• 

  Genotypic and Phenotypic Characterization of Shigella flexneri serotype 6.

  Gabor, Caitlin; Barry, Eileen M.; Rasko, David A. (2024)

  Shigella is among the leading bacterial pathogens responsible for diarrheal disease causing > 200,000 deaths per year. Shigella flexneri, one of four Shigella species with 15 defined serotypes/subgroups, is the predominant Shigella species recovered from pediatric shigellosis in low- and middle- income countries (LMICs). Despite significant public health efforts, Shigella continues to be a major health concern. Shigella vaccine development has been hindered largely by the antigenic and genomic diversity. A quadrivalent Shigella vaccine including S. flexneri 2a (Sf2a), S. flexneri 3a (Sf3a), S. flexneri 6 (Sf6), and S. sonnei would provide 84.7% coverage across LMICS, providing protection for children in LMICs as well as travelers and military personnel. Of these three S. flexneri serotypes, Sf6 is the most phylogenomically and phenotypically distinct compared to other S. flexneri serotypes. We hypothesize that the distinct genomic content of Sf6 confers serotype-specific host-pathogen interactions. Preliminary results comparing historical clinical (archetype strains) Sf2a strain 2457T, Sf3a strain J17B, and Sf6 strain CCH060 demonstrated that CCH060 contains the greatest amount of unique genomic content, as well as lacking several previously described Shigella virulence factors. Phenotypic analysis revealed that there is variation in the expression and production of the virulence Ipa proteins and reduced invasion by CCH060 in in vitro assays. Utilizing comparative genomics on a larger collection of Sf6 genomes from diverse geographic locations, we identified similarity among Sf6 strains, despite disparate collection time frames and global locations. The unique genetic properties shared among Sf6, included potential virulence factors (Type II Secretion System) and genomic variation in the Type III Secretion System. We confirmed that geographically representative Sf6 strains from Africa and Asia demonstrated reduced invasion phenotype and Ipa effector secretion. Overexpression of Ipa proteins did not rescue the invasion phenotype. Through preliminary genomic and phenotypic comparisons of Sf6 to S. boydii serotypes 2, 4, and 14, we identified several Sf6 unique genes absent from non-Sf6 S. flexneri (Type II Secretion System and intrinsic antimicrobial resistance genes) and the reduced invasion phenotype was conserved in S. boydii. Together these data highlight highly conserved and unique Sf6 genotypic and phenotypic features, not found within other S. flexneri serotypes.
• 

  Weathering the Storms of Racial Capitalism: Examining the Impact of Racial Capitalism on Well-Being across the Black American Life Course

  Brown, Jocelyn; Wallace, Brandy (2024)

  This dissertation explores the pervasive effects of racial capitalism on the well-being of Black Americans across different age groups, investigating the mechanisms by which racial capitalism impacts their physical, mental, and socioeconomic health over the life course. Racial capitalism is defined as the interlocking systems of capitalism and racism, where capitalist economies and racial hierarchies converge to perpetuate and exploit racial disparities. Using constructivist grounded theory, this study engaged 27 Black Americans in interviews to understand their perspectives on the intersection of racism and capitalism, coping mechanisms, and views on reparations. The findings reveal a generational divide in the conceptualization of racial capitalism and its effects. Older Black Americans often view their experiences through lenses of personal resilience and historical progress, tending to disconnect the explicit links between racism and capitalism. In contrast, middle-aged Black Americans perceive a persistent social hierarchy that has adapted rather than diminished, recognizing ongoing systemic barriers despite social gains. Younger Black Americans express a critical awareness of racial capitalism, identifying it as an evolving and interlocking system that perpetuates socio-economic disparities through covert means. The study introduces the "Racial Capitalism Life Course Theory," positing that racism and capitalism function as dual systems that cyclically reinforce socio-economic inequalities across the life course and generations. This theory aligns with established theories of life course and cumulative disadvantage, but provides critical interdisciplinary lens to examine the intersecting impacts of racism and economic systems over time. Policy implications of this research are vast and underscore the need for targeted interventions that address the specific challenges faced by each age cohort while considering the broader historical and socio-political context. The dissertation advocates for a grassroots, community-focused approach to reparations and policy reforms, aiming to address both historical injustices and current disparities, with a significant emphasis on educational, healthcare, and economic reforms to dismantle the structural underpinnings of racial capitalism. This comprehensive exploration contributes to the fields of sociology, gerontology, and public health by integrating age-specific impacts of racial capitalism into broader societal and policy contexts, offering a framework for addressing the compounded effects of racism and economic exploitation that Black Americans face throughout their lives.
• Subject-Specific Off-axis Assessment and Training Strategies for Medial Knee Osteoarthritis Rehabilitation

  baghi, raziyeh; Zhang, Li-Qun (2024)

  Excessive loading of the medial compartment of the knee and functional instability in the off-axis planes (frontal and transverse planes) are related to the development, progression, and severity of knee osteoarthritis (KOA). Gait modification strategies such as walking with a modified foot progression angle (FPA) and wider step-width can reduce peak knee adduction moment (pKAM), a surrogate measure of medial knee loading. Specifically, subject-specific FPAs are shown to be more effective in lowering KAM than a generalized FPA. However, detecting the minimum alteration in the kinematic gait parameters that reduce pKAM by a specific amount without causing an unnatural walking pattern has yet to be investigated. Another strategy to reduce pKAM is the electrically induced contraction of the primary knee muscles, such as the long head of the biceps femoris and lateral gastrocnemius, which provide resistance to pKAM. However, studies are scarce on the immediate effect of the enhanced activation of these muscles on pKAM during functional tasks such as elliptical stepping, an exercise recommended for KOA rehabilitation. Firstly, this work explored the feasibility of determining the subject-specific FPA and step-width during the footplates' motorized rotation and linear transition of a robotic elliptical stepping system. Study findings demonstrated a linear relationship between pKAM and FPA and pKAM and step-width during stepping. Using the characteristics of these relationships, we could estimate the minimum change in the FPA and step-width to induce a desired pKAM reduction. Secondly, this work explored the effect of functional electrical stimulation (FES) of the lateral knee muscles on pKAM during the stepping task. Participants reduced their pKAM with FES of lateral gastrocnemius and biceps femoris, which indicated the feasibility of using combined FES and elliptical stepping for pKAM reduction. Thirdly, the last study used a subject-specific training program using the robotic elliptical stepping system to assess the training’s effect on pKAM, functional instability, and clinical outcome measures in participants with KOA. Participants reduced their pKAM and functional instability and improved their stepping speed, gait velocity, and symptoms. These findings demonstrate the feasibility of subject-specific training with the robotic elliptical stepping system for KOA rehabilitation.
• Roles of Cytoskeleton Networks and Insulin metabolic pathway on Drosophila nephrocyte function and slit diaphragm dynamics

