of cancer cells to other parts of the body. Common metastatic sites for breast cancer include the bone,

brain, liver, and lungs.

Treatments for metastatic breast cancer such as chemotherapy, hormone therapy, targeted drug therapy, and immunotherapy, often result in significant reductions in quality of life and do not guarantee survival (“Metastatic Cancer: When Cancer Spreads”). These limitations highlight the need for a better understanding of the disease and more effective disease management.

To address these limitations, researchers have investigated three-dimensional (3D) models in an attempt to better understand the underly-

ing mechanisms for breast cancer metastasis. In comparison to traditional two-dimensional (2D) cultures, 3D models can more accurately mimic the structure, mechanical properties, and cell-cell interactions of human tissue. Nonetheless, current models fail to fully mimic the complexities of breast cancer cell interactions with surrounding microenvironments.

Recent advancements in tissue engineering have paved the way for the development of more physiologically accurate 3D modeling systems. Tissue engineering is an area of research that aims to develop biological substitutes for damaged or diseased tissues, most often using a combination of cells and biomaterial scaffolds that support cell growth and subsequent tissue formation. Tissue engineering-based approaches are increasingly being adapted for the development of 3D tissues that could be used as models of diseased tissue. Tissue-engineered 3D systems hold great potential for replicating the conditions of metastatic sites, enhancing our understanding of breast cancer metastasis, and providing a better model for testing novel therapeutics.

With so many unknowns surrounding cancer metastasis, researchers are working to fill these gaps by more accurately modeling breast cancer bone metastasis in vitro. Breast cancer spreads to bone in 70% of metastatic breast cancer cases which makes it a critical area of study (Pulido et al.). Accurate in vitro models bring science closer to understanding the key cells, proteins, and biochemical signals involved in this process, improving our grasp of the mechanisms that drive metastasis.

3D In Vitro Breast Cancer

Bone Metastasis Models

Bones are a dynamic organ, periodically adjusting to the demands of the body. Throughout one human lifetime, bones break, fracture, or micro-crack under the stresses of daily life. Despite the damage, bone stays healthy by

removing the damaged tissue and replacing it with new healthy bone in a process known as remodeling. Bone tissue is made up of three types of bone cells: osteoblasts, osteoclasts, and osteocytes. Osteoblasts are responsible for forming new bone, osteoclasts are responsible for breaking down old or damaged bone, and osteocytes act as a mechanical sensor that regulates the process of remodeling (Katsimbri).

Bone remodeling happens in five phases: activation, resorption, reversal, formation, and termination. Once remodeling is activated via hormone or mechanical cues, osteoclasts are recruited and signaled to differentiate into mature osteoclasts during the activation phase (Yang and Liu). Once mature osteoclasts have attached to the bone, the resorption phase begins. Osteoclasts resorb bone tissue through the creation of an acidic resorptive pit. Carbonic anhydrase catalyzes the reaction within the cell, shown in Equation (1) which produces the hydrogen ions needed to create an acidic environment.

(1) CO2+ H2 O --> H++HCO3-

ATPase then pumps the hydrogen atoms out of the cell to create an acidic environment with a pH of approximately 4 (Kennedy). This environmental acidity will cause protonation of amino acid side chains causing disruptions in protein folding, known as protein denaturation. Bone matrix protein denaturation will cause matrix degradation and resorption. Once resorption is complete, osteoclasts undergo apoptosis.

During the reversal phase, reversal cells are recruited to clear the debris made by osteoclasts (Bolamperti et al.). Osteoblasts are recruited to initiate the bone formation stage. During this time, osteoblasts synthesize a new matrix of proteins, primarily made of Type I collagen (Zhu et al.). This protein matrix is then mineralized by osteoblasts to produce bone over a

period of 4 to 6 months. Once osteoblasts are done forming new bone, remodeling is terminated. 50-70% of osteoblasts will undergo apoptosis, while the rest transform into bone lining cells or become buried in the bone tissue and slowly differentiate into osteocytes (Katsimbri).

The remodeling process can be triggered by hormonal or mechanical cues. Hormonal signals from parathyroid hormone (PTH) or oestrogen (also known as estrogen) commonly signal bone remodeling. Remodeling can also be activated by mechanical cues. When damage occurs, normal forces acting upon bone will cause asymmetric loading on the weakened parts of the tissue. In bone, this physical deformation is converted to electrical signaling because bone tissue is piezoelectric. In piezoelectric materials, asymmetric mechanical force displaces ions unevenly leading to polarization; this polarization generates electricity (see Fig. 1). It is thought that osteocytes can sense the electric gradient and respond by stimulating bone remodeling (Mohammadkhah et al.). Understanding the bone remod-

eling process is crucial for gaining insight into how diseases, like breast cancer, exploit these mechanisms and for identifying potential therapeutic targets.

