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.

Alzheimer’s disease (AD), currently the seventh leading cause of death in the United States, is a neurodegenerative disease characterized by amyloid beta (Aβ) plaques and chronic inflammation in the brain. Microglial cells, which act as the immune cells of the central nervous system (CNS), function in response to Aβ by secreting pro-inflammatory cytokines and reactive oxygen species (ROS). Microglial activation is a healthy response in the brain, but chronic activation of these cells and thus chronic secretion of neurotoxic factors creates a cyclic process that leads to neuronal cell death. In order to protect against oxidative stress, cells activate the nuclear factor erythroid 2-related factor (Nrf2) pathway. Nrf2 is a transcription factor that regulates the expression of antioxidant enzymes, which can protect the cell from ROS. Here we focus on the therapeutic potential of cannabidiol (CBD) to mitigate oxidative stress in both microglial and peripheral macrophage cell lines. We show that CBD can activate the Nrf2 pathway and thus increases the expression of several antioxidant proteins such as Heme oxygenase-1 (HO-1). This research is significant because it could provide evidence for the use of CBD as a potential therapy in AD patients.

Zebra mussels are an introduced species that has spread throughout much of the eastern United States and recently invaded Texas. These freshwater mussels cause ecological damage by reducing food available and outcompeting native clams. They cause significant economic damage by attaching to hard surfaces in the water such as pipes to factories and water treatment plants. Understanding where they might spread is an important step in controlling their invasion. Predicting their distribution can be challenging; however, several factors are indicative of where zebra mussels may spread (pH levels, temperature, calcium). Of these factors, calcium is currently viewed as the most significant. Zebra mussels needing calcium for general blood physiology, creating their calcium carbonate shells, and by developing larvae which have small shells. The working model is that zebra mussels will thrive in waters with calcium levels greater than 27mg/L, zebra mussel adults may survive but the larvae may not survive in calcium levels between 27-12 mg/L, and less than 12mg/L of calcium is too low for any stage of mussels to survive for an extended period. My projected look at survival of zebra mussels at varying concentrations of calcium in waters on zebra mussels. Differing calcium levels of 0, 5, 10, 20, and 30 mg/L in artificial pondwater were be used to determine at what levels of calcium zebra mussels are able to survive.

Cryptococcus neorformans is a fungal pathogen that mainly affects immunocompromised patients and is opportunistic as it invades the central nervous system. In the Wormley research lab, we are currently working with multiple genes that have been shown to be involved in lipid metabolism. C. neorformans. Using the TRACE procedure; Transient CRISPR-Cas9 coupled with Electroporation is hypothesized to be a reliable method in order to knock out genes in C. neoformans. This specific project we have been working on will lead to a knockout by using CRISPR methodology to create a Cryptococcus neoforman deletion construct for an associated gene. We are analyzing identified genes that have been found to be upregulated in C. neoformans, multiple of which have been shown to be involved in lipid metabolism and virulence. By characterizing the role of these genes and certain proteins this project aims to deepen the knowledge of the roles of lipids in pathogenesis and hopefully develop ways to combat infection of people with weakened immune systems. To further characterize the role of these genes in virulence we focus on the gene CNAG_00474, which was upregulated in C. neoformans in the presence of arachidonic acid. To achieve this we will generate a KO using the TRACE technique. The overall problem in this study is the implications these fungal proteins may have and the lack of understanding surrounding their involvement which is essential to research in order to create a pathway leading toward potential drug targets. The methodology of this project includes the amplifying promoter and end sequences from the unidentified protein to fuse the primer with sgRNA to create a construct ultimately. From here, amplifying the ‘arms’ of the target protein and the selection marker from a plasmid (in this case NAT) will use PCR to fuse the marker and arms together and create the deletion construct. Once we confirm the gene has been knocked out we will analyze its role in virulence by assessing phenotypic characteristics in vitro and in vivo. I am hoping in the near future to have the specific KO ready to be able to confirm it via PCR.

Marine environments are at risk of contamination from oil refinery effluents, major oil spills, and wastewater runoff. To identify and mitigate such risks, the EPA requires toxicity testing of marine effluents. The larval growth and survival (LGS) test, featuring either sheepshead minnows (SHMs) or inland silversides (INS), is currently used to screen marine effluents for acute toxicity; however, the use of fish larvae represents an animal welfare concern, especially in light of legislation calling for adherence to the 3Rs of animal research. The fish embryo toxicity (FET) test and mysid growth and survival (MGS) test may represent viable alternatives to LGS tests as the FET test uses fish embryos (which are thought to experience less pain than older fish), while the MGS test uses invertebrates. The objective of this study was to determine if the FET and/or the MGS tests produce similar results as the LGS. To accomplish this, INS LGS, SHM LGS, INS FET, SHM FET, and MGS tests were run using phenanthrene, an environmentally-relevant component of crude oil. Results revealed that the LC50 values obtained from the MGS and INS LGS tests were comparable and that both were significantly lower than that of the other test types, suggesting that the MGS test may be a viable replacement for the LGS tests. This was further substantiated when growth metrics were evaluated. In contrast, the LC50 values obtained from both FET tests were significantly higher than those of the other test types indicating a relative lack of sensitivity. However, when hatchability was included as a test metric, the sensitivity of the INS and SHM FET was enhanced indicating that the inclusion of hatch may improve FET test performance.

Due to our rapidly aging population, 6.5 million Americans currently have Alzheimer’s disease (AD), and this is predicted to increase to almost 14 million in the next 40 years. AD is more prevalent in western societies, and researchers suggest that this may be due to the typical Western diet. In contrast, AD prevalence is lower in Mediterranean regions, where a healthier diet could be a contributing factor. Therefore, this research examined the neuroprotective potential of a Mediterranean diet against AD pathologies and inflammation in mice. Our lab designed two experimental rodent diets, one that mimicked a typical Western-style diet, and another that mimicked a typical Mediterranean diet. We examined the lifelong effects of diet on biological markers of AD, including amyloid beta, a protein that aggregates together to form plaques in the AD brain, and pro-inflammatory cytokines, which are associated with increased inflammation. We hypothesized that the Mediterranean diet has the potential to mitigate these AD pathologies and therefore, could potentially be used as a future preventative strategy for AD.

Bacillus anthracis is the causative agent of anthrax. Previously, our lab identified the clpX gene as critical for virulence in B. anthracis. The ΔclpX mutant exhibited decreased cell wall integrity and increased susceptibility to cell-envelope active antibiotics. ClpX is one component of the intracellular caseinolytic protease ClpXP that degrades multiple proteins including transcriptional regulators. To understand changes in gene expression in ΔclpX, a microarray comparing WT and ΔclpX was conducted. This project focuses on msrA, an upregulated gene in ΔclpX. MsrA is an antioxidant enzyme that reduces methionine-S-sulfoxide to methionine but also impacts cell wall strength in S. aureus. This study will determine if loss of the msrA gene impacts antibiotic susceptibility. We hypothesized that since ΔmsrA is upregulated in ΔclpX, ΔmsrA would exhibit the opposite phenotype. Surprisingly, we find that ΔmsrA has significant growth inhibition in the presence of penicillin. However, we do not find susceptibility with other antibiotics, such as daptomycin, nor does it appear to be more susceptible to other clpX-related stress responses such as heat or acid stress. Future research will test ΔmsrA susceptibility to additional antimicrobials, such as the antimicrobial peptide LL-37 and the antibiotic vancomycin, as well as ΔmsrA virulence in vivo with the Galleria mellonella infection model. We will also complement ΔmsrA to confirm the phenotypes are due to loss of the msrA gene. This research is important as it aids our understanding of bacterial defenses and may provide new drug targets to help combat rising antibiotic resistance.