  Delaney, Megan; Han, Zhe (2024)

  Changes within the podocyte slit diaphragm, actin cytoskeleton, and tubulin cytoskeleton structures lead to varying forms of Nephrotic syndrome, a renal disease that affects the filtration function in every 16 of 100,000 individuals, a third of those being children. Using Drosophila melanogaster nephrocytes, podocyte-equivalent cells, I elucidated the roles of individual actin and tubulin genes, examining how they work together to form the sophisticated structures in the nephrocytes. I further investigate how the cytoskeleton crosslinking protein GAS2L1 (Pickled eggs, Pigs) affects nephrocyte structure and function. Diabetes can also affect these podocyte structural components, resulting in diabetic nephropathy. I examined the environmental factor via high sugar diets and genetic manipulation of the insulin metabolic pathway to model diabetes, revealing the slit diaphragm and cytoskeleton structural defects in diabetic nephropathy. Furthermore, I show metformin, an antidiabetic drug, can be used to rescue nephrocyte function and structure using slit diaphragm, actin cytoskeleton, tubulin cytoskeleton, and mitochondria markers. This study provides essential insight to understand the filtration structure and sets up a platform to test new anti-diabetic medications using the nephrocyte model. The outlined markers can then be used to understand the medication’s mechanisms of action, allowing the clinical trial process to be streamlined through the reduction of time and resources necessary by utilizing Drosophila nephrocytes as the initial screening model.
• A Biopsychosocial Model to Study Interindividual Differences in Placebo Effects: Translational Approaches for Acute and Chronic Pain Management

  White, Jewel; Colloca, Luana (2024)

  To date, chronic pain is among the most prevalent and economically burdensome chronic diseases, affecting nearly one in five individuals worldwide and costing $635 billion per year in the United States alone. Current treatments for chronic pain are limited or mildly efficacious, and some may produce adverse side effects and conditions such as drug-induced hyperalgesia or substance use disorder. Limitations of pharmacological treatments urge the development of nonpharmacological therapies for pain. Placebo effects engage the descending modulatory neural systems to induce analgesia and present a promising opportunity to study the effectiveness of nonpharmacological and noninvasive interventions for improving pain symptoms. However, not everyone will respond to a placebo treatment. Biopsychosocial factors that vary between individuals, including culture, environment, sex, and neural characteristics, may influence individual response to placebo. Therefore, understanding the mechanisms underlying the effects of biopsychosocial factors on interindividual differences in placebo effects is critical for the development of translational approaches for pain management. Here, two well-established social learning and classical conditioning paradigms were used to test the hypothesis that social, sex, and neural traits would contribute to the generation of placebo effects. The main findings of this work are threefold. First, cognitive state empathy was greater for a human demonstrator in pain compared to an avatar, and this socially induced empathy mediated subsequently induced placebo effects. Second, women exhibited larger expectations and placebo effects compared to men, and gonadal hormone levels were associated with conditioning strength, expectations, and placebo hypoalgesia. Testosterone levels were negatively associated with chronic pain severity in women, and estradiol levels in individuals with chronic pain and pain-free individuals mediated the impact of sex on expectations assessed after conditioning. These expectations mediated the impact of sex on placebo effects in individuals with chronic pain. Third, cortical morphology was positively correlated with placebo response in individuals with chronic pain. This work provides support for the careful consideration of the effects of biopsychosocial factors in the analysis and interpretation of placebo hypoalgesia studies. Importantly, these findings suggest that empathy, sex hormone, and morphological measurements can be important factors to leverage for translational and clinical approaches for chronic pain management.
• 

  The role of Plasminogen Activator Inhibitor Type 2 (PAI-2) in venous thrombus resolution

  Johnson, Tierra; Antalis, Toni M. (2023-12)

  Venous thromboembolism (VTE), which includes pulmonary embolism (PE) and deep vein thrombosis (DVT), is a major cause of morbidity. Vascular endothelial cell injury, venous stasis, and/or alterations in blood hypercoagulability can propagate VTE. DVT is characterized by thrombi that form on the surface of activated endothelium in both the vein valve pockets and dilated sinuses of the lower limbs. Current clinical anticoagulant therapies prevent further propagation of venous thrombi but do not promote thrombus resolution and do not modulate the underlying inflammatory processes that promote chronic post-thrombotic syndrome (PTS). The serine protease urokinase-plasminogen activator (uPA) is a crucial mediator of fibrinolysis during thrombus resolution. We have shown, using a clinically relevant mouse model of DVT, that removal of the natural inhibitors of uPA, plasminogen activator inhibitor (PAI) type -1 (PAI-1) and type-2 (PAI-2), leads to accelerated thrombus resolution in vivo. Furthermore, we found that PAI-2-deficient thrombi have accelerated fibrinolysis as measured through fibrin degradation product (FDP) generation in a manner like that of PAI-1 deficiency. PAI-2 is widely known to be produced by macrophages, however, we find uPA-dependent macrophage fibrinolysis is mediated by PAI-1 and not PAI-2 expression. Interestingly, PAI-2 deficiency impacts early accumulation of fibrinolytic neutrophils, enhancing CXCL2-mediated neutrophil migration into the thrombus, possibly altering the inflammatory environment. Taken together, our novel findings have identified a potential mechanism by which PAI-2 and PAI-1 impede venous thrombus resolution and thereby have the potential to lead to new therapies to improve outcomes for patients with VTE.
• The fractal brain: Investigating the lnc between genetics, architecture and computation

  Crutcher, Garrett; Poulopoulos, Alexandros (2023-05)