In the multi-step process of breast cancer metastasis (see Fig. 2), cells from a cancerous tumor detach from the primary tumor in the breast, circulate through the body via lymph fluid or blood, and settle on a new tissue, in this case, bone. Once localized at these new tissues, the tumor cells release growth factors triggering osteoclast proliferation and inhibiting osteoblast formation.

Bone Tissue Microenvironment and Breast Cancer Metastasis

Currently, animal models are used to provide preliminary data for clinical trials. It is widely known that animal models differ significantly from humans in terms of physiology and biology, yet they remain our primary tool for collecting preclinical data. There is a large movement to develop highly accurate in vitro models that can mimic human physiology better than animal models and traditional 2D

Fig. 1. Mechanical remodeling of bone tissue. Electrical signaling is observed in bone under applied mechanical loading. Created with biorrender.com.

in vitro models (see Fig. 3). The development of 3D in vitro models would minimize the need for animal testing, decrease the cost per model, address ethical concerns, and provide a better understanding of experimental outcomes before moving to clinical trials.

The goal of three-dimensional (3D) in vitro models is to most accurately mimic the physiological environment in a lab setting. With biomimetic models, we can start to answer some of the lingering questions that exist related to breast cancer metastasis. As of now, it is largely unknown why breast cancer preferentially spreads to the bone, lung, liver, and brain tissue. By successfully mimicking bone tissue, we can learn more about the spread of breast cancer cells to bone tissue without the use of animal models and clinical trials.

It is known that breast cancer affects the

process of bone remodeling (Tahara et al.) (see Fig. 4). With so many ties between bone tissue remodeling and disease, it is important to capture this dynamic activity in vitro. To do this, we propose that introducing bioelectricity into a novel 3D breast cancer bone metastasis model will increase the physiological accuracy of the modeling system. To achieve this effect, our model consists of a 3D printed scaffold made of polycaprolactone (PCL), demineralized bone matrix (DBM), and polyvinylidene fluoride (PVDF). PVDF is a widely known piezo-polymer that produces an electrical output when mechanically stimulated. In this model, ultrasound will be used to deliver mechanical stimulation. Through this convention, users can modulate how frequently this biomimetic bone tissue is experiencing mechanical loading. This gives researchers the ability to investigate how differing amounts of piezo-

Fig. 2. Breast cancer metastisis to the bone microenvironment. Created with biorender.com

electricity impact bone remodeling and consequently breast cancer migration.

Bone is remodeled through a dynamic process facilitated by biophysical cues that support signaling pathways. For bone tissue, this signaling is regulated by cells and the ECM and transmitted via electrical synapses. The mechanosensitive property of bone cells has been shown to play an important role in breast cancer metastasis to bone. In vivo, bone tissue experiences mechanical loading and creates electrical signaling as a result of this piezoelectric effect. Breast cancer cells take advantage

Fig. 3. 3D in vitro models bridge the gap between traditional 2D in vitro models and 3D in vivo animal models. Created with biorender.com

Fig. 4. Effects of breast cancer on bone homeostasis. Created with biorender.com

of this nutrient-rich, remodeling environment when invading bone tissue. Metastatic breast cancer cells have recently been shown to exhibit varying levels of electrical excitability depending on metastatic potential (Ribeiro et al.). The mechanisms breast cancer cells use to interact with the bone microenvironment and piezoelectricity are not fully understood, and existing bone metastasis models lack this component. Thus, by incorporating a piezoelectric component into our model, we will be able to observe these interactions to further understand the underlying mechanisms. It is known from previous work that electrical stimulation will result in increased osteogenic differentiation and calcium deposition for bone cells integrated within the scaffold (Dixon et al.). It is hypothesized that ultrasonic treatment will result in increased proliferation and migration of breast cancer cells with the most migration observed for highly aggressive cell lines.

Materials and Methods

To evaluate breast cancer cell migration in the

novel piezoelectric model, bone scaffolds were fabricated using 3D pneumatic printing. Briefly, bone cells were differentiated in the piezoelectric models to develop ECM and mature osteoblasts; then, breast cancer cell migration was measured within the model.

Composite Scaffold Preparation

Human femurs were used to create the demineralized bone matrix (DBM) powder (<125 μm) for this study. Briefly, diaphyseal cortical/ cancellous sections (~1 cm thick) were first prepared using a chop saw before being further segmented into blocks (~1×1×1 cm). Bone segments were defatted, ground, and demineralized using an established lab protocol (Dixon et al.). Bone particles were then sorted. Particles <125 μm were collected and used for this study.

