Alzheimer’s disease (AD) is ranked as the seventh leading cause of death in the US with over 6 million Americans currently diagnosed, and that number is projected to reach about 13 million by 2050. AD is currently believed to be caused by numerous factors ranging from genetics, lifestyle, and environmental conditions. The exact pathogenesis of AD remains uncertain, but the pathology of the disease includes the presence of amyloid beta (Aβ) plaques and neurofibrillary tangles composed of the protein tau in the brain. These are two proteins are normally found in the brains of healthy individuals, but amyloid-beta peptides are often degraded under normal conditions, while tau plays a role in stabilizing our cell’s cytoskeletal structures. In Alzheimer’s however, these proteins are misfolded and accumulate, causing disruptions in cell signaling and neuronal death, therefore worsening the disease. Aβ plaques also activate microglial cells, which produce cytokines and induce inflammation. Cytokines are signaling molecules produced by immune cells that mediate inflammatory signaling. Activation of an inflammasome complex found in microglial cells, NLRP3, leads to the production of the cytokine IL-1β which has been implicated in Alzheimer’s due to its ability to induce and maintain this chronic cycle of inflammation, and possibly results in more amyloid-beta deposition. Our research looks into the mode of action of novel anti-inflammatory drugs and their potential to reduce inflammation at the level of the NLRP3 inflammasome as a mechanism to slow down the progression of AD.
 

Understanding the diversity and distribution of species on Earth is crucial in the face of contemporary threats to biodiversity, such as climate change and unsustainable economic practices. Unfortunately, the process of documenting and describing biodiversity often cannot keep pace with habitat loss and species extinction, especially in tropical regions where the number of undescribed and poorly known species is highest, and where biodiversity is most severely threatened. If this diversity is not documented, it will mean a loss of valuable understanding of the natural world and a failure to recognize species whose societal values remain undiscovered or underappreciated. This research will assess the extinction risk of selected fern species to understand their conservation status. The focus lies on understanding the classification, distribution, and conservation status of a group of species within the fern genus Elaphoglossum, the Elaphoglossum dendricola Clade, consisting of around 12 species distributed in the Tropical Andes, mostly at high altitudes (over 2400 m). This assessment aims to serve as a baseline for future conservation studies of this neotropical group of ferns.

Mercury contamination is of increasing concern. As the earth’s temperature continues to rise, it is vital to study the trends of MeHg absorption. Continuing to gather MeHg absorption data in the Northwest part of Greenland will help to grow our understanding of MeHg trends in Arctic territories. This study will increase the amount of data collected on MeHg levels, allowing a more accurate comparison between MeHg level patterns and species behavior, breeding success, and death rates in Arctic bird species (Chastel et al., 2022). Understanding how MeHg contamination affects health and prosperity is critical, not only for the environment and animals but people as well, as these birds are often part of the native Greenlander diet (Hong et al. 2012; Johansen et al. 2004). Temporal monitoring is also highly beneficial for evaluating the efficacy of policies aiming to reduce anthropogenic Hg emissions (AMAP 2021).

Due to widespread anthropogenic emissions and a global atmospheric cycle, mercury contaminates all aquatic ecosystems, including in the Arctic, at concentrations above pre-industrial baselines. Many seabirds nest in large colonies in the Arctic and are at elevated risk of mercury contamination due to their planktivorous and piscivorous diets and long lifespan. We investigated temporal trends of mercury contamination in five species of seabirds from northwest Greenland. Blood samples were collected regularly since 2010 from adult Atlantic puffins (Fracterula arctica), black guillemots (Cepphus grylle), black-legged kittiwakes (Rissa tridactyla), dovekies (Alle alle) and thick-billed murres (Uria lomvia). Samples were analyzed for total mercury using direct mercury analysis. All species had average blood mercury concentrations between 212 and 769 ng/g-wet weight, concentrations associated with a low risk for mercury toxicity. Individual black guillemots and thick-billed murres had blood mercury concentrations >1,000 ng/g wet weight, concentrations associated with moderate risk for mercury toxicity. Preliminary analyses suggest an overall increase in mercury concentrations in black-legged kittiwakes, dovekies and thick-billed murres over the study period. Comparable temporal studies in the Arctic have shown wide variation in mercury contamination trends. The results of the present study contribute to the understanding of regional mercury trends in the Arctic and efforts to assess the impact of the Minamata Convention.

