Now showing items 1-20 of 1002

    • Mast Cell Modulation of the Developing Hippocampus

      Blanchard, Alexa; McCarthy, Margaret M., 1958- (2023)
      Brain development requires orchestration of overlapping critical periods defined by responsiveness to endogenous or exogenous stimuli (i.e. hormones, light, touch). Simultaneously, sensitive periods represent heightened risk to perturbations by abnormal stimuli (i.e. inflammation). Though neuro-immune cooperation between resident microglia and neurons is essential during critical periods of brain development, the contribution of infiltrating immune cells to neurodevelopmental processes is largely elusive. We discovered a critical and sensitive period created by a population of peri-hippocampal mast cells (phMCs) in the lateral ventricles abundant from birth through 2-weeks-old in the neonatal rat but absent thereafter. This epoch of phMCs is maintained by self-replication inside the brain, creating a mix of mature and immature mast cells which constitutively piecemeal degranulate. phMCs harbor a unique transcriptomic identity to skin, bone marrow and other brain mast cells including transcripts encoding colony-stimulating factors (Csf1, Csf2) essential for microglial development. Pharmacological inhibition of phMC degranulation and secretion stunts microglial maturation, illuminating the role of phMC-derived factors in driving hippocampal microglial development. In contrast, activation of phMCs releases proinflammatory chemokines, compromises the blood-brain-barrier (BBB), and recruits peripheral immune cells across the CNS for at least two days. Together, these findings indicate that a transient population of peri-hippocampal mast cells creates a critical period for microglial maturation via piecemeal degranulation in the healthy brain and a sensitive period to inflammatory stimuli. The dual role of phMCs in homeostasis and inflammation highlight an essential need to understand how the immune system can adapt its function to serve neuronal populations and prevent inflammatory disturbances that lead to neurologic disease.
    • Unraveling Cochlear Otic Mesenchyme Cells: The Role of POU3F4 During Cochlear Development

      Rose, Kevin; Hertzano, Ronna P. (2023)
      The cochlea consists of diverse cellular populations working in harmony to convert mechanical stimuli into electrical signals for the perception of sound. One such cell type are otic mesenchyme cells (OMCs), which are a specialized type of neural crest and cranial paraxial mesoderm that express multiple unique transcription factors (e.g., POU3F4), all of which are known deafness genes, highlighting the importance of OMCs in auditory function. OMCs are known to terminally differentiate into spatially and functionally distinct cell types, including fibrocytes of the lateral wall and spiral limbus, modiolar osteoblasts, and specialized tympanic border cells of the basilar membrane. Interestingly, consequences of Pou3f4 mutations are diverse and include a complete loss of endocochlear potential, shortening of the cochlear duct, and defective pathfinding and survival of spiral ganglion neurons (SGNs), indicating diverse roles of POU3F4 in each OMC-derived cell type. Here, we aim to illuminate the molecular distinctness and functionality of OMCs and show how loss of Pou3f4 impacts cochlear development. By utilizing scRNA sequencing, we elucidated that OMCs divide into four transcriptionally distinct subpopulations well before the onset of hearing, each of which corresponding to one of the OMC-derived cochlear structures. Furthermore, we show OMC subpopulations display distinct functional roles corresponding to their spatial localization. We also unravel the cochlear cellular communication pathways showcasing OMCs are the main contributors of outgoing signaling during cochlear development, including both global and subpopulation specifying signaling pathways. Finally, we indicate how Pou3f4 expression regulates gene expression in each OMC subpopulation and which signaling pathways are lost in Pou3f4 mutants which may be the cause of the defects in surrounding cell types. Our data suggest that OMC diversification occurs not long after the formation of the otocyst with further refinement until the onset of hearing, well before terminal differentiation. Our data also suggests OMCs are the main contributors of paracrine signaling during cochlear development, showcasing their importance in influencing surrounding cochlear cell types. Finally, we show how loss of Pou3f4 affects each OMC subpopulation differently, leading to diverse phenotypes in Pou3f4 mutants. Without cochlear OMCs and their later terminally differentiated cell types, normal auditory function would not be feasible highlighting the importance of tissue specific mesenchymal cells in cochlear development.
    • Bacterial enzymatic combinatorial chemistry (BECC) enabled targeted lipid A modification of Shigella vaccine strains to reduce endotoxicity without compromising immunogenicity or invasiveness