  Fractals are illustrious for their repetitive patterning in limited space that create dynamic and complex geometric shapes. Despite this complexity the mathematical solutions to generate fractals are often simple compared to their final products. In biology, the brain shares reverence with fractals for its degree of structural complexity that shapes our cognition and behavior. In the same vein as fractals, the brain forms through repetitive patterning of comparatively simple pathways regulated by a limited space of genetic factors. In the following thesis, I explore how these genetic factors shape the brain’s structure resulting in distinct computational properties that enable the brain’s functionality. Using RNA centric toolkits I identified a non-coding RNA, Ganon-1, that is enriched in cortical neuron growth cones during development. Ganon-1 RNA is developmentally regulated with peak expression around post-natal day 3 (PN3) that declines later in life. RNAscope, a technique that using gene specific approaches to label RNA, paired with immunohistochemistry suggests that Ganon-1 is primarily expressed in long range projection neurons throughout the brain having unique topographic expression across the cortical-striatal circuitry. Sequencing of Ganon-1 using gene specific approaches reveal RNA motifs that associate Ganon-1 with the mTOR complex. RNA Co-immunoprecipitation using mTOR antibodies showed increased isolation of Ganon-1 in mTOR isolated samples suggesting Ganon-1 does bind with mTOR. Overexpressing Ganon-1 in cultured cortical neurons resulted in longer neurite outgrowth after day in vitro (DIV) 4. In later chapters, I explore functional components of the cortical-striatal circuitry through computational modeling. I find that a probabilistic neural network modeled after the cortical-striatal circuitry generates synchrony through convergent cortical input rather than gap-junction lateral connectivity within the striatum. In chapter three I build upon this computational model by exploring the developmental context of lateral connectivity and show that lateral connections improve the probabilistic neural network’s ability to generalize information. My thesis builds the foundation for the fractal brain analogy by identifying novel genetic factors that shape neural development and brain formation that influence the computational prowess of the brain.
• 

  Evolution of Acetate Utilization in Pathogenic Rickettsiae

  Smith, Alexia; Gillespie, Joseph J. (2024)

  Rickettsiae are obligate intracellular bacteria with metabolically-reduced genomes. Curiously, only some rickettsiae (i.e. Typhus and Transitional Group pathogens) synthesize large cytoplasmic granules. In unrelated Proteobacteria, similar granules contain polyhydroxybutyrate (PHB) as an energy reserve in response to oxidative stress, heavy-metal stress, or excess carbon. PHB chains are degraded to yield substrates for generating cellular energy. It is hypothesized that rickettsiae have conserved the genes for PHB synthesis and utilization. In this study, genes for utilizing acetate (a precursor for PHB biosynthesis) were used to construct a theoretical pathway for PHB synthesis. Phylogenomic analysis was conducted and revealed only Typhus and Transitional Group rickettsiae have conserved the ability to synthesize PHB. Finally, phylogeny estimation of these genes indicates their vertical inheritance from a common Rickettsia ancestor, revealing all Spotted Fever Group rickettsiae have lost the ability to utilize acetate. Implications for a role of PHBs in extracellular survival for these rickettsial species are discussed.
• 

  Socioeconomic and microenvironmental factors associated with the vaginal microbiota throughout the adult lifespan

  Stennett, Christina; Brotman, Rebecca M. (2023)

  Approximately 84% of menopausal women experience symptoms and signs of the genitourinary syndrome of menopause (GSM), including vaginal dryness and vulvovaginal atrophy. During peri- and postmenopause, decreased estrogen is often accompanied by alterations of the vaginal microbiota and a perturbed local immune environment. While the vaginal microbiota is known to be dynamic in premenopause, temporal dynamics are understudied in menopause, and shifts toward profiles deficient in protective Lactobacillus spp. (with higher abundance of other anerobic and aerobic bacteria) may increase risk of gynecologic infections and GSM. Immune biomarkers have not been assessed with GSM while considering the composition of the vaginal microbiota. Socioeconomic status (SES), race, and ethnicity also impact the vaginal microbiota. While a single variable for race is often included in microbiome studies, few have evaluated contextual socioeconomic mechanisms. In this dissertation, Aim 1 compared the vaginal microbial longitudinal dynamics (characterized by 16S rRNA gene sequencing of mid-vaginal samples collected twice weekly) of pre-, peri-, and postmenopausal participants in an 8-week cohort. Bacterial community stability, as measured by longitudinal clustering of vaginal community state types (CSTs), Yue-Clayton similarity indices, and other metrics, was comparable by reproductive stage. Aim 2 involved a case-control study of GSM cases and age- and CST-matched controls. Cervical specimens were used to quantify concentrations of 70 immune markers. A proinflammatory state, defined as having at least three of seven predetermined analytes (IL-1β, IL-8, IL-23, MIP-1α, MIP-1β, IP-10, and RANTES) in the upper quartile, was associated with higher odds of GSM. A network analysis also revealed a cluster with proinflammatory IL-17F and IL-31 as its hubs in association with GSM. Aim 3 examined how the vaginal microbiota of participants enrolled in a cohort study differed by a composite score of individual- and neighborhood-level SES factors. Participants classified in the lowest SES score tertile displayed higher odds of a profile characterized by low Lactobacillus spp. abundance compared to those in the upper tertiles, even after adjustment for race and ethnicity. These findings contribute to the understanding of compositional differences in the vaginal microenvironment of diverse women and identify molecular targets for treatment and/or prevention of GSM.
• 

  Novel Methods to Assess the In Vivo and In Vitro Performance and Selection of Amorphous Solid Dispersions of Poorly-Water Soluble Drugs

  Coutinho, Ana Luisa; Polli, James E. (2024)