As the number of antibiotics in development dwindles and antibiotic resistance continues to rise,
there is a need for novel, non-traditional antibiotics such as zinc oxide nanoparticles (ZnO NPs).
While the broad-spectrum antimicrobial properties are well established, the mechanism of action
is still unknown. Previous work has proposed that reactive oxygen species (ROS), toxic Zn2+ ions,
and electrostatic interactions with the cell envelope may be implicated in the mechanism. To
evaluate which of these mechanisms are involved, we characterized the physical and genetic
properties that confer resistance to ZnO NPs in three novel ZnO resistant strains of
Staphylococcus aureus (ZnOR). These strains possess comparable growth rates and are at least
four times more resistant than the parental strain against ZnO NPs acquired from multiple
sources. This suggests that all ZnO NPs, regardless of morphology, size, or method of synthesis
share a mechanism of action. We found that cell charge, measured by cytochrome c, was not
different between the parental and resistant strains, indicating that electrostatic interactions
with the membrane are not involved in the mechanism. Additionally, the ZnOR strains shared a
similar susceptibility to H2O2, a ROS commonly suggested to be generated by ZnO. We have also
found that internalization and physical contact with the bacterial envelope are not necessary for
ZnO mediated growth inhibition suggesting that ZnO produces a soluble species that is
responsible for the antibacterial action. Future work includes sequencing the genome of the
parental and ZnOR strains to identify mutations that led to gain of resistance.

There are numerous known chemicals, present in surface waters, which may pose a significant risk to the health of fish living in aquatic environments. These chemicals can impair a variety of physiological process and to date, screening assays for evaluating acute, chronic, reproductive toxicity, and endocrine disruption have been well developed. However, screening assays capable of identify chemicals that can adversely impact immune function have yet to be developed. Developing such assays is necessary given that immune system disruption can increase the incidence of disease and death. Thus, the purpose of this study was to develop and validate a fish-based neutrophil migration assay that can be utilized to rapidly screen chemical for immunotoxicity. The specific objective of this study was to develop a neutrophil migration assay featuring adult fathead minnows (a commonly-used toxicity testing model organism). Specifically, this study sought to optimize methods for two key steps of the neutrophil migration assay – tail injury and depigmentation of fish tails for neutrophil visualization. Three tail injury methods were evaluated including partial amputation, a tail nick, and a biopsy punch. The methods of depigmentation evaluated were H2O2/KOH treatment alone, H2O2/KOH and acetone, and H2O2/KOH treatment in combination with acetone and FlyClear solution 1.1 (Triton X, THEED, and urea). Results showing which of these methods is best suited for neutrophil migration assays featuring adult fathead minnows will be presented.

Oxidative stress is an imbalance of reactive oxygen species (ROS) and antioxidant defenses resulting in cell damage and chronic inflammation. It contributes to many pathologies including neurodegenerative disorders, cardiovascular disease, diabetes, and cancer. Macrophages and microglia are phagocytic immune cells that destroy pathogens while releasing inflammatory mediators, such as pro-inflammatory cytokines and ROS. While inflammation is initially a protective mechanism, chronic inflammation is damaging to tissues. To counter oxidative stress, cells express nuclear factor-erythroid 2-related factor (Nrf2) to mitigate excess ROS production. Nrf2 is a transcription factor that promotes the expression of numerous antioxidant enzymes. Our study targets the expression and activation of Nrf2 in cells treated with L2, a compound created by Dr. Kayla Green (TCU Chemistry). Our lab is attempting to determine the molecular mechanism in which L2 may protect phagocytic cells from oxidative stress, and if this mechanism involves the Nrf2 pathway. This research could provide preliminary evidence for the efficacy of this compound as a treatment option for diseases involving oxidative stress.