      Sherman, Matthew; Ernst, Robert K. (2023)
      Shigella spp. are Gram-negative bacteria that cause severe diarrheal disease, contributing significantly to morbidity and mortality worldwide. High transmissibility and increased antibiotic resistance have propelled the development of Shigella vaccine candidates; however, no FDA-approved vaccine exists to date. We have collaborated with Walter Reed Army Institute of Research (WRAIR) to improve their live-attenuated vaccine candidates, whose only drawback during clinical trials was febrile symptoms experienced in a few individuals. In this study, we sought to resolve those unwanted side effects by detoxifying the lipid A moiety (also known as endotoxin) that is thought to contribute to the febrile symptoms observed. To do so, we employed bacterial enzymatic combinatorial chemistry (BECC), whereby lipid A modification enzymes were ectopically expressed to induce structural alterations known to dampen lipid A signaling capacity. The enzymes LpxE (phosphatase) and PagL (deacylase) were expressed in both virulent and attenuated vaccine strains of Shigella to modify the bis-phosphorylated hexa-acylated lipid A structure ordinarily present in the Shigella outer membrane. The expected modifications were confirmed using mass spectrometric and gas chromatographic analyses when the enzymes were expressed individually or in combination (both LpxE and PagL) using a construct we refer to as “Dual”. These enzyme/enzyme combinations were subsequently integrated into the chromosome using Tn7 transposition to avoid the possibility of plasmid loss during production. The impact of the induced lipid A structural alterations on innate immune signaling was assessed by stimulation of NF-?B reporter cell lines and human PBMCs with the extracted lipopolysaccharide (LPS). Additionally, in vivo reactogenicity of the LPS in a murine acute endotoxemia model was assessed. We found that dephosphorylation, but not deacylation, of lipid A, was a powerful tool to reduce LPS-mediated signaling in live-attenuated Shigella vaccines, resulting in reduced toxicity of Shigella LPS in vivo. Additionally, we found that dephosphorylation of the lipid A moiety did not impair invasion of colonic epithelia or immunogenicity in a mouse pulmonary model. Overall, this study generated Shigella vaccine candidates with reduced endotoxicity, which upon oral ingestion in humans, will ultimately have reduced reactogenicity.
    • The predictive accuracy of Shock Index in trauma outcomes in older injured patients

      Bulatao, Ilynn; Smith, Gordon S., M.B., Ch.B., M.P.H. (2023)
      The elderly is an increasing proportion of all cases treated at trauma centers. Shock index (SI) calculated as heart rate (HR) divided by systolic blood pressure (SBP), has been shown to be a good predictor of mortality and transfusion in injured patients. One limitation of SI is that its accuracy in different age groups, especially the elderly has not been fully evaluated. We studied the accuracy of admission SI in predicting early, 48-hour and in-hospital mortality, and major interventions (massive transfusion, ICU admission and surgery in 24 hours) in trauma patients admitted to a major trauma center. We examined whether age, injury severity, injury type, blood alcohol and comorbidities affected the predictive accuracy of SI. Of particular interest is the accuracy of SI in the elderly. We also compared the predictive accuracy of SI, HR and SBP. Optimal cut-points for SI were determined. SI had acceptable accuracy in predicting mortality outcomes, and ICU admission overall. Accuracy was good in the prediction of massive transfusion, and poor in the prediction of surgery in 24 hours. SI was better than HR or SBP in predicting mortality outcomes (all ages, elderly, and younger patients). However, in older patients, accuracy of SI in predicting major interventions was not different from that of SBP. Accuracy of SI in predicting 48-hour and all in-hospital mortality, and ICU admission was better in younger patients. Accuracy was also better among those with lower injury severity than in those who were more severely injured. Accuracy of SI in predicting massive transfusion was similar in older and younger trauma patients. Optimal cut-offs for predicting outcomes were lower for older patients (0.5-0.7 for mortality and major interventions) than in younger patients (0.6-0.9 for mortality and 0.6-0.8 for major interventions). Accuracy of SI in predicting all in-hospital death and massive transfusion was less among patients with elevated blood alcohol while comorbidities did not affect accuracy. In conclusion, SI is less accurate in in predicting mortality among older patients and is less accurate in predicting mortality and massive transfusion among blood alcohol-positive patients, potentially affecting its utility in triage and clinical management.
    • TLR4-SNP Mice Reveal the Role of M2a Macrophages in Resolution of Chemically-Induced Colitis