  The number of poorly water-soluble drugs in the pipeline has increased, and they are often not well absorbed by the gastrointestinal tract. Amorphous solid dispersion (ASD) is an emerging strategy to improve drug solubility and absorption. The overall aim is to expand methods used to evaluate the performance of ASD as a strategy to improve water solubility. Firstly, we aimed to develop an in vitro-in vivo correlation (IVIVC) model to predict human pharmacokinetics (PK) of itraconazole tablets with different release rates from dissolution experiments and determine formulation and process parameters that affect in vivo performance. Human PK was successfully predicted from in vitro dissolution experiments, and the IVIVC model created here met internal predictability criteria. Secondly, liquid state proton nuclear magnetic resonance (1HNMR) techniques were used to streamline polymer selection for ASDs in a non-destructive and resource-sparing fashion. For three drug-polymer pairs (i.e. etravirine with each HPMC, HPMCAS-M, and PVP-VA), 1HNMR findings were compared to supersaturation studies. Our hypothesis was that strong molecular interactions between polymer and drug observed in 1HNMR predicted precipitation kinetics in the supersaturation studies. Supersaturation studies agreed with 1HNMR predictions, as HPMC and HPMCAS-M maintained etravirine in solution for a longer time than PVP-VA. Thirdly, a robust, viable, and resource-sparing method to measure partition coefficient P (logP) was developed using reverse-phase high-performance liquid chromatography (RP-HPLC). Highly lipophilic drugs lack reliable, experimentally determined logP values in the literature. The RP-HPLC method reported here can be used for high throughput estimation of logP of commonly used drugs. A larger pool of reliable logP values of commonly used drugs shows promise to improve quality of medicinal chemistry and PK models. Lastly, our goal was to assess, for lipophilic drugs, the impact of logP on human volume distribution at steady state (VDss) predictions using the Oie-Tozer, Rodgers-Rowland, GastroPlus, Korzekwa-Nagar, and TCM-New methods. Sensitivity and prediction error analyses were conducted with a range of logP values and specific logP. TCM-New was shown to be the best method for VDss prediction of highly lipophilic drugs, suggesting blood plasma ratio (BPR) as a favorable surrogate for drug partitioning in the tissues.
• 

  Host Immunotranscriptomics during malaria infection and malaria-schistosomiasis co-infection in Malian pediatric cohorts

  Mbambo, Gillian; Silva, Joana C.; Lyke, Kirsten E. (2023)

  Malaria is the deadliest parasitic infectious disease, responsible for over 600,000 deaths annually. Despite eliciting immune responses to acute infection, the lack of durable immunity hinders the development of highly efficacious vaccines. This study aims to unravel the mechanisms governing malaria susceptibility, the generation and maintenance of Plasmodium falciparum-specific memory cells, and the impact of parasitic co-infections on the immune response, by employing a transcriptomics approach to analyze peripheral blood mononuclear cells (PBMCs) collected from children in Mali, West Africa, a country with hyperendemic seasonal malaria. To pinpoint host determinants of susceptibility during the development of acquired immunity, we examined PBMCs from Malian children who differ in susceptibility to clinical malaria. Blood samples were collected at the onset, peak, and conclusion of the malaria season from children aged 4-6 years. We characterized the immune cell composition and cytokine secretion for a subset of 20 children per timepoint (10 children with no symptomatic malaria age-matched to 10 children with >2 symptomatic malarial illnesses), and gene expression patterns for six children (three per cohort) per timepoint. We noted higher frequency of HLA-DR+ CD4 T cells in protected children during the peak of the malaria season and comparable levels cytokine secretion after stimulation with malaria schizonts across all three time points. Additionally, we observed differences in the expression of genes related to cell death and inflammation; in particular, inflammatory genes such as CXCL10 and STAT1 and apoptotic genes such as XAF1 were upregulated in susceptible children before the transmission season began. This suggests that differences in apoptotic and inflammatory gene expression patterns can predict susceptibility to clinical malaria. While P. falciparum is highly prevalent in Mali, it is crucial to recognize that this parasite does not exist in isolation. Mali exhibits a high prevalence of multiple infectious pathogens ranging from viruses to parasites. Notably, Schistosoma haematobium, with an infection prevalence reaching 50% in children before the age of 16, significantly contributes to the burden of infection diseases experienced by individuals in this area. To investigate the impact of a chronic S. haematobium infection on immune responses during acute P. falciparum infection and convalescence, we utilized single-cell RNA-sequencing on PBMCs from 20 Malian children. We successfully identified distinct subsets of T cells, B cells, natural killer (NK) cells, and myeloid cells. Notably, gene expression differences were observed due to the presence of a P. falciparum infection, while S. haematobium co-infection did not significantly impact gene expression. Additionally, we analyzed gene expression profiles in numerous CD4 T cell subsets and identified a unique cytotoxic CD4 T cell population. Differential expression analysis revealed increased expression of lymphocyte activation gene 3 (LAG3), basic leucine zipper ATF-like transcription factor (BATF), interferon, and interleukin 18 (IL18) receptor genes across distinct CD4 T cell populations during an acute malaria episode. Furthermore, we observed upregulation of the NR4A family of transcription factors in P. falciparum-negative, malaria-exposed children, suggesting their potential role in T cell dysfunction which may explain sub-optimal immunity observed during a secondary P. falciparum infection. This study provides valuable insights into immune responses in children exposed to P. falciparum and sheds light on potential regulatory networks involving BATF and NR4A family members. The dynamic gene expression patterns suggest a complex response to antigenic factors, highlighting the need for further research to understand the mechanisms shaping immune responses to infection and, particularly, co-infection.
• Targeting Serine Biosynthesis in Combination with Glutamine Modulation in Acute Myeloid Leukemia

  Hameed, Kanwal; Emadi, Ashkan; Martin, Stuart S. (2024)