Bioactive composites were created via ambient wet mixing of the DBM powder and polymer solution at an optimized weight ratio of 50:50. Firstly, a PCL solution and a separate PVDF solution were created (10% wt/vol in dichloromethane and dimethylformamide, respective-

Fig. 5. Steps taken for composite formation. Created with biorender.com

Fig. 6. Piezoelectric bone model fabrication. 3D porous scaffolds were made by dissolving polycaprolactone (PCL) in dichloromethane (DCM) and polyvinylidene fluoride (PVDF) in dimethylformamide (DMF) then fabricated using a BIO X pneumatic bioprinter and cut to size using a metal punch.

ly). PCL and PVDF solutions were then mixed together on a stir plate until uniformity was reached (see Fig. 5). DBM powder was added to the solution (50% wt/vol in polymer solution) in an open-air environment (i.e., chemical fume hood) and sonicated for 30 minutes at 30°C until the solution reached an ideal viscosity through solvent evaporation.

3D Model Fabrication

Model scaffolds were fabricated using a Cellink 3D pneumatic BIO X ™ printer (see Fig. 6).

A volume of 3 mL of the bioactive composite solution was loaded into a printing cartridge and printed at 110 kPa, 50 mm/s, and room temperature. The composite solution was submerged in deionized water during the critical drying period. Scaffolds were left to dry overnight. After full solvent and water evaporation, films were cut into circles (16-mm diameter) with a metal punch and press fit into 24-well Ultra-low Attachment plates (Corning). Films were pre-conditioned in culture media at 37°C and 5% CO2 in an incubator for 2 hours prior to cell seeding.

Cell Culturing and Seeding

A multi-step approach was used for preparing the bone model niche and then evaluating breast cancer cell migration. Firstly, bone cells were seeded onto the fabricated films and differentiated for 7 days to develop an extracellular matrix within the models. Breast cancer cells were subsequently seeded into the top chamber of a 6.5 mm Transwell® with 8.0 μm Pore Polycarbonate Membrane Insert (Corning) and then monitored over the next 12 hours.

Murine preosteoblast bone cells (MC3T3-E1) were cultured in alpha Minimum Essential Medium (α-MEM) with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (P/S) until experimental numbers were reached. MC3T3-E1 cells were harvested and seeded onto pre-conditioned films in Corning 24-well

Ultra-low Attachment plates at 90% confluency. MC3T3-E1 cells were differentiated for 7 days in high glucose Dulbecco’s Modified Eagle’s Medium (DMEM, high glucose) with 10% FBS, 1% (P/S), 8 mM beta-glycerophosphate disodium salt hydrate (β-GP), and 50 mg/mL ascorbic acid. A breast cancer cell line with low metastatic potential (MCF-7) and another with high metastatic potential (MDA-MB-231) were cultured in low glucose DMEM complete (10% FBS, 1%P/S) with and without 0.01mg/mL bovine insulin, respectively. Before seeding, MCF-7 and MDA-MB-231 cells were stained with CellTracker Green fluorescent probes (25 μM solution) and incubated overnight. MCF-7 and MDA-MB-231 cells were incubated in the CellTracker Green solution overnight.

Xylenol Orange Mineral Staining

Xylenol Orange (XO) was used to stain calcium deposits from differentiated MC3T3-E1 cells.

A 0.1% XO solution was added by volume and incubated overnight (at 37° C and 5% CO2). Once the models were ready for viewing, the XO stain was aspirated, and the scaffolds were rinsed with Dulbecco’s Phosphate Buffered Saline (PBS) before viewing. Calcium deposits were viewed and imaged under a TRITC filter at 10× magnification.

Cell Counting Kit-8 Assay

Cell Counting Kit-8 (CCK-8) was used to quantitatively determine the total number of cells in each well over the 21-day cell study. 10% by volume (50 μL for a 24 well plate) of the pre-prepared CCK-8 solution was added to each well and incubated (at 37 ° C and 5% CO2) for 1 hour and 30 minutes. Two replicate samples were taken per well and absorbance was read at 450 nm.

Quantification of Cell Migration

To quantitatively determine the number of cells that migrated to the bone model, a transwell migration assay was used (see Fig. 7). Breast cancer cells were seeded onto transwell

inserts on Day 7 of MC3T3-E1 differentiation. Directly prior to placing the transwell inserts seeded with cancer cells into the wells containing bone scaffolds, the piezoelectric group received ultrasonic treatment for 30 minutes at 37°C. Then, the transwell inserts containing breast cancer cells were placed into the wells containing bone models. After a 6-hour period of attachment, transwell inserts were rinsed twice in room temperature Dulbecco’s phosphate buffered saline (DPBS). NucBlueTM Live ReadyProbesTM was used to stain the cell nuclei. One drop of NucBlueTM reagent was added to 0.5 mL of cell culture media and swirled to mix. The transwell inserts were placed into cell culture wells containing the reagent and left to sit protected from light, at room temperature, for 20 minutes. After staining the nuclei, the top chamber of the transwell inserts were swabbed to remove cells from the top of the membrane, effectively removing all cancer cells that had not migrated. After swabbing, the inserts were transferred to a new 24-well plate for imaging.

The underside of the transwell membranes were imaged using DAPI and GFP fluorescent filters at 10× magnification using a Cytation 1 imaging reader (BioTek, Agilent Technologies). A cell count was performed using the Gen5 software (Agilent) to count the number of migrated cells for each sample (Appendix 1).

Results and Discussion

Material characterization was performed to determine the chemical properties of the modeling systems. Cells were counted to quantita-

tively analyze cell proliferation. Models were stained using a calcium stain to qualitatively observe differentiation. Cancer cells were stained using fluorescent probes to qualitatively observe migration.

Xylenol Orange Mineral Staining

Images of Xylenol Orange staining from Days 3 and 7 are compared in Fig. 8. A significant increase in stained calcium deposits can be observed between the two timepoints.

When bone cells differentiate, they start producing calcium when they become mature osteoblasts. The increase in calcium deposits (see Fig. 8) confirms osteogenic differentiation and the development of the ECM, completing our model.

Fig. 7. Timeline of transwell-migration study. After differentiating bone cells within the model for 7-14 days, breast cancer cells were introduced, and migration was recorded using a cell count.

Fig. 8. Calcium deposits observed after 3 (top) and 7 (bottom) days of MC3T3-E1 cell differentiation

Cell Counting Kit-8 (CCK-8)

The CCK-8 assay provided quantitative data of the total number of cells in each well throughout the study. The total number of bone cells during osteogenic differentiation is shown in Fig. 9.

Fig. 9. Cell Counting Kit-8 was used to quantify the relative number of bone cells within the bone model during the 7 days of differentiation. No significant increase is observed.

Statistical analysis shows no significant change in the number of bone cells within the model over 7 days of differentiation. When bone cells differentiate, they initially grow rapidly until confluency is reached then, proliferation stops as osteogenic expression increases (Quarles et al.). These results lead to the conclusion that bone cells are present and entering osteogenic lineage within the model, quantitatively confirming the results seen in Xylenol Orange imaging.

Quantification of Cell Migration

A cell count was performed to quantitatively determine the number of breast cancer cells that had migrated toward the piezoelectric bone model. The number of migrated breast cancer cells (MCF-7 and MDA-MB-231) in both treatment groups (piezoelectric and non-piezoelectric) is shown in Fig. 10.

At 6 hours after introducing the breast cancer cells into the modeling system, a significant difference was shown between cancer cell lines. The aggressive breast cancer cell line displayed significantly more migration than the cancer line with low aggression, just as we see in vivo. Without the piezoelectric component, the two cancer types did not behave differently. At 12 hours after introducing the breast cancer cells into the modeling system, significant differences are seen between the cell lines in both treatment groups. These results can be explained by one of two reasons: Transwell migration is affected by gravity which is a common limitation in this type of experiment, or ultrasonic treatment was delivered only once before the cancer cells were introduced.

Fourier Transform Infrared Spectroscopy (FTIR)

To assess the piezoelectricity of the 3D models, FTIR was used to analyze the chemical properties of prepared samples. Figure 11 shows the FTIR spectra of scaffolds fabricated with pneumatic printing at different pressures and solvent casting with and without solvent exchange with DI water.

The FTIR spectra of the films and scaffolds show beta-phase absorption peaks at 1275 cm−1 and 840 cm−1 and α-phase absorption peaks around 795 cm−1 and 760 cm−1. The FTIR spectra of the scaffolds used in this experiment (printed at 110 kPa) show strong beta-phase absorption peaks at 1275 cm−1 and 840 cm−1 and weak α-phase absorption peaks around 795 cm−1 and 760 cm−1.

Strong β-phase peaks can be explained by the interaction between PVDF and water during the critical drying period. The hydroxyl group (O-H) in deionized water molecules and the fluorine atoms in PVDF interact to form an all-trans chain formation (TTT). All-trans chain formation is shown in the β-phase

peaks at 1275 cm−1 and 840 cm−1 in Fig. 11. All-trans formation increases the piezoelectric properties of PVDF necessary for creating a biomimetic bone scaffold (Tu et al.). The shear stress caused by pneumatic printing also explains strong beta-phase peaks. High pressures through a small diameter nozzle during fabrication expose the polymer to high levels of shear stress. Shear stress has been shown to increase the beta-phase content of PVDF (Dixon).

Conclusion

Bone metastasis of breast cancer is a prevalent disease in women, resulting in long-term pain and poor patient outcomes. Over the last 50 years, promising treatments have emerged to

treat bone metastasis. Targeted treatments, like those targeting hormone receptors estrogen and progesterone, are used to attack cancer cells with specific characteristics. Meanwhile, chemotherapy and radiation therapy are common non-targeted therapeutics and remain a standard of care for metastatic cancers, despite major side effects, like nausea, vomiting, hair loss, fertility issues, etc. (DeMarco; “Chemotherapy”). Metastasis of breast cancer remains the leading cause of mortality among patients, prompting scientists to focus on preventing its spread to improve survival rates and patient outcomes.

In recent years, 3D in vitro models have emerged as a promising platform for studying

Fig. 10. Number of migrated breast cancer cells with (+) and without (-) piezoelectricity.

Fig. 11. FTIR spectra of solvent cast films fabricated with and without critical drying in DI water and scaffolds printed at 8 kPa and 110 kPa (both exposed to DI water). Solvent cast film without exposure to DI water is shown by the red line; the solvent cast film with DI water exposure is shown in green; the scaffold printed at 8 kPa is shown in dark blue the scaffold printed at 110 kPa is shown in purple.

the spread of breast cancer cells to bone tissue. These models are highly tunable and provide unlimited opportunity for treatment testing unlike traditional 2D models and 3D in vivo, or animal models. While recent advancements have greatly improved the efficacy of 3D in vitro models, many fail to completely mimic the complex biochemical and biomechanical environment of human bone. Most notably, current models fail to recreate the piezoelectric properties of bone tissue, which convert mechanical forces into electrical signals that regulate bone remodeling. The remodeling process is critical to bone homeostasis and is disrupted during breast cancer invasion (Tahara et al.). To recapitulate the biomechanical properties of bone tissue, a novel scaffold was created using PVDF to mimic the piezoelectric properties of bone. Mechanical stimulation (ultrasonic treatment) can effectively activate PVDF, generating an electrical field in response to applied stress. This process mimics the natural electrical signals produced by bone tissue under mechanical loading, which are critical for bone remodeling and cellular signaling.

Overall, this work has demonstrated that piezoelectricity in a bone model can affect the migration of breast cancer cells. The findings suggest that the piezoelectric properties of bone tissue play a significant role in breast cancer metastasis and should be considered when designing biomimetic modeling systems aimed at investigating the mechanisms of breast cancer spread to bone.

Acknowledgements

The authors acknowledge Dr. Damion Dixon and Kyndra Higgins for technical assistance. The authors gratefully acknowledge MTF Biologics for providing tissue samples through their Non-Transplantable Tissue Program. Funding for this work was provided by the National Science Foundation CAREER Award #2145521.

Appendix

The Cell Count feature within Agilent’s Gen5 imaging software was used to perform a cell count via stained nuclei. The membrane pores can be visualized in Fig. 12 as small green rings. The top of the membrane was swabbed prior to imaging to remove non-migrated cells. Migrated cells were subsequently imaged on the underside of the membrane. Cell bodies were probed with Cell Tracker Green (Invitrogen) and used to confirm the presence of a cell, while cell nuclei are shown in blue using NucBlue probes (Invitrogen). The cell count was performed by counting the number of cell nuclei. The pores of the transwell membrane reflected light on both the DAPI and GFP channels, causing the pores to sometimes be recognized as a cell nucleus. To remedy this and increase overall efficacy, a minimum size filter was set to 12 μm; only objects larger than an 8 μm pore could be counted as a cell nucleus.

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Fig. 12. Image of migrated breast cancer cells (MDA-MB-231). Cell bodies are probed with Cell Tracker Green and used to display the presence of a cell. Cell nuclei are shown in blue using NucBlue probes. Cell count software is used to count the number of nuclei.

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This edition of the Journal for Undergraduate Research Opportunities would not be possible without the help and support of:

Andrea Silletti Morehead Honors College Staff

Stephanie Schupska

Research Mentors and Principal Investigators

James Byars

Dr. Charles Geier

Dr. Cheryl Gomillion

Dr. Erin Little

Dr. Asaaf Oshri

Morrighan Wingate

Dr. Nina Wurzburger

With special thanks and recognition to President Jere W. Morehead