      Vlk, Alexandra; Vogel, Stefanie N. (2023)
      Toll-like receptor 4 (TLR4) is an innate immune receptor responsive to Gram negative lipopolysaccharide (LPS). Single nucleotide polymorphisms (SNPs) in human TLR4 that encode an A896G transition at SNP rs4986790 (D299G) and a C1196T transition at SNP rs4986791 (T399I) render individuals hyporesponsive to LPS. In humans, these SNPs have also been associated with increased susceptibility to inflammatory bowel disease (IBD). IBD, including Crohn’s Disease (CD) and Ulcerative Colitis (UC), impacts millions of individuals worldwide and severely impairs quality of life for these patients. While multiple treatments are available for IBD, there are several problems: (1) not all patients respond, (2) responses may diminish over time, and (3) treatments often have undesirable side effects. Some patients with IBD express these SNPs and, using knock-in mice engineered to express the murine homologues of these human TLR4 mutations (“TLR4-SNP” mice), we have shown that TLR4-SNP mice develop significantly more severe colitis induced by dextran sodium sulfate (DSS) than wild-type (WT) mice. Previous studies have provided indirect evidence for a role of “tissue repair” M2 macrophages (Mφ) in the resolution of colitis. Signaling through the shared IL-4/IL-13 receptor, IL-4Rα, leading to activation of the transcription factor peroxisome proliferator-activated receptor (PPARγ) has been shown to be required for induction of M2a Mφ and our data provide direct evidence for the involvement of both in repair of DSS-induced colonic damage. In response to DSS, colons of TLR4-SNP mice produced reduced levels of M2a Mφ marker mRNA and protein. Additionally, PPARγ protein levels were reduced in colons from DSS-treated TLR4-SNP mice and therapeutic administration of the PPARγ agonist ligand, rosiglitazone, ameliorated colitis in TLR4-SNP mice. Together, these data indicate that the failure of TLR4-SNP mice to resolve DSS-induced colitis may be secondary to their failure to induce “tissue repair” M2a Mφ. Our findings provide insight into the potential development of novel therapies targeting Mφ signaling pathways that aim to alleviate the debilitating symptoms experienced by individuals with IBD.
    • From What to How: An Exploration of How Genetic Counseling Program Directors Learn to Teach