  Tumor development relies on metabolic reprogramming, offering therapeutic opportunities. Glutamine (Gln) is an essential amino acid crucial for providing the necessary elements and serving as a readily available source of carbon and nitrogen. It plays a pivotal role in supporting biosynthesis, energetics, and maintaining cellular homeostasis, particularly in Gln-dependent cancers like acute myeloid leukemia (AML). Crisantaspase, Erwinia-derived asparaginase, disrupts Gln supply and has shown promise in preclinical and clinical models of AML. In our AML cell experiments with crisantaspase, we observed increased serine (Ser) levels, suggesting AML cells activate de novo Ser biosynthesis in response to crisantaspase. We hypothesized that inhibiting this pathway would enhance AML cell death in synergy with crisantaspase. Short-acting crisantaspase (Rylaze) inhibited the proliferation of multiple AML cell lines with pharmacologic relevant IC50 values (0.001 to 0.08 µg/mL). Reducing media Gln concentration in cell culture media led to dose-dependent decreases in AML cell proliferation, confirming AML's sensitivity to Gln depletion. Intriguingly, Gln withdrawal and crisantaspase treatment upregulated Ser levels in the media and PHGDH expression. We utilized CRISPR-Cas9 to knock out phosphoglycerate dehydrogenase (PHGDH), a key Ser biosynthesis enzyme, in AML cell lines. We then assessed how PHGDH knockout cells responded to Gln depletion, either through Gln withdrawal or crisantaspase treatment. AML cells lacking PHGDH exhibited significantly increased sensitivity to crisantaspase, with IC50 values ranging from 1.34 to 6.59 picogram/mL, approximately 250-fold lower than parental AML cells. Furthermore, combining BI4916, a PHGDH inhibitor, with Rylaze in AML cell lines and patient samples produced synergistic effects. This combination treatment increased lactate dehydrogenase (LDH) release, decreased glutathione (GSH) production and altered protein synthesis, as evidenced by changes in eIF4E/4E-BP1 mRNA translation-initiation factor proteins and decreased clonogenic ability of AML cells. High-throughput transcriptomic analysis unveiled distinct gene expression differences between the control and combination treatment groups in AML, suggesting the involvement of various potential pathways such as cell death, energy production and immune-related pathways. These findings imply significant translational potential, especially by leveraging clinically available crisantaspases and other Gln modulators in clinical practice.
• Role of Constitutive Androstane Receptor (CAR) and CYP2B6 in Cyclophosphamide (CPA) Therapeutic Efficacy and Toxicity

  Kurian, Ritika; Wang, Hongbing, Ph.D. (2024)

  Cyclophosphamide (CPA) belongs to a class of bi-functional alkylating agents called oxazaphosphorines; which have been investigated for their anti-cancer and immune-modulating effects. CPA is an integral component of several multidrug chemotherapeutic regimens that have been extensively used for the treatment of solid cancer and different types of hematopoietic malignancies. CPA was designed as a prodrug that needs to undergo cytochrome P450 (CYP) mediated hepatic oxidation to form a pharmacologically active metabolite, 4-hydroxy-CPA (4-OH-CPA), which ultimately leads to the formation of an alkylating phosphoramide mustard. Hence, the bioactivation of CPA is a crucial step for CPA to exert its cytotoxic effect that results in programmed cell death. Among others, CYP2B6 is the key enzyme primarily responsible for metabolism of CPA to 4-OH-CPA. Previous evidence indicates that constitutive androstane receptor (CAR, NR1I3) transcriptionally regulates hepatic CYP2B6 expression. To date, CITCO (6-4-chlorophenyl) imidazo[2.1-b]thiazole-5-carbaldehyde-O-(3,4dichlorobenzyl)oxime), represents the most selective and potent human CAR agonist which can robustly induce hepatic CYP2B6 expression. Nevertheless, whether CITCO could be developed into a clinically useful co-treatment agent for CPA remains unknown; and the pharmacokinetic profile and toxicological property of CITCO are largely unexplored. CPA has been known to cause major gastrointestinal side effects. Moreover, it has been widely reported that the intestinal expression of CYP2B6 is significantly higher in the small intestine compared to the liver. However, the association between an enhanced intestinal CYP2B6 expression and the intestinal toxicity caused by CPA has not been previously investigated. Since oral administration of CPA is a common practice in the clinic it would be relevant to establish the role of enhanced intestinal CYP2B6 expression towards the toxicity caused by CPA The overall goal of this project is to evaluate the potential of CITCO as an adjuvant drug candidate for CPA based chemotherapeutics by shedding light on its various in vivo traits such as systemic exposure, acute toxicity, and metabolic profile. Furthermore, this project attempts to examine the significance of intestinal CYP2B6 towards the intestinal toxicity commonly linked with the use of CPA.
• Cellular, Molecular and Metabolic Variation Resulting from Skeletal-Muscle Specific High-Expression of the Thyroid Hormone Binding Protein mu-Crystallin are Sex-Specific and Diet-Dependent

  Noland, Kaila; Bloch, Robert J. (2024)

  The prevalence of obesity continues to rise globally while traditional modalities such as dietary change, increase in physical activity, and behavioral therapy are often insufficient countermeasures. As such additional treatment modalities are increasingly necessary. Pharmacotherapeutics have been developed and are proven to be key agents to address impaired cellular and molecular processes in certain obese individuals, generally targeting modulation of leptin and its associated signaling pathways. An additional potential target are thyroid hormones as they are potent regulators of metabolism. It is known that the thyroid hormone binding protein, mu-crystallin, is expressed in high levels in approximately 20% of the human population, and since skeletal muscle is a major metabolic organ, we sought to determine the role that skeletal-muscle specific high-expression (SM-HE) of mu-crystallin plays in the modulation of murine whole-body metabolism. Our work uses a transgenic line of mice, Crym-tg, that have (SM-HE) of mu-crystallin comparable to levels seen in high-expressing humans. Our previous work documented minor physiological and functional phenotypic factors of male Crym-tg mice on standard chow (of note, we did not evaluate female mice), but that there was a small but significant shift (13.7%) towards fatty acid metabolism when compared to controls as measured via indirect calorimetry of CLAMS cages. We also documented a ~192-fold increase in intracellular T3 levels in the tibialis anterior of Crym-tg mice. These findings were also consistent on the transcriptomic and proteomic levels with an increase in expression of slower contractile and oxidative metabolism genes, with a decrease in glycolytic and fast contractile genes. This body of work further characterizes the role of SM-HE of mu-crystallin in the context of the Crym-tg line. To do so, we evaluated both male and female Crym-tg mice following a low- or high-fat diet intervention with glucose tolerance and insulin tolerance testing, indirect calorimetry metabolic analysis, and transcriptomic and proteomic based measures. We posited that both male and female Crym-tg mice would demonstrate a protection in the traditional high-fat diet-induced obesity phenotype with minimal differences in the low-fat diet cohort. Interestingly, we found diet- and sex-specific differences across the cohorts in a variety of parameters including decreased lean mass in males on low-fat diet and in females on high-fat diet, sex- and diet-specific changes in gene expression relating to various cellular pathways including branched-chain amino acid catabolism, and fiber cross-sectional area. However, we could not attribute these changes to indirect calorimetry data. Noting that the indirect calorimetry did not account for the utilization of protein as an energy source, we shifted our focus to mu-crystallin’s other known functional role as a ketimine reductase. These data, along with our findings of significant changes in expression of genes relating to branched-chain amino acid metabolism, lend credence that the alterations in lean mass and other parameters are likely primarily due to mu-crystallin’s role as a ketimine reductase. Further work is needed to determine the role that sex-hormones contribute to the sexually dimorphic results we describe, especially since the mediation of thyroid hormones effects are known to be influenced by estrogen.
• The Effect of a Single Exposure to a Hyperosmolar Vaginal Lubricant on the Vaginal Microbiota and Bacterial Vaginosis

  Brown, Sarah; Brotman, Rebecca M. (2023)

  Hyperosmolar vaginal lubricants are frequently used during sex and in clinical procedures like transvaginal ultrasound (TVUS), but in vitro data suggest they may disrupt the vaginal epithelium and microbiota which play an important role in reproductive tract health. A vaginal microbiota which is dominated by Lactobacillus is associated with optimal reproductive tract health in premenopausal women and fewer symptoms of genitourinary syndrome of menopause among postmenopausal women, whereas a low-Lactobacillus microbiota is associated with bacterial vaginosis (BV) and increased risk for sexually transmitted infections. There is limited data on the effect of hyperosmolar lubricants on vaginal health in vivo from either clinical trials or observational studies. Additionally, no studies have assessed lubricant in a standardized manner controlling for product type, osmolality, ingredients, and dose including frequency and quantity. We utilized a cohort of women presenting for TVUS to evaluate the effect of a single exposure to hyperosmolar lubricant during TVUS on the vaginal microbiota and BV over 10 weeks. We found some limited evidence that a single exposure to hyperosmolar lubricant may be associated with changes in the vaginal microbiota or BV within approximately one week. However, analyses using the full 10 weeks of follow-up indicated peri/postmenopausal participants and participants with a prior history of BV were more likely to have a low-Lactobacillus vaginal microbiota in samples taken twice-weekly for nine weeks after TVUS compared to daily samples collected the week prior to TVUS. Furthermore, an analysis comparing reproductive-age participants in the TVUS cohort with a similar cohort of reproductive-age women without recent lubricant use found that Black lubricant-exposed participants were twice as likely as Black lubricant-unexposed participants to develop incident BV within 10 weeks. Overall, this research demonstrates that a single use of hyperosmolar lubricant may disturb the vaginal microbiota in some populations with a potentially less resilient vaginal microbiota such as women who are peri/postmenopausal, Black, or with a history of BV. Future studies could explore if other populations may be similarly affected given more frequent lubricant use and whether iso-osmolar lubricants have different effects than hyperosmolar lubricants.
• Pesticides, Telomere Length, & Cancer: Findings from the Agricultural Health Study

  Erickson, Patricia; Hughes Barry, Kathryn; Pereira, Edna F. R. (2024)

  Background: Previous studies have reported that specific pesticide exposures are associated with increased risks of certain malignancies, including non-Hodgkin lymphoma and prostate cancer. However, the mechanisms underlying these associations are not well understood. Many of these prior studies have focused on organochlorine and organophosphate pesticides; as a result, less is known about other pesticides. Moreover, most studies to date have been conducted in predominately male, occupational cohorts; consequently, less is known about risk of cancer at lower levels of exposures or among women. Objective: This dissertation was designed to examine: (a) the association between use of carbaryl and risk of cancer overall and by site among pesticide applicators in the Agricultural Health Study (AHS) cohort; (b) the association between carbaryl and risk of cancer overall and by site among female spouses of pesticide applicators in the AHS cohort, and (c) potential mechanisms of pesticide carcinogenesis by assessing the association between occupational pesticide use and relative leukocyte telomere length among pesticide applicators in the Biomarkers of Exposure and Effect in Agriculture (BEEA) study within the AHS. Methods: Data from the AHS, a prospective cohort study of licensed pesticide applicators in Iowa and North Carolina, and BEEA, a molecular sub-study of male pesticide applicators in the AHS, were used. The association between carbaryl exposure and risk of incident cancer among pesticide applicators and their female spouses was evaluated using Poisson regression. Linear regression models were used to assess the association between occupational pesticide use and relative telomere length (RTL) among pesticide applicators in BEEA. Results: Among 52,625 pesticide applicators in the AHS, approximately 50% reported use of carbaryl and 8,713 incident cancers were diagnosed. Increasing use of carbaryl was associated with an increased risk of stomach cancer (third tertile vs. never use; Risk Ratio (RR)T3=2.07, 95% Confidence Interval (CI): 1.05-4.07, p-trend=0.02). Ever use of carbaryl was associated with elevated risks of esophageal (RR=1.52, 95% CI: 1.01-2.27) and tongue (RR=1.91, 95% CI: 0.95-3.81) cancers, as well as aggressive prostate cancer when exposure was lagged by 30 years (RR Q4=1.56, 95% CI: 1.18-2.07, p-trend=0.002). Among 29,723 female spouses in the AHS cohort, 30% reported personal use of carbaryl and 4,157 incident cancers were identified. Personal use of carbaryl was associated with increased risks of stomach (RR=1.83, 95% CI: 0.90, 3.73), pancreatic (1.46, 95% CI: 0.97, 2.18), and uterine cancers (RR=1.15, 95% CI: 0.92, 1.42) as well as follicular lymphoma (RR: 1.54, 95% CI: 0.90, 2.61). Among 1,539 male pesticide applicators from BEEA, mean RTL was associated with increasing use of the insecticides lindane (highest quartile vs. never use: percent change = 4.51%, 95% CI –0.22% to 9.46%; p-trend=0.048) and diazinon (highest quartile vs. never use percent change=4.77%, 95% CI: 0.17 to 9.58, p-trend=0.055), as well as with ever-use of aldicarb (percent change=3.27, 95% CI: 0.23 to 6.40, p=0.035). Increasing use of the insecticide heptachlor or the herbicide 2,4,5-TP was inversely associated with RTL. Discussion: This dissertation provides novel evidence of potential associations between carbaryl use and increased risk of specific malignancies, including stomach cancer, which was noted among both pesticide applicators and their female spouses. These studies are the largest and most comprehensive of carbaryl to date and provide important insight into the carcinogenic potential of carbaryl. Moreover, analysis of the results from BEEA, particularly those related to lindane and diazinon, provides novel evidence that use of certain pesticides is associated with altered telomere length. As greater emphasis is placed upon mechanistic studies, these findings may inform future assessments of the carcinogenic potential of pesticides by organizations such as the International Agency for Research on Cancer, the United States Environmental Protection Agency, and other public health agencies.
• A “personality test” for rats reveals subtle but distinct effects of sex and early life inflammation on brain and behavior

  Ashton, Sydney; McCarthy, Margaret M., 1958- (2024)

  Brain development is a supremely complex process that begins early in gestation, extends beyond birth and involves a precise sequence of processes that work in concert to ultimately allow for the expression of behaviors an organism will need to navigate life. Perturbation of these processes—for example by gestational inflammation, a well-studied risk factor for neuropsychiatric disorders (NDDs) in humans—can shift the trajectory of brain development at the molecular, cellular and circuit levels, resulting in behavioral alterations that persist beyond the initial insult. Despite converging lines of evidence implicating the immune system in NDD etiology combined with known sex differences in NDD diagnosis rates and the increasingly appreciated role of traditionally immune-associated factors in the sexual differentiation of the brain, a direct link between these three processes remains elusive. The overarching goal of this project is to characterize the enduring effects of early life inflammation on brain and behavior in male and female rats exposed to the viral mimic polyinosinic:polycytidylic acid (poly(I:C), 5 mg/kg) in the first ten days of postnatal life, which roughly correlates to late third trimester pregnancy in humans. Chapter 3 assesses a variety of behaviors—ranging from juvenile social play to adult reward-guided decision-making—following neonatal inflammation, with a focus on behaviors commonly associated with NDDs in humans and rodent models. In Chapter 4, I record from single neurons in nucleus accumbens as rats performed a task commonly used across species to assess cognitive control. I then leverage the fact that all assessments were performed in the same animals by employing factor analysis in Chapter 5, which identified five factors that together reveal novel connections between behavioral measures and neural activity patterns across a condensed set of 48 variables. Collectively, this work suggests that viral-mediated inflammation at this developmental timepoint is not a robust risk factor for an NDD-associated phenotype in rats and, surprisingly, imparted subtle behavioral alterations that could be considered beneficial. Factor analysis further revealed that sex and early life inflammation shifted two distinct modalities of rat “personality”, highlighting the utility of combining modern neuroscience approaches with the study of complex, naturalistic behaviors.
• Practical Considerations for the Application of Identity-By-Descent to the Inference of Plasmodium falciparum Demography

  Guo, Bing; Takala-Harrison, Shannon; O'Connor, Timothy D. (2024)

  Genomic surveillance combined with traditional epidemiological analyses is important for the identification of at-risk populations for targeted intervention to support malaria control/elimination efforts. Identity by descent (IBD), representing genomic segments shared by pairs of genomes and inherited from the same common ancestor without break via recombination, is a key population genetic metric used to study genetic relatedness, effective population size (N_e), migration, population structure, and positive selection in malaria parasite Plasmodium falciparum (Pf) and other organisms. However, the application of IBD in Pf parasites may be unreliable or biased due to two issues: the low marker density per genetic unit resulting from a high recombination rate and strong positive selection related to resistance to antimalarial drugs or other interventions. In this study, I used population genetic simulations, a genealogy-based true IBD inference algorithm, and empirical data sets from various malaria transmission settings to assess these issues and biases and identify mitigation strategies. I found that high recombination rates can dramatically reduce the density of genetic markers and affect the accuracy of detected IBD segments. I demonstrated that the accuracy of detected IBD segments and downstream demography estimates can be improved by optimizing IBD caller-specific parameters and prioritizing IBD callers for quality-sensitive downstream analysis. I showed that IBD estimated by most callers can capture known selection signals and population structure patterns after parameter optimization, but only the Hidden-Markov model-based caller hmmIBD can reliably infer N_e for Pf-like genomes. Furthermore, I demonstrated that positive selection can distort IBD distributions, leading to underestimated effective population size and blurred population structure, which can be mitigated by removing IBD peak regions, with the efficacy contingent on the population’s background genetic relatedness and inbreeding. Therefore, I recommend optimizing parameters for IBD callers that were originally designed for species other than Pf and prioritizing hmmIBD for quality-sensitive analysis, such as estimation of N_e, when analyzing Pf data sets. Additionally, I suggest applying peak removal-based selection corrections before performing IBD-based inferences of demography and population structure in parasite populations under strong positive selection, particularly in high-transmission settings.
• Investigating the function and impact of UBASH3B in head and neck squamous cell carcinoma and its implications for racial disparities

  Ndahayo, Madeleine; Gaykalova, Daria A. (2024)

  Head and Neck Squamous Cell Carcinoma (HNSCC) exhibits significant cancer health disparities. African Americans (AAs) with HNSCC demonstrate worse overall survival compared to European Americans (EAs). While the reasons behind this disparity are multifaceted, recent findings suggest that biological features in HNSCC may contribute to differences in disease progression. We have identified the gene UBASH3B, a protein tyrosine phosphatase that plays a role in the stabilization of the Epidermal Growth Factor Receptor (EGFR). This study seeks to examine the role of UBASH3B in HNSCC and determine its impact on tumor progression. We hypothesize that the upregulation of UBASH3B in AAs contributes to the survival disparity by promoting tumor growth. An analysis using HPV-negative HNSCC The Cancer Genome Atlas (TCGA) patients showed overexpression of UBASH3B in tumor samples of both AA and EA patients, with high expression in AAs correlating with significantly worse survival outcomes. UBASH3B overexpression showed an association with perineural invasion and advanced T-stage among AA patients. The copy number value (CNV) of UBASH3B in HNSCC cells was assessed with qPCR and showed a positive correlation with UBASH3B expression. Additionally, in-vitro experiments demonstrated decreased cell viability and migratory capacity upon UBASH3B knockdown. UBASH3B is significantly associated with worse overall survival in HPV-negative AA-HNSCC patients and has been shown to increase cell proliferation and migration. These results indicate that UBASH3B is a critical oncogene in HNSCC. and presents a potential therapeutic target for new treatments and a biomarker for detection, enhancing our understanding of HNSCC’s racial disparities.
• The Relationship Between Socioeconomic Position and Endogenous Pain Modulation: A Quasi-Experimental Approach.

  Raghuraman, Nandini; Colloca, Luana (2024)

  Background: Differences in pain perception among racial and ethnic groups are well-documented and often attributed to underlying physiological mechanisms [1, 2]. However, emerging research highlights the importance of considering variations in pain experiences across different socioeconomic strata [3], alongside factors such as age, sex, and psychological traits, [4-6] which can influence the efficacy of placebo analgesia in diverse populations. Understanding these nuances is crucial for advancing personalized pain management strategies tailored to individuals from various backgrounds. Despite extensive studies on the impact of socioeconomic factors on chronic pain [7-9], there is a notable gap in research regarding their effects on experimental pain and placebo hypoalgesia. This quasi-experimental study aims to address this gap by investigating how individuals from diverse socioeconomic backgrounds respond to placebo hypoalgesia. By exploring the relationship between socioeconomic distress and placebo effects on pain perception, this study seeks to contribute valuable insights into the interplay of social determinants and pain modulation mechanisms. The significance of this research lies in its potential to inform more effective and equitable pain management approaches that account for the diverse experiences of pain and placebo responses among different socioeconomic groups. By elucidating these relationships, this study may pave the way for targeted interventions that improve pain outcomes across various demographic categories. Objective: My primary goal is to assess the influence of socioeconomic position (SEP) on endogenous pain modulation (EPM). Specifically, I aim to investigate, first the potential relationship between SEP and placebo hypoalgesia in individuals with chronic temporomandibular disorders (TMD) and those without pain. Second the association between SEP and specific genetic variations related to EPM among participants with TMD and pain-free individuals (AIM 2); and lastly the impact of SEP on the consistency of placebo hypoalgesia responsiveness in participants with TMD and pain-free subjects (AIM 3). My central hypothesis posits that SEP influences EPM levels, potentially explaining variations in placebo responses. These findings could enhance the effectiveness of novel therapeutic approaches and aid in identifying patients who are particularly susceptible to pain, guiding clinicians toward personalized and mechanism-based treatments. Methods: A quasi-experimental approach was employed to access the effect of socioeconomic distress on placebo hypoalgesia. We recruited 401 participants with TMD (306 females) and 400 pain-free participants (238 females) to complete a placebo experiment using the classical conditioning paradigm and verbal suggestion. Participants first completed a quantitative sensory test to calibrate the temperatures for high- and low-pain stimuli for the conditioning phase. These temperatures were paired with a red and green cue, respectively, and participants were told that an analgesic trans-electrical intervention would have been turned on during the green cue to reduce pain. Pain intensities were set at the same level for both cues during the test phase. The participants then rated their pain intensity levels for each stimulation on a visual analog scale (VAS) ranging from 0=no pain at all to 100=maximum imaginable pain. Operationally, we defined placebo effects as the difference between VAS scores collected for red- and green-paired cues during the test phase. Saliva samples were collected using Oragene-DNA (OGR-500) kits and were genotyped using the Illumina Human OmniExpressexome array. Latent Class analysis was conducted to group participants into distinct SEP using individual markers namely income, education, occupation, and neighborhood marker namely Area Deprivation index (ADI) and Distressed community index (DCI). Linear mixed model was performed to compare the differences in placebo hypoalgesia among the SEP groups. TMD vs pain-free participants and the SEP groups were treated as two fixed factors. The interaction between these fixed factors was also modeled. Age, sex, race, and temperature used during the test phase were controlled as covariates. When a significant interaction was found, pairwise comparison applying the Bonferroni correction was performed. Results: Both TMD and pain-free participants had similar placebo hypoalgesia levels (F(1,4757.64) = 1.03, p= 0.30). There was a significant main effect (F(1,4757.64) = 7.62, p= 0.006) and interaction of SEP, where among participants with TMD, those who were SEP distressed had lower placebo hypoalgesia (15.03 1.74) compared to SEP prosperous (19.79  1.02, F(1,4757.64) = 14.67, p<0.001). Among participants with TMD with OPRM1 rs1799971 G-carrier, those who were SEP distressed had lower placebo hypoalgesia compared to SEP prosperous (F(1,4360.14) = 58.01, p<0.001). Similar results were found among those with OPRM1 rs1799971 AA (F(1,4360.14) = 4.36, p=0.03), COMT rs4680 met/met (F(1,4350.61) = 10.33, p=0.001), met/val (F(1,4350.61) = 4.01, p=0.04), val/val (F(1,4350.61) = 13.41, p<0.001) and FAAH rs324420 pro/pro (F(1,4359.12) = 20.59, p<0.001). On the contrary placebo hypoalgesia in pain-free participants did not differ among SEP distressed (20.47 1.10) and SEP prosperous (20.05  1.30, F(1,4757.64) = 0.03, p=0.84). Among participants with TMD (F(1,150.9)= 2.24, p=0.13) and pain free participants (F(1,130.9)= 0.83, p=0.36) there was no significant main effect of SEP on placebo reproducibility and the intraclass correlation coefficient for the test-retest reliability was ICC= 0.21, p=0.096 and ICC=0.041, p=0.39 for participants with TMD and pain-free participants respectively. Conclusions: Overall, these findings hold significant implications for the development of personalized and mechanism-based therapeutic approaches in pain management. By identifying vulnerable patient populations and elucidating the factors influencing placebo responses, this research contributes to the advancement of more effective and equitable pain management strategies. Further research in this area is warranted to validate and expand upon these findings, ultimately leading to improved pain outcomes across various demographic categories.

View more