With increasing global industrialization and subsequent pollution, there are mounting concerns regarding the presence and impacts of reproductive endocrine disrupting chemicals (REDCs), including environmental estrogens. These concerns have led to new international regulations (i.e. REACH) which require that chemicals be screened for endocrine disrupting activity. A variety of in vivo and in vitro screening currently exist; however, the in vivo methods are time-intensive and expensive and the in vitro methods may fail to detect estrogenic compounds with unique modes of action. Thus, there is a need for in vivo estrogen screen assays that are quick and inexpensive. The objective of this study is to validate the newly-developed Rapid Estrogen ACTivity In Vivo (REACTIV) Assay as a reliable approach for the detection of chemicals with estrogenic activity. This assay employs Japanese Medaka (Oryzias latipes) that have been genetically modified to co-express green fluorescent protein and choriogenin (an egg precursor protein). In the assay, the transgenic medaka embryos are exposed to a chemical of interest for 24 hours after hatch and then imaged under a fluorescent microscope. To validate the performance of the assay, tests were performed using two chemicals with known estrogenic activity (i.e., bisphenol A, estradiol) and two inert chemicals (i.e., saccharin and cefuroxime). Results showed that larvae exposed to the estrogenic compound experienced dose-dependent increases in liver fluorescence, while those exposed to the inert chemicals did not. Overall, these results indicate that the REACTIV assay produces predicable results and thus, may be appropriate for use as a standardized estrogen screen method.

BRCA1 is a gene found in humans that, when mutated, has been linked to breast and ovarian cancer. A homolog version of this gene, known as brc-1, exists in an organism called the Caenorhabditis elegans. This is a species of nematode worm that has the potential to be used as a model organism to study this homolog gene that is associated with human breast cancer. Previous studies with C. elegans have shown links between the brc-1 gene and DNA damage responses, cytochrome p450, or cyp, transcription levels, and ratios of male phenotype worms. This project focused on studying whether these brc-1 functions are dictated by the enzymatic activity of the protein made by this gene. To measure these phenotypes, we used a strain of C. elegans with a brc-1 mutation engineered to lack enzymatic activity of the BRCA1 protein toward nucleosomes. In order to determine how this lack of enzymatic activity affects brc-1 functions, we measured levels of reactive oxygen species (serving as a proxy for DNA damage), numbers of male offspring, and cyp levels in the mutant and wild-type C. elegans. Our initial results indicate the effects of enzymatic activity towards nucleosomes on the aforementioned phenotypes.

Staphylococcus aureus is the causative agent of many skin infections and the leading cause of death due to infectious disease in the United States. Additionally, S. aureus is known to rapidly gain antibiotic resistance, as seen with methicillin resistant Staphylococcus aureus (MRSA). Zinc oxide (ZnO), a nontraditional antibiotic, demonstrates antimicrobial action against S. aureus. While the exact mechanism of ZnO antibacterial action is currently unknown, production of reactive oxygen species (ROS) is a commonly proposed mechanism. We find that S. aureus ΔkatA, a mutant susceptible to hydrogen peroxide (H2O2) due to a deletion in the catalase gene, exhibits comparable growth to wild type S. aureus in ZnO. This suggests that production of H2O2 is not vital to the antimicrobial action of ZnO. To further test this, we generated a ZnO resistant mutant (ZnOR) that demonstrates less susceptibility to ZnO. We find that the ZnOR mutant demonstrates comparable growth to wild type S. aureus in H2O2, making H2O2 production an unlikely toxicity mechanism of ZnO. To evaluate the role of ROS besides H2O2, susceptibility of ZnOR and wild type S. aureus to two other ROS, bleach and paraquat was evaluated. We are currently investigating whether N-Acetyl-Cysteine (NAC), a compound that stimulates production of antioxidants and is protective against a wide range of ROS, protects S. aureus from ZnO mediated toxicity. Our data suggests that ROS formation is not the dominant mechanism of antimicrobial action by ZnO and future studies should focus on other potential mechanisms of action.

Cryptococcus neoformans is an ubiquitous fungal pathogen that is detrimental for immunocompromised patients, leading to pneumonia and fatal meningoencephalitis. Fungal signaling lipids termed eicosanoids have been associated with increased virulence in this pathogen. Since C. neoformans lacks common enzymes associated with eicosanoid biosynthesis in humans, this pathway presents novel genes which could be used as potential drug targets. Our study focuses on EncT, a poorly characterized gene which encodes an efflux pump and is involved in the production of eicosanoids in C. neoformans. To evaluate the potential role of this gene in virulence, we used CRISPR technology to knock out (KO) the EncT gene, followed by screening for stable mutants and confirming the gene deletion via DNA amplification. After constructing the KO, we conducted in-vitro virulence assays of the KO strain and the wild-type strain (H99), including tests to assess sensitivity to temperature and changes in virulence factors including the production of melanin and capsule. These tests will help us characterize the potential role of the EncT gene in the virulence of this pathogen. Future directions include using a similar gene-editing method to generate an EncT reconstituted strain for use as an additional control in the in-vitro assays. Further, H99, the KO strain, and the reconstituted strain will be given to mice to evaluate the pathogenicity of the KO strain in an in-vivo model. Additionally, we will evaluate the presence of the fungi in the lung and the dissemination in other organs, and we will analyze the host immune response. By knocking out a gene involved in virulence-associated lipid production and characterizing the role of this gene in pathogenicity, this project will broaden the knowledge of the role of lipids in fungal pathogenesis and provide information that could potentially assist in developing therapies against this pathogen.

In a remarkable work of symbiosis, the gut microbiota coordinate with the brain to regulate multiple bodily functions, including those of the immune system, through bidirectional communication with the gut-brain axis. This symbiotic process has been shown to affect human health and disease pathology as certain inflammatory responses correlate with the composition and general disruption of the gut microbiome. To name a few, neurological disorders, gut-based inflammatory disorders, and cancer have been linked, in part, to dysfunction of the gut-brain axis. Previous literature on the gut-brain axis stems from in vivo and in vitro models, which have worked to understand the connection between the microbiome and disease pathology. Emerging evidence from these studies has continued to become more convincing regarding the importance of the bidirectional relationship in human health. In this review, evidence focusing on the intricate connections between the gut-brain axis and several inflammatory diseases, including irritable bowel syndrome, celiac disease, Crohn's disease, cancer, lupus, and Alzheimer’s disease, will be discussed. How this information can be utilized, including what has been or could be done in the clinic to improve the outcomes of patients with inflammatory-related diseases, will be highlighted so that continued advances in this newer aspect of medicine might lead to direct benefits for human health.

Bacillus anthracis is the causative agent of the fatal disease anthrax, and its virulence is of great interest due to its potential as a biological weapon. B. anthracis causes disease by both escaping immune defenses and acquiring nutrients. A necessary nutrient that pathogens must acquire from its host is iron. To discover novel genes essential for iron acquisition, we screened transposon mutants in iron-deficient media with hemoglobin as the sole source of iron. We further prioritized the mutants discovered in our in vitro screen by assessing for attenuated virulence using our in vivo G. mellonella infection model. We found one mutant that has a disruption in the first gene of a two-gene operon containing putative dUTPase and aminopeptidase genes known as 9F12 Tn. Neither of these genes have been previously linked to iron acquisition. To confirm the role of the dUTPase gene in the observed 9F12 Tn phenotype, we created an independent insertional mutant in the dUTPase gene (dUTPase IM). We found that both of our mutants, 9F12 Tn and dUTPase IM, could not use hemoglobin as a source of iron. We also found that G. mellonella injected with 9F12 Tn and dUTPase IM had higher survival rates than those injected with the parent strain. Our results indicate that the dUTPase gene is necessary for iron-acquisition and virulence in B. anthracis. This study furthers our understanding of iron acquisition in a bacterial pathogen and increases our knowledge of how B. anthracis causes disease.

Bacillus anthracis is a gram-positive bacterial pathogen that causes the deadly infectious disease anthrax. B. anthracis contains over 5,000 chromosomal genes, and we believe there are unidentified chromosomal genes important for virulence. Our lab constructed a transposon mutant library with random disruptions in the B. anthracis Sterne genome to screen for novel virulence factors, and we have previously identified two virulence genes, clpX and yceGH, using this library. In this screen, we used hydrogen peroxide, a reactive oxygen species involved in innate immune defense, and screened around 1000 mutants. We obtained three mutants that were susceptible to hydrogen peroxide in vitro: 11F11, LV1, and LV2. To determine whether they also had phenotypes in vivo, we infected Galleria mellonella to study their virulence in an invertebrate animal infection model. LV2 showed reduced virulence in the in vivo survival assay, and all three mutants showed reduced virulence in the in vivo competition assay. I have determined the site of the transposon insertion in 11F11 and LV1, and the transposon has inserted in the genes for catalase and a collagenase-like protein, respectively. I am currently creating an independent insertional mutation in LV1 to confirm that the observed phenotypes are linked to the disruption of the collagenase-like protein. Future directions include creating a complementation plasmid for LV1 and determining the insertion site of LV2. The findings of this research could be used as potential therapeutic drug targets and will offer insight into the mechanisms that B. anthracis uses for its pathogenesis.

Bacillus anthracis is a gram-positive bacterium that causes the deadly anthrax disease. ClpX is a subunit of ClpXP protease that is known to be essential in virulence as well as providing resistance to cell-envelope targeting antibiotics such as penicillin, daptomycin, and the antimicrobial peptide LL-37. While clpX is critical for virulence in B. anthracis, it is unlikely to be directly mediating the effect. Hence, our lab investigated the genes that are differentially expressed in the ΔclpX mutant compared to the wild type B. anthracis through microarray analysis. We found 119 genes that were highly differentially expressed in the ΔclpX mutant. In this study, we focused on two genes sigM and glpF, which are downregulated in the ΔclpX mutant, because sigM and glpF confer resistance to cell-wall targeting antibiotics in the closely related gram-positive bacterial species, Bacillus subtilis and Staphylococcus aureus respectively. We wanted to determine whether loss of sigM and glpF will lead to similar phenotypes as loss of clpX in B. anthracis Sterne. We found that sigM mutant is more susceptible to penicillin and daptomycin, although in a growth phase dependent manner, but glpF mutant is not. Future studies will examine the susceptibility of these mutants to LL-37 and other stressors such as acid and heat stress. Complementation of these mutants will serve to further support the importance of these genes for the roles we examined. This research will aid in understanding the mechanism of antibiotic resistance and virulence in the ClpX regulatory network in B. anthracis.

Salmon hatcheries are widely used across the Pacific Northwest to enhance fisheries and supplement declining wild populations. However, substantial evidence suggests that hatchery fish have reduced fitness compared to their wild counterparts. Domestication selection, or adaptation to the hatchery environment, poses a potential risk to wild populations if introgression between hatchery and wild fish occurs. While few studies have investigated domestication selection on a genomic level, none have done so in parallel across multiple hatchery-wild population pairs. In this study, we examined three separate hatchery populations of Chinook salmon, Oncorhynchus tshawytscha, and their corresponding wild progenitor populations using low-coverage whole genome sequencing. We sequenced 192 individuals from populations across Southeast Alaska and estimated genotype likelihoods at over six million loci. Each hatchery population, which was reared in a hatchery for approximately seven generations, was then compared to its wild progenitor population using multiple metrics of genomic divergence. While evaluating population-level genomic differentiation (FST), we discovered numerous outlier peaks in each hatchery-wild pair, although no outliers were shared across the three comparisons. Further analyses indicated that these relatively small (5 – 10 kilobase) peaks are likely due to genetic hitchhiking on hatchery-selected alleles, though the effects of these peaks on fitness are unknown. Overall, our genome-wide analyses demonstrate that domestication selection is prevalent in all hatchery facilities, but the genetic pathways differ across populations, possibly due to a polygenic basis of fitness related traits. These results provide fine-scale genetic evidence for domestication and highlight the need to assess if certain management practices, such as integration of wild broodstock, can universally mitigate genetic risks despite multiple pathways of domestication.

Alzheimer's Disease (AD) affects approximately 6.5 million Americans, and there is currently no cure. Prior research has shown that a key pathology of AD is amyloid beta, a protein that aggregates and form plaques in the brain, under pathological conditions. If amyloid beta is not cleared by the body, resultant plaques may disrupt proper cognitive and neuronal function. As the liver plays a crucial role clearing amyloid beta, liver damage may jeopardize the efficacy of the liver to clear amyloid beta in the periphery of the body, enabling it to reach the brain.

One way liver function can be disrupted is through diet, specifically the Western diet (WD), which has been shown to cause non-alcoholic fatty liver disease (NAFLD) and inflammation, both of which are associated with AD. A WD is classified as one that contains high amounts of refined sugars and saturated fats derived from animals. Conversely, the Mediterranean Diet (MD), a largely plant-based diet, contains high amounts of monounsaturated fatty acids and polyunsaturated fatty acids. These dietary factors have been shown to decrease inflammation and increase antioxidant effects, further protecting the brain from AD pathology. Therefore, we hypothesize that the MD could protect the liver and be used as a potential prevention strategy for NAFLD and AD.

The current study examined the effects of WD and MD on the relationship between the liver and the brain in wild type mice. During tissue collection, livers were taken and histologically analyzed. The livers from each experimental group were processed, stained, and evaluated for their overall composition.

Oxidative stress caused by the imbalance between antioxidants and oxidative species is a major component of several chronic diseases such as cardiovascular disease, cancer, and some neurodegenerative diseases. Potential therapeutics have previously been explored to address the role of oxidative stress in disease, but many have been unsuccessful or only target one aspect of this multifaceted disease pathway. To address this, Dr. Green’s lab at TCU created the L2 compound to act as a multimodal antioxidant therapy. Specifically, preliminary in vivo studies have demonstrated L2 can increase the cellular level of nuclear factor-erythroid 2-related factor (Nrf2), the natural antioxidant pathway of the cell. Normally this pathway is activated due to oxidative stress, allowing Nrf2 to migrate to the nucleus where it acts as an important transcription factor to produce antioxidant and detoxifying enzymes. This data was unexpected as the addition of antioxidant compound L2 should mitigate the need to activate the Nrf2 antioxidant pathway. Therefore, it is the purpose of this study to confirm that treatment of cells with L2 results in translocation of Nrf2 into the nucleus of cells. Further experiments will determine if this translocation leads to antioxidant effects as proposed.

Wind-energy production has expanded due to interest in increasing energy production and decreasing reliance on fossil fuels. Unfortunately, collisions and fatalities are unintended consequences of wind-energy production for many bat species. The Mexican free-tailed bat (Tadarida brasiliensis) has a non-migratory population in California that has an assumed sex ratio of 50:50, as seen in other nonmigratory bat species, and migratory sex-skewed (9:1 Female:Male) population in Texas that arrives in the summer to form maternal colonies. Knowing how males and females are impacted by collision mortality at wind turbines can provide insights into population-level effects. We determined the sex of bat carcasses discovered at wind turbines using DNA extracted from wing tissue samples collected during post-construction surveys in California (n = 502, 5 years) and Texas (n = 437, 3 years). Preliminary analysis of bats from California suggests that the sex ratio of fatalities did not differ significantly from 50:50 from 2016 to 2020 (p>0.05). In contrast in bats from Texas, the sex ratio of fatalities was significantly female-skewed in 2017 (6.8:3.2, z=3.25, p<0.001), became less female-skewed in 2018 (4.8:5.2) and 2021 (4.4:5.6), with neither 2018 nor 2021 being significantly different from 50:50 (p>0.05). This change in sex ratio in Texas might be demographically relevant if the loss of females from previous years is causing the migratory population to become less female-skewed over time. Studies of sex ratios at summer and winter colonies would allow determination of whether this same pattern is observed at the population level.