      Dixon, Shannan; Kulo, Violet; Cestone, Christina (2023)
      The pathway from medical practitioner to academic teacher is not-well defined, specifically, there is a lack of clarity in how clinicians learn to teach, particularly within the field of genetic counseling. While there is a defined curricular pathway for entry into clinical practice there is not a defined pathway for instruction of health professionals who want to teach. While healthcare professionals enter an academic role with a defined set of clinical skills, they often lack formal training in how to teach. This qualitative phenomenological study, using a community of practice (CoP) theoretical model, explored the pathway to teaching for genetic counseling education program directors. Thirteen study participants shared their professional journey of learning to teach. Data from this study support the three phases of CoP in genetic counseling education program director development: engagement, imagination, and alignment. Participants reported content, instructional, and pedagogical reflection as they progressed in their understanding and development of their own teaching practice. Engagement with genetic counseling education programs was reported in three different ways: planned, sequential, and unplanned. All respondents acknowledged content reflection as a part of their early practice of learning to teach. The imagination phase is demonstrated as individuals expanded their understanding of teaching, program leadership, and responsibilities required to serve as a program director – in other words they began a practice of instructional reflection. Those who reported alignment did so based on mentorship and contribution to the community of genetic counseling education program directors and shared instances of pedagogical reflection. The findings of this study support the premise that genetic counseling education program directors, although not formal scholars in education prior to entry into their educator role, practice the scholarship of teaching and learning through a CoP. They are committed to the practice of evaluating how students learn to improve their own teaching. Further, they are committed to modeling professional development and learning as a member of a CoP. What they lack is the formal understanding of educational theory as it relates to genetic counseling instruction – without this knowledge it is difficult to conduct theoretically grounded educational research and advance the profession.
    • Evaluation of Salmonella enterica serovar Typhimurium Vaccines in the Context of Immunosenescence

      Allen, Jessica; Tennant, Sharon M. (2023)
      Non-typhoidal Salmonella (NTS) is responsible for a high burden of foodborne infections and deaths worldwide. In the United States, NTS infections are the leading cause of hospitalizations and deaths due to foodborne illnesses, and older adults (≥65 years) are disproportionately affected by Salmonella infections. Due to this public health concern, we have developed a live attenuated vaccine, CVD 1926 (I77 ΔguaBA ΔclpP ΔpipA ΔhtrA), against Salmonella enterica serovar Typhimurium, a common serovar of NTS. The effects of age on parenteral vaccination are well documented, however, how advanced age impacts oral vaccine responses is less understood. In this dissertation, systemic and mucosal immune responses to CVD 1926 were evaluated in adult (six-to-eight-week-old) and aged (18-month-old) mice. We demonstrated that aged mice immunized with CVD 1926 failed to reduce bacterial burden upon challenge with wild-type S. Typhimurium, which was associated with lower vaccine-induced antibody titers and weaker T cell responses. Notably, characteristics of a successful mucosal vaccine response were weak in aged mice, suggesting that mucosal responses to oral vaccines decrease with advanced age. In efforts to develop a NTS vaccine that is effective for older adults, two vaccine approaches were evaluated: (i) a novel live-attenuated vaccine strain, CVD 1926 ΔsteD and (ii) heterologous prime boost strategy involving a mucosal prime with CVD 1926 that was followed by a parenteral boost with a conjugate S. Typhimurium vaccine. SteD is a Salmonella effector that suppresses CD4+ T cell responses during infection with wild-type S. Typhimurium. Using in vitro and in vivo assays, we demonstrated that CVD 1926 ΔsteD immunization elicits enhanced MHC-II expression, increased flagellin-specific CD4+ T cells, robust serum IgG and fecal IgA responses, and protection against S. Typhimurium colonization of the spleen, cecum, and small intestine upon challenge in aged mice. While the heterologous prime boost strategy induced robust Salmonella-specific antibody responses in aged mice, only modest protection against S. Typhimurium colonization was observed, suggesting that this vaccination approach cannot overcome immunosenescence. Taken together, these studies identify the age-associated deficits in mucosal vaccine responses and presents a promising prototype vaccine strain that may be effective for older adults.
    • Predictors of Traumatic Stress in Hospital Nurses in the Third Year of COVID-19 and Their Lived Experiences: A Mixed Methods Study

      Storm, Asha; Friedmann, Erika (2023)
      Background: Research on nurses early in the COVID-19 pandemic shows elevated severity of trauma-related stress, depression, anxiety, and poorer well-being than before the pandemic. Fewer studies examined nurses’ experiences three years into the pandemic and the relationships of the experiences that predict increased post-traumatic stress severity. Objectives: This study evaluates the relationships among peritraumatic distress, moral distress, resilience, and post-traumatic stress severity as its primary objective. The study also examined the relationships of depression, anxiety, sleep quality, and nurses’ perceived work environment to post-traumatic stress as its secondary objective. The overall goal is to understand targets for intervention on the path to the development of post-traumatic stress that could potentially reduce the impact of crises on nurses. Methods: This study evaluates the interrelationships of these variables using a concurrent triangulation mixed methods framework. Nurses participated in multiple surveys, and a subset of these nurses participated in semi-structured interviews. A structural equation model (SEM) examined the relationships of the primary outcomes, and multiple regression analyses investigate the independent predictive ability of the variables on post-traumatic stress. The interviews utilized a descriptive phenomenological methodology to describe the lived experiences of traumatic stress during the pandemic for these nurses. Results: In the SEM, moral distress partially complementarily mediated the direct positive effect of peritraumatic distress on post-traumatic stress and negatively moderated the direct negative effect of resilience on post-traumatic stress. A multiple regression with all variables excluding resilience and sleep quality (not significant at p < .20) accounted for 62.6% of the variability in post-traumatic stress symptom severity. The interviews revealed that nurses exist in three interrelated worlds: their “Internal World” (emotions and personal well-being), their ‘Hospital World” (coworkers, leadership, environment, etc.) and the “Outside World” (the public, social media, current events, etc.). Conclusion: Nurses require support for their mental health at work and outside the hospital during a crisis, and hospital policies must consider all three. Reduction in peritraumatic stress and moral distress and support for nurse resilience are some of the most critical areas to focus on to reduce the post-traumatic stress severity in nurses during a long-term crisis.
    • RTS,S/AS02A/1B induces antibodies to a novel Plasmodium falciparum circumsporozoite protein epitope with sequence similarities to the junctional region

      Friedman-Klabanoff, DeAnna; Berry, Andrea A. (2023)
      Background: Plasmodium falciparum circumsporozoite protein (CSP) is the target of multiple vaccines in development. New promising monoclonal antibodies target the CSP junctional region, which is not contained in the RTS,S vaccine. Methods: We compared CSP antibody responses on a diversity-reflecting peptide array between adults receiving a full-length CSP (rCSP) vaccine and unprotected after controlled human malaria infection (CHMI), adults receiving RTS,S and protected after CHMI, and adults receiving RTS,S and unprotected after CHMI. Results: Overall, the rCSP group had lower anti-CSP antibody responses compared to the RTS,S-protected group. An epitope of interest in the C-terminal region was identified for both RTS,S groups that had sequence similarities to junctional region epitopes, and the RTS,S-protected group responded to more diverse peptides at this epitope than the RTS,S-unprotected group. Conclusions: Sequence similarities between the identified C-terminal epitope and junctional region epitopes warrant further investigation into whether these are cross-reactive antibodies driving protective responses.
    • Analysis of Novel Mass Spectrometry Diagnostics for Clinical Microbiology

      Smith, Richard Daniel; Johnson, J Kristie; Ernst, Robert K. (2023)
      To optimally manage bloodstream infections, quick and appropriate antimicrobial treatment is critical. Rapid diagnostic testing (RDT) can help guide antimicrobial therapy in a timely and reliable fashion. Use of RDT in clinical microbiology is associated with improved patient outcomes including decreased mortality, morbidity, time of hospital stay, and patient costs. The ideal RDT is accurate, rapid, identifies antimicrobial resistance, is easy to use, and low cost. For septic patients, time to appropriate antimicrobial therapy is inversely related to the patient’s survival; therefore, fast, and accurate RDT is especially important. Several different types of RDT assays exist, including polymerase chain reaction, nanoparticle probe technology, and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). MALDI-TOF MS is a mainstay diagnostic technology that has revolutionized the world of clinical microbiology. MALDI-TOF MS methods are high-throughput and have extensive pathogen panels. Microorganisms can be identified by MALDI-TOF MS once growth in culture is visible, thereby providing a decrease in both pathogen identification and time to effective antimicrobial therapy. Limitations of current MALDI-TOF MS diagnostics include limited ability in the identification of antimicrobial resistance and time to identification of organism, typically requiring 24-48 hours of additional ex vivo culture after positive blood culture. Recently, novel MALDI-TOF MS based technologies have been developed to identify pathogens direct from positive blood culture bottles, eliminating the need for further culture; however, novel technologies need to be evaluated and compared to current methods to provide valuable insight about the use of these diagnostics in clinical microbiology laboratory settings. This study addresses current limitations of MALDI-TOF MS diagnostics. In Specific Aim 1, a novel lipid-based technique termed Fast Lipid Analysis Technique (FLAT) was evaluated for its clinical utility in identifying colistin resistant Enterobacter species and Klebsiella aerogenes utilizing MALDI-TOF MS. For Specific Aim 2, positive patient blood cultures were prospectively collected from the University of Maryland Medical Center (UMMC) clinical microbiology laboratory to evaluate and compare the FDA-approved Bruker MBT Sepsityper® to other FDA-approved direct-from-blood culture RDTs. Finally, Specific Aim 3 compared the use of FLAT for direct-from-blood culture identification to the other previously compared RDTs using the novel Benefit-risk Evaluation for Diagnostics Framework (BED-FRAME).
    • The Role of Parabrachial in Nociception and Pain in Awake Mice

      Smith, Jesse; Keller, Asaf (2023)
      The parabrachial nuclear complex is a nexus for aversion, and for the sensory and affective components of pain perception. It has been previously shown that, during chronic pain, parabrachial neurons have increased activity and respond to noxious stimuli with prolonged after-discharges – responses that far outlast the stimulus. This phenomenon—like most of what we know about the electrophysiology of pain—has only been observed in anesthetized animals. Anesthesia profoundly alters neuronal responses to nociceptive stimuli and masks their responses to the affective component of pain. I have developed a method to investigate parabrachial neurons in awake, behaving animals by recording single units in vivo from head restrained mice, and applying reproducible noxious stimuli. This offered opportunities to study the time course of changes in parabrachial activity by recording repeatedly from the same animals. It also allows me to correlate parabrachial activity with the animal's behavioral state, by using pupil changes as a proxy for internal states. I report that, in parabrachial neurons from both male and female mice, anesthesia leads to decreased activity, including a decrease in spontaneous activity and reduced magnitude of responses to noxious stimuli. I also demonstrate that, in awake mice, evoked response after chronic pain results in a lasting amplification of parabrachial activity. Finally, I show that changes in parabrachial activity are related to changes in arousal, measured as increases in pupil diameter states.
    • The Impact of Genetic Ancestry on Mutation Frequency and Histopathologic Characteristics in High-Grade Endometrial Cancer

      Bremseth-Vining, Ryan; O'Connor, Timothy D. (2023)
      Endometrial cancer, like numerous other cancer types, exhibits clear racial disparities in the United States for both the incidence and outcomes of the disease. This study seeks to interrogate the role that germline genetic influences, specifically genetic ancestry, may play in contributing to endometrial cancer disparities. This is accomplished by examining the relationship between local ancestry inferences and somatic mutation frequency as well as histologic subtypes. This study highlights three regions along the genome wherein African local ancestry segments were found to significantly correlate with higher tumor mutation burden. A relationship between heterozygous ancestry combinations and increased mutation frequency is also described. Finally, this study identified a single region wherein African local ancestry was found to significantly correlate with a higher incidence of the serous histologic subtype. These findings help to uncover the complex relationships between genetic ancestry and multiple factors underlying the incidence and outcomes of endometrial cancer.
    • The claustrum: A novel cortical network hub

      Qadir, Houman; Mathur, Brian N. (Brian Neil) (2023)
      The human brain is an exceedingly complex biological system with billions of neurons that allows us to process and respond to a constant changing physical environment. Examining the interconnectivity of each of these regions at a circuit level provides insight to brain functions that dictate cognition and action. The work from this dissertation stands to expand our current knowledge of cerebral cortical systems and introduces a heavily understudied cortical-subcortical loop network that bridges cortical network components critical for performing cognitive functions. Using viral tract-tracing and ex vivo electrophysiology methods, I found that a subcortical structure, called the claustrum, connects specific cortical network components. In doing so, I revealed the bulk of the input-to-output circuit map of the murine claustrum. These findings support a model wherein the claustrum supports cortical networks for cognitive control. Understanding the neural circuit mechanisms underlying cortical networks is critical for treating cortical network dysfunction, and resultant cognitive impairment, across a multitude of neuropsychiatric diseases.
    • Enhanced myogenesis through lncFAM-mediated recruitment of HNRNPL to the MYBPC2 promoter

      Chang, Ming-Wen; Gorospe, Myriam; Wilson, Gerald M. (2023)
      Long non-coding RNAs (lncRNAs) are potent transcriptional and posttranscriptional regulators of mammalian gene expression programs. By interacting with different molecular partners, particularly proteins and other RNAs, lncRNAs serve diverse roles in biological processes. One such process is myogenesis, wherein mononucleated, undifferentiated myoblasts proliferate and further differentiate into multinucleated, mature muscle fibers capable of contractile activity. Through myogenesis, muscle is created during development and is regenerated in adult life. As myogenesis is impaired with advancing age and in certain pathologies, understanding it in molecular detail can uncover therapeutic targets. However, the specific molecular mechanisms by which lncRNAs regulate human myogenesis are poorly understood. In this thesis, we have uncovered a novel muscle-specific lncRNA, lncFAM71E1-2:2 (lncFAM), which increased robustly during early human myogenesis. Silencing lncFAM attenuated differentiation of human myoblasts into myotubes, while overexpression of lncFAM promoted this process. As lncFAM resides in the nucleus, chromatin isolation by RNA purification followed by mass spectrometry (ChIRP-MS) analysis was employed to identify the molecular mechanisms whereby it might promote myogenesis. Analysis of lncFAM-interacting proteins revealed that lncFAM recruited the RNA-binding protein HNRNPL to the promoter of MYBPC2, in turn increasing MYBPC2 mRNA transcription and enhancing production of the myogenic protein MYBPC2. These results highlight a mechanism whereby a novel ribonucleoprotein complex, lncFAM-HNRNPL, promotes MYBPC2 expression transcriptionally to increase myogenesis.  
    • Striatal fast-spiking interneurons regulate compulsive alcohol consumption

      Patton, Michael; Mathur, Brian N. (Brian Neil) (2022)
      Persistent drinking in the face of a negative consequence, or compulsive drinking, is a prominent feature of alcohol use disorder. This behavior is directed, in part, by habitual actions encoded by the dorsolateral striatum, or the putamen in humans and nonhuman primates. How ethanol targets the dorsolateral striatum to drive compulsive drinking is poorly understood. However, parvalbumin-expressing striatal fast-spiking interneurons, which comprise ~1% of the total neuronal striatal population, are enriched in the dorsolateral striatum, regulate habit formation, and are functionally modulated by ethanol. To determine whether fast-spiking interneurons are necessary for compulsive ethanol consumption, we selectively ablated these neurons in adult male and female C57BL/6J mice undergoing a voluntary chronic intermittent ethanol consumption paradigm followed by a compulsive ethanol drinking assay. Fast-spiking interneuron ablation curtailed the development of organized ethanol lick sequence behavior, reduced ethanol consumption, and abrogated compulsive consumption of ethanol with the added bitterant quinine. We subsequently investigated how chronic ethanol exposure modