Little is known about how male and female Pieris rapae behave during territorial behaviors, making this project particularly significant. Through our previous experiment, we have uncovered intriguing insights into the unique behaviors exhibited by male P. rapae when interacting with pseudo-females (males in disguise). Our observations suggest a complex interplay between competitive instincts and courtship behaviors. Specifically, male Pieris rapae display distinct responses when encountering pseudo-females, indicating a potential recognition of the sex of the interacting butterfly. This recognition may trigger competitive behaviors like a territorial move if perceived as a rival male or foster a display of courtship if perceived as a potential mate. Additionally, we aim to study how female P. rapae interact with other females to determine if there are any behavioral differences in these interactions. By studying the intricacies of both male and female courtship and/or territorial behaviors in P. rapae, this project seeks a deeper understanding of the factors shaping mating systems and reproductive success in this species and beyond.

As climate change increases temperature, Pieris rapae caterpillars' feeding habits may be affected. As their feeding habits are altered, P. rapae may begin to migrate to different plants; this can be detrimental for agriculture because these defoliating caterpillars are pests. P. rapae caterpillars are known to feed on Brassica species, including collards and kale. It is also shown that caterpillars increase their feeding rate at higher temperatures (Kingsolver 2000). However, little is known about how temperature influences their feeding preferences. In this experiment, we find the consumption rate of P. rapae 4th instar larvae eating kale (Brassica oleracea var. sabellica) and collards (Brassica oleracea var. viridis) to find consumption preference between these two plants at 14°C, 23°C, and 35°C. Larvae were placed on a moist filter paper in petri dishes containing 2 collard and 2 kale leaf disks placed in an alternating fashion. Petri dishes were placed in three separate incubators set to the three temperatures. We predicted that P. rapae would have a preference for collards since they are reared on collards in the lab and they would increase their consumption of the preferred plant. We also examined the percentage per hour of each leaf eaten and compared this data between types of leaves and temperatures. The data showed that as temperatures increased, the consumption rate of P. rapae caterpillars also increased. Though, there was no change in preference as the P. rapae caterpillars consistently preferred collards over kale. This suggest that higher temperatures from climate change will increase the rate at which caterpillars eat, but will not affect preference. It is important to consider the change in consumption rate of caterpillars with temperature when aiming to prevent crop damage in the face of climate change.

The focus of this research was to test the effectiveness of a silicone-based paint in the marking of Pieris rapae in a manner that was non-invasive and durable. Previous studies have tested other marking methods but have faced challenges such as harm to the organism. By using a paint made from red cabbage (Brassica oleracea), I aim to minimize the harm to larvae in current marking methods while retaining durability under moist conditions. This experiment examined both the durability and health effects of cabbage-based paints on P. rapae caterpillars. Preliminary experiments tested a water-based version, which did not appear to affect survival but faded under moist conditions, and a silicone-based version, which withstood moisture but raised concerns about potential effects on health due to the additional ingredients required for the silicone base. My research continued testing the cabbage paint with a cosmetic-grade dimethicone base and aimed to determine the extent that the cabbage paint may have on caterpillar health and survival. Survival experiments were conducted on 4th and 5th instar caterpillars to determine larva mortality rates when exposed to the pigment. Weight change experiments were conducted from the 4th instar to pupation to be used as a metric of the overall health of the larvae. I conclude that the silicone-based cabbage pigment is a promising marking method for larger caterpillars, offering improved durability and minimal impact on overall health compared to many conventional methods. These findings contribute to the development of safe durable marking techniques suitable for ecological research on soft-bodied insects.

Mycoplasma genitalium (MG) is a common sexually transmitted bacterium that causes serious health problems such as pelvic inflammatory disease, urethritis, and pregnancy complications. The efficacy of antibiotics has significantly decreased due to antibiotic resistance; about 50% of US strains are resistant to azithromycin, a common treatment path, and resistance reaches 100% in high-risk populations. Preliminary research done in our lab has shown that MG is susceptible to tinidazole (another antibiotic) in vitro. We hypothesize that tinidazole is effective against MG because it creates superoxide radicals that MG cannot detoxify. To test this, the sodA and katA genes, encoding enzymes that detoxify reactive oxygen species, were introduced into the MG genome. The insertion site was determined by whole genome sequencing, and we selected two mutants with insertions unlikely to affect other genes. These two strains were compared to the parent strain in time-kill experiments to measure susceptibility to tinidazole. Cultures of these strains were incubated for 10 days with two-fold dilutions of tinidazole, plating aliquots onto agar plates each day to quantify surviving MG. The individual colonies present on the plates are counted and graphed, allowing us to compare the efficacy of tinidazole on the separate strains. To confirm the enzymes are being expressed, we used a hydrogen peroxide assay to measure the levels of hydrogen peroxide, which is formed from the radicals released from the cells. In conclusion, we hypothesize that the radicals produced by tinidazole kill MG by inducing DNA damage. We, therefore, measured the susceptibility of 10 DNA repair mutants to tinidazole and found that deletion of MG_360 enhances susceptibility. Results from these experiments can be used to understand the mechanism by which tinidazole and other nitroimidazoles kill MG. This data is critical in the battle against antibiotic resistance and can improve treatment options globally. 

Traumatic brain injury (TBI) results from a blow to the skull that causes shearing forces in the brain. Elevating intracranial pressure (ICP) at the moment of impact may protect the brain from TBI by stiffening the brain tissue and decreasing shearing. When they expect an impact, humans naturally brace and perform a Valsalva maneuver (exhaling against a closed airway), which momentarily elevates ICP. In a ferret TBI model, we conducted abdominal compression using a blood pressure cuff to induce a Valsalva-like response (VLR) and determine whether VLR resulted in neuroprotection. The ferret model was chosen for its gyrified brain structure and white to grey matter ratio that closely resembles the human. TBI was induced using a CHIMERA (Closed-Head Impact Model of Engineered Rotational Acceleration) device, which is designed to deliver high-energy, controlled skull impacts. Initial work showed that the abdominal compression procedures increased ICP. The TBI study involved a total of 36 adult ferrets of both sexes randomized into three groups: (1) a sham control group exposed to isoflurane with a cuff but no compression, (2) a TBI group with a cuff but no compression, and (3) a TBI group with a cuff and abdominal compression. Baseline behavioral assessments (CatWalk, Novel Object Recognition, Swim Test, and Open Field) were conducted one week prior to injury. Post-injury behavioral testing, using the same assessments, was performed at 24–48 hours and 8 days post-TBI to evaluate functional outcomes. On day 8, ferrets were euthanized, and their brain tissue was collected and assessed for neuropathological outcomes. We hypothesize that abdominal compression will mitigate deleterious TBI outcomes. If these findings are supported, this intervention could improve the lives of those at risk of TBI and contribute to ongoing research in the field.

Neonatal hypoxic-ischemic encephalopathy (HIE) occurs when the brain receives insufficient oxygen and blood supply before or during childbirth. HIE is a leading cause of neonatal mortality and morbidity that may also affect later brain development, specifically gyrification - folding of the cerebral cortex creating gyri and sulci. The nonhuman primate (NHP) brain is gyrified, similar to humans, making NHPs a highly translatable model to examine brain development after injury, which has not been well-studied in HIE. In our nonhuman primate (NHP) model of neonatal HIE, we induced injury through in utero umbilical cord occlusion (UCO) for 20 minutes, mimicking the cause of HIE in humans. Twenty-two term-equivalent pigtailed macaques (Macaca nemestrina) underwent UCO and were randomized to no treatment (n = 11) or treatment with therapeutic hypothermia and erythropoietin (TH + Epo [5x1000 U/kg]; n = 11), while non-UCO animals served as controls (n = 7). All animals were delivered via cesarian section. Injury severity was determined by physiological parameters (Apgar score), lactate, and pH levels after resuscitation. To evaluate the impact of injury on gyrification, we will utilize magnetic resonance imaging (MRI) taken 6-months post-injury to measure the gyrification index (GI). GI will be calculated by taking brain’s inner-to-outer hemispheric ratio; the inner trace following the contours of the gyri and sulci, and the outer trace following the circumference of the cerebral cortex. We hypothesize that global and regional GI will be altered in animals exposed to UCO, corresponding with decreased brain volume and greater injury. We also hypothesize that treatment will mitigate some of these changes, leading to a GI closer to control. These results will help determine whether hypoxia-ischemia alters the trajectory of cortical development, as well as the association between injury severity, brain volume, and gyrification.

Advancements in HIV prevention include pre-exposure prophylaxis strategies (PrEP), which are not as effective for women due to poor partitioning of antiretrovirals (ARVs) to the female reproductive tract. Integrating ARV-releasing reservoirs with intrauterine devices (IUDs) offers a strategy for local sustained delivery to overcome the partitioning issue. Our lab investigates reservoirs containing polymer-drug conjugates (drugamers), where the HIV integrase inhibitor raltegravir (RAL) is covalently attached to a polymer through a hydrolyzable linker. A previously characterized RAL-polymer exhibited release over 30 days, which is insufficient for the targeted 1-3 years of IUD-mediated delivery. To address this kinetic problem, the drugamer linker chemistry was modified from an ester to an acetal carbonate. Since the rate-determining step of the acetal carbonate linker hydrolysis does not depend on the acidic RAL hydroxyl (pKa = 6.6), it was hypothesized that this acetal carbonate linker will slow the RAL release rate as opposed to the ester linker. An acetal carbonate-linked monomer of RAL was synthesized and led to a 30-fold reduction in hydrolysis rate. The corresponding drugamer was then synthesized via RAFT polymerization and characterized via NMR. In hydrophilic media, RAL released from the novel polymer significantly slower than in the current lab polymer, showing potential for lengthened duration of action in in vivo models. Future work includes measuring release from RAL-polymer in a matrix device for future IUD incorporation, assessing potential polymer cytotoxicity, and evaluating release rates in mouse models. These findings lay the groundwork for the development of long-acting formulations for sustained HIV prevention.

The Acanthocephala are a phylum of parasitic worms commonly found in the gastrointestinal tracts of mammals and birds. They are dioecious, having distinct male and female sexes. Despite their frequent occurrence, there is little research describing the ratio of sexes in acanthocephalans infecting marine mammals. Understanding the life cycles and life history traits of parasites is important to understanding the ecosystem as a whole. This study aims to determine the sex ratio of Corynosoma spp. infecting harbor seals, which will increase knowledge of transmission and reproduction within harbor seals and potential intraspecific competition between acanthocephalans. The results from this study will be used within a larger project to calculate the energetic burden that endoparasites have on their hosts and help inform seal and parasite conservation efforts. To do this, we will first determine key morphological differences (size, weight, body shape, and number of spines) between the sexes, then count the numbers of males and females present in each seal’s gastrointestinal tract. We will calculate the ratio of male to female acanthocephalans within each individual harbor seal and use those values to generate an average sex ratio for acanthocephalans across all sampled harbor seals. Finally, average weights will be determined for each sex to help confirm if there is a size difference between sexes and determine relative biomass differences across the sexes within harbor seal hosts.

Preterm birth is a leading cause of under-5 morbidity and mortality. No treatments exist to address the neurological complications of premature birth, which include loss of oligodendrocytes and activation of microglia, leading to white matter injury and inflammation, respectively. Our study explored repurposing azithromycin, an FDA-approved antibiotic with anti-inflammatory properties, to mitigate preterm brain injury caused by hypoxia-ischemia. We used a postnatal day (P)14 neonatal ferret model, equivalent to extremely preterm infants. We induced brain injury through a combination of inflammatory stimulus, bilateral carotid artery ligation, and oxygen fluctuations (hypoxia/hyperoxia). Ferrets were randomized into control, vehicle (saline)-treated, and azithromycin-treated groups. Littermate controls were not exposed to injury. Body weights and ex-vivo brain measurements (sulci and gyri widths) were recorded at P21, seven days after injury. Quantitative immunohistochemistry (qIHC) was performed to analyze microglia (Iba-1) and oligodendrocyte (Olig-2) density, and data were analyzed using Kruskal-Wallis tests. In our preliminary findings, post-surgical weights from the azithromycin-treated ferrets were similar to those of vehicle-treated animals. Azithromycin-treated ferrets also showed similar global microglia and oligodendrocyte staining compared to the vehicle group. The vehicle group had lower summed gyri measurements than controls (p=0.04), while azithromycin-treated ferrets had more similar gyri widths to controls (p=0.21). We will continue investigating microglial and oligodendrocyte density using qIHC across additional brain regions using pathology software (VisioPharm), including subregions of each gyrus (cortex, subcortical white matter, and coronal radiata), corpus callosum, hippocampus, and upper and lower thalamus. This will allow us to identify the brain regions most impacted by the injury and investigate if there are regional neuroprotective responses to azithromycin. By deepening our understanding of preterm brain injury and azithromycin-mediated neuroprotection, these findings could lay the groundwork for advancing azithromycin toward clinical trials, offering new hope for saving the lives of the tiniest neonates.

Traumatic brain injury (TBI), characterized by a physical impact to the skull, is a significant health concern among veterans, athletes, and the elderly, with over 200,000 TBI-related hospitalizations in 2020. TBI causes shearing forces and physical damage to the brain, resulting in increased risk of neurodegeneration and mental health problems. When they expect an impact, humans brace, exhaling against a closed airway in what is known as a Valsalva maneuver. This prevents venous return from the head, pressurizes the vascular network in the brain, and increases intracranial pressure (ICP) in a way that may protect the brain from TBI. We aim to mimic a Valsalva-like response (VLR) through external abdominal stimulation and measure corresponding ICP changes. First, we performed a 3mm-wide craniotomy in anesthetized ferrets and implanted a pressure transducer inside the brain to collect baseline pressure readings. After skull closure, VLR was performed both supine and upright (body at 45°), either physically (pVLR, 80-120mmHg by abdominal compression using a blood pressure cuff, n=4) or electrically (eVLR, bilateral 25-30mA stimulus of the rectus muscles, n=4). pVLR resulted in a 2-4mmHg increase in ICP over 2-5 sec. By comparison eVLR resulted in a larger and faster ICP increase - 3-7mmHg with an onset of 250-750ms. Consequently, we will utilize eVLR to modulate ICP in a TBI model to determine whether it is neuroprotective. Ferrets will be assigned to control or randomized to receive a TBI impact with either sham eVLR or eVLR. Animals will be subjected to baseline (pre-TBI), acute, and long-term behavioral testing. Additionally, we will perform brain cell specific histological staining. Results from behavioral testing and histology will inform us of the potential neuroprotective effects of eVLR against TBI and provide future direction towards translating the findings into a wearable device for at-risk individuals.

Multidrug resistant Gram-negative bacteria are an emerging threat to public health, continuously evolving to survive under an increasing number of antibiotics and evade the immune system. A major feature of these bacteria is a polysaccharide capsule, which prevents their immune detection. Thus, there is a need to therapeutically restore an effective immune response against them. The Woodward Lab verified that bacteriophage tail spike proteins (TSPs) act as opsonins, which coat and increase phagocytosis of bacteria by macrophages as part of a novel phagocytic pathway. To expand on these data, I am assessing how the adaptive immune system is influenced by the TSP opsonization pathway, analyzing markers of T cell activation and macrophage polarization as starting points. I hypothesize that this pathway has distinct effects on antigen presentation, costimulation, and cytokine expression, compared to better known opsonization pathways like complement and immunoglobulins, and that some of these effects are conserved across bacterial species. To first assess this, I infected macrophages in tissue culture with bacteria, with or without TSP, and measured MHC-II and costimulatory marker expression, an increase which would be associated with enhanced ability to induce T cell responses. I did not observe any differences when TSP was added to the infection. To characterize macrophage cytokine expression, I am treating cultured macrophages with TSP and bacteria-specific antibodies, with the latter serving as a point of comparison between the TSP and antibody opsonization pathways, and quantifying proinflammatory and anti-inflammatory cytokines resulting from this treatment. These studies will reveal whether the TSP opsonization pathway promotes or inhibits adaptive immune responses, which would implicate their utility as a therapeutic and contribute to our understanding of the interaction between bacteriophages, bacteria, and the immune system.

Hypoxic-ischemic encephalopathy (HIE) is a leading cause of neonatal morbidity and mortality worldwide. The ferret provides a highly translational model to investigate HIE; the gyrified ferret brain has a similar grey-to-white matter ratio to humans, allowing for better assessment of white matter injury and impairment of cortical development compared to rodents. Our previous work has suggested that ferret brains also show greater resilience to hypoxia-ischemia (HI) than rats. Ferrets tolerate exposure to much longer and more significant HI, and 100-fold larger doses of inflammatory stimuli, than rats do. We seek to identify signatures of the ferret's protective mechanisms by comparing differentially regulated genetic pathways in the ferret versus the rat when exposed to identical insults. Whole-hemisphere organotypic brain slices were obtained from term-equivalent ferrets and rats and cultured for 72 hours. Slices were randomly assigned to control or oxygen-glucose deprivation (OGD), an in-vitro model of HIE. Cytotoxicity was assessed by lactate dehydrogenase (LDH) release, while global transcriptomics were analyzed via a 770-gene digital transcriptomics panel. Preliminary results show significantly lower LDH release in ferrets compared to rats, reaffirming the ferrets' resilience to OGD. We identified 90 differentially expressed genes in ferrets following OGD, and 11 genes in the rat. Ferrets upregulated CCL2 and LGALS, genes associated with inflammatory responses, and downregulated ADRB1 and NOS2, suggesting reduced oxidative stress. Rats downregulated KIR3DL1/2 and TGM1, which suppress natural killer cells and form the cell envelope, respectively. The experiment will be repeated with double the sample size and region-specific analysis of gene regulation. We hypothesize the ferret will display lower injury markers globally, which will be associated with regional differences in gene expression compared to the rat. We hope this will enable us to identify potential treatment targets for infants with HIE that can increase resilience and repair after injury. 

Hypoxic Ischemic Encephalopathy (HIE) is a brain injury caused by a lack of oxygen and blood flow in the peripartum period. Cardiac dysfunction occurs in up to 80% of infants with HIE and is associated with worse neurodevelopmental outcomes. The current standard of care for HIE is whole body therapeutic hypothermia (TH). The expected physiologic response to TH is a decrease in cardiac output by 10%, and heartrate (HR) by 10bpm, per 1-degree Celsius decrease in body temperature. However, neonates with cardiac dysfunction tend to have normal or elevated HR to compensate for decreased cardiac output. Therefore, normal or elevated HR during TH may indicate compromised cardiac function. We hypothesize that in neonates with HIE, HR trends during TH reflect cardiac function, and a sustained HR above 100bpm is indicative of cardiac dysfunction. Using echocardiograms performed within the first 2 days after birth in babies with HIE treated with TH at the Seattle Children's neonatal intensive care unit (NICU; n=19), we categorized neonates by cardiac function: normal, right ventricular (RV) dysfunction, and RV plus left ventricular (LV) dysfunction. We then extracted continuous HR data and compared median HR during TH across groups using linear regression during specific periods: 12-24h, 24-36h, and 36-48h after birth. Results showed that infants with RV+LV dysfunction had a higher HR than those with RV dysfunction only or normal function. Across all time periods, infants with any kind of cardiac dysfunction had an average HR above 100bpm, while those without dysfunction had average HRs less than 100bpm. Therefore, it appears that HR can be utilized as a proxy for cardiac dysfunction in neonates with HIE. Utilizing HR as screening biomarker for cardiac dysfunction may allow improve optimal resource utilization of echocardiograms as well as real-time, cost-effective monitoring and targeted treatment initiation. 

Mycoplasma genitalium (MG) is a sexually transmitted bacterial pathogen commonly associated with urethritis in men and cervicitis, endometritis, pelvic inflammatory disease, infertility, and preterm birth among women as it invades the upper reproductive tract. Due to antimicrobial resistance, infections can persist for months to years, and first-line drug choices fail in over half of all patients. Whole-genome sequencing reveals that natural nitroimidazole (NDZ)-resistant mutants have mutations in or near MG_342, which encodes a flavin mononucleotide-dependent oxidoreductase required for activation of NDZs to the toxic form. We hypothesize that these mutations reduce oxidoreductase expression or activity, impairing drug efficacy. To determine if these mutations reduce MG342 protein expression, I used molecular techniques to engineer MG strains expressing FLAG-tagged alleles of MG342 including wild type and four spontaneous resistance mutations in or near the MG_342 start codon. FLAG-tags are peptide tags that bind to commercially available, high-affinity antibodies for protein quantification. My study aims to examine how these MG342 mutations affect (1) protein levels using quantitative immunoblots and (2) NDZ susceptibility using qPCR-based minimum inhibitory concentration (MIC) assays. As MG_342 is an essential gene, we hypothesize that an alternate downstream start codon allows sufficient expression for viability of MG while reducing activation of NDZs, leading to resistance. Future RNA sequencing will examine how mutations, particularly a 92 base pair deletion upstream of MG_342, impact transcription. Developing this RNA sequencing method will help define mechanisms of resistance as new mutations are identified. Since physicians are already beginning to treat MG patients with NDZ drugs, insight into the resistance mechanisms could help determine which mutations to screen for to prevent drug-bug mismatch and treatment failure.

Acute myeloid leukemia (AML) is an aggressive hematologic malignancy with poor long-term survival rates. Cytarabine (Ara-C) is a standard chemotherapy used to treat AML patients. However, many patients relapse due to refractory disease, highlighting the need for new therapeutic strategies. Heparan sulfate proteoglycans (HSPGs) are glycoproteins that regulate key signaling pathways by interacting with growth factors and receptors. HSPG glycan chains are modified by the addition of negatively charged sulfate groups. HS2ST1 and HS6ST1 catalyze sulfate addition at the 2-O and 6-O positions of heparan sulfate chains, respectively. In AML, increased HS6ST1 expression correlates with worse patient survival, while low HS2ST1 expression is linked to adverse outcomes in certain AML subtypes, suggesting distinct roles in disease progression. To investigate the contribution of HS modifications to chemotherapy response, we generated CRISPR-edited (sgHS2ST1, sgHS6ST1, or sgControl) MOLM-13 AML cells. Compared to sgControl cells, sgHS6ST1 cells displayed increased sensitivity to Ara-C, suggesting that 6-O heparan sulfation may contribute to chemoresistance. To test whether MOLM-13 AML cells alter the expression of HS-modifying enzymes in response to chemotherapy, I performed RT-qPCR analysis at 24 and 72 hours after Ara-C treatment. Upon Ara-C treatment, HS2ST1 expression increased by 1.5-fold and HS6ST1 transcript increased by 4-fold at 24- and 72-hours post-treatment. In contrast, sulfatase 2 (SULF2) removes 6-O sulfate modifications at the cell membrane. Strikingly, compared to vehicle treatment, SULF2 expression was increased by sixfold at both time points. Our results highlight HS sulfation as a dynamic regulator of AML chemoresistance and suggest that targeting HS-modifying enzymes could enhance chemotherapy efficacy. In the future, I will create an sgSULF2 cell line to characterize the functional role of SULF2 in AML disease progression and chemotherapy resistance.