BRCA1 and BARD1 are proteins involved in the repression of genes associated with increased risk for breast and ovarian cancers. This is accomplished through ubiquitination of H2A and subsequent changes in chromatin compaction. BRCA1 and BARD1 form an E3 ligase (BCBD complex), and mutations affecting the enzymatic functions of this complex can predispose women to these cancers. The model organism C. elegans contains orthologs of these proteins, BRC-1 and BRD-1, which makes it a useful organism for studies of protein function; however, little is known about the mechanism of ubiquitination in C. elegans as compared to humans. This project used nucleosome assays to provide more insight on the ubiquitination of H2A by the BCBD complex in C. elegans. The objectives of this project included characterizing the interaction of the BCBD complex with H2A and identifying a specific lysine target in C. elegans. The conserved lysine targets were mutated out of H2A and nucleosome assays were performed to identify potential reductions in ubiquitination activity. In addition, we hypothesized that enzyme-substrate interactions, specifically between H2A and BRD-1 in C. elegans, are important in directing ubiquitin to the target site. Amino acid residues in BRD-1 thought to be important for these interactions were mutated out, and assays were performed to assess changes in ubiquitination activity. The H2A nucleosome assays showed that the mutations of conserved lysines in the H2A N-terminus and C-terminus in C. elegans did not significantly reduce ubiquitination activity, and a definitive target could not be identified. However, the BRD-1 assays identified amino acid residues in C. elegans that participate in directing the ubiquitination process. Further studies are needed to determine if C. elegans has any preferential lysine targets at a non-conserved residue or if it is truly nonspecific in its activity. Currently, mass spectrometry analysis is being performed as a complementary method to attempt to pinpoint the location of lysine ubiquitination.

Oxidative stress is the imbalance between reactive oxygen species and antioxidants in a cell. Often this imbalance is caused by an increase of reactive oxygen species (ROS) leading to dyshomeostasis of the cellular redox balance. Oxidative stress is a major component of several chronic diseases including cardiovascular diseases, cancer and neurodegenerative diseases like Parkinson’s and Alzheimer’s diseases. To mitigate the damage caused by oxidative stress our cells are capable of producing their own antioxidants. One cellular mechanism involves the nuclear factor-erythroid 2-related factor (Nrf2) antioxidant pathway which can be activated in the presence of ROS. To better understand how this pathway works, it is important to track Nrf2 during activation of this pathway. Here we test three different plasmids designed to either force expression of “tagged” proteins in the Nrf2 pathway, or to provide a readout mechanism for the level of Nrf2 activation. These experiments lend support for the efficacy of using these tools to better understand the Nrf2 pathway.

Alzheimer’s Disease is a neurodegenerative disease characterized by cognitive, functional, and neuronal loss. Its core pathology includes beta-amyloid protein plaque formation, neurofibrillary tangles of tau protein, and loss of microglial cell function, all of which may be facilitated or exacerbated by a prolonged neuroinflammatory response. The inflammatory signaling pathway culminates in the activation of transcription factor NF-κB, which then goes on to activate the expression of cytokines and other signaling molecules such as TNFα. One of the points of regulation for this pathway is the constitutive binding of the IκBα protein to NF-κB that prevents NF-κB from entering the nucleus. However, when the appropriate stimulus triggers the pathway, a downstream effect is the phosphorylation of IκBα by the IκB kinase, and its subsequent degradation which then releases NF-κB for translocation into the nucleus.
This project aims to elucidate the mechanism of action of novel anti-inflammatory drugs (provided by P2D Biosciences company). Previous in vivo studies with the compound have shown a reduction in inflammation and improved cognition, but the drug’s exact point of interference in the pathway remains unclear. Therefore, this project aims to assess if the drug reduces inflammation by reducing IκBα degradation, thus preventing NF-κB from being able to turn on cytokine expression.
BV-2 mouse microglial cells were exposed to the drugs, followed by exposure to LPS for various time intervals, then harvested and lysed. A Western blot procedure was performed on the lysates to visualize the amount of IκBα present, then those bands were quantified to compare against control cells that were not incubated with the drug. It follows then, that if the drugs’ mechanism of action is inhibition of NF-κB release into the nucleus, then there will be increased amounts of IκBα in the treatment cells compared to the control cells as IκBα degradation is prevented.

According to Rita Charon, founder of the developing field of narrative medicine, "medicine practiced with narrative competence, called narrative medicine, is proposed as a model for humane and effective medical practice," which "offers fresh opportunities for respectful, empathetic, and nourishing medical care" (Charon, 2001). Narrative medicine is composed of three key practices: close reading, reflective writing, and active listening. Developing each of these skills, Sharon proposes, can foster compassion and empathy in medical providers. The demonstration of these practices has been shown to "facilitate an authentic partnership by building empathy and trust," as well as "promote physician well-being and prevent burnout" (Khawand-Azoulai, et. al. 2022, Stumbar, S. E et. al. 2020). Medical education currently is striving to incorporate humanistic training to develop a holistic approach to patient care, but narrative medicine training has yet to be extensively explored in undergraduate pre-health education (Pentiado, J. A. et. al., 2016, Barron, L., 2017). Narrative competence, defined by Charon as "the ability to acknowledge, absorb, interpret, and act on the stories and plights of others," has been proposed as a pre-requisite to developing good patient-care skills (Charon, 2001, Baron, L., 2017). The incorporation of humanistic training for undergraduate pre-medical students in the form of narrative medicine practices can prepare future professional school students to begin developing a patient-centered perspective of healthcare. The undergraduate years have the "potential to shape the kinds of caregivers we want for our patients, for friends, for our families, and for ourselves" (Barron, L. 2017). It is therefore important to gain an understanding of how medical humanities education can impact undergraduate students because foundational knowledge of these concepts prepares students for later development of humane medical practice in professional school. The three avenues of narrative medicine training that I have chosen to analyze include a narrative medicine workshop series, a group of pre-health students called the Illness Narrative Listening Project that gathers regularly to intentionally listen to patients stories, and lastly, my own reflective writing from experiences in medical contexts. Data is collected through a mixed methods approach, gleaning insight through both qualitative and quantitative research methods, in the form of interviews and surveys respectively, will illuminate the complexities of the research question. The goal of the workshops and listening project is to teach these practices to undergraduate students in order to explore if the same benefits seen in clinical practice and medical education could be demonstrated in undergraduate pre-health education.

Alzheimer’s disease (AD) currently afflicts well over six million people in the United States, and this number is projected to increase exponentially in the coming years. While much remains to be understood about the causes and pathogenesis of AD, two potential risk factors are chronic insufficient sleep and long-term consumption of an unhealthy diet. Both of these lifestyle factors are often studied separately, and evidence suggests that each has negative impacts on brain health and cognitive function, perhaps due to increases in inflammation, which itself is associated with increased anxiety and cognitive dysfunction. The current study investigated the combined effects of long-term consumption of a typical American-style diet (TAD) and six weeks of chronic sleep restriction on locomotor activity and anxiety-like behavior in male and female wild-type mice not otherwise predisposed to disease pathology. Female mice that underwent sleep restriction and consumed the TAD displayed greater anxiety-like behavior compared to mice that the TAD and did not undergo sleep restriction. This difference was not observed in male mice. Furthermore, male mice that underwent chronic sleep restriction displayed greater locomotor activity compared to controls. These differences were not observed in females. Given the prevalence of AD and the projected rise in AD cases, understanding how controllable lifestyle or environmental factors can increase AD risk is essential. Importantly, as AD is more prevalent in women compared to men, it is imperative that research efforts utilize male and female animals seek to understand the mechanisms driving this phenomenon.

There is an oxidative stress component to a wide range of neurobiological diseases. In Alzheimer’s disease (AD), secondary brain injury is associated with an imbalance between oxidant and antioxidant agents. This imbalance contributes to the pathophysiology of AD through the oxidation of macromolecules, destabilization of neuronal cells, and generation of ROS that upregulates synthesis and deposition of p-tau and Amyloid-β (Aβ). The expression of antioxidant defense enzymes can decrease damaging reactive oxygen species, so some efforts to alleviate secondary injury focus on this mechanism of reducing oxidative stress. One pathway that is activated in response to oxidative stress is the Nrf-2/ ARE pathway. Under stress conditions, the protein sensor for oxidation levels Keap1 that is bound to Nrf2 is oxidized, and Nrf2 levels are stabilized and subsequently increased in the cell. The Nrf2 transcription factor then translocates into the nucleus and binds to the antioxidant response element (ARE) promoter to turn on the expression of downstream antioxidant genes. The genes that are expressed include heme-oxygenase (HO-1) and NADPH quinine oxidoreductase 1 (NQO1). These antioxidants can then regulate the redox balance in the internal environment and reduce oxidative stress. The goal of my research is to design an assay to measure Nrf2 activation, so we can test drugs shown to reduce oxidative stress in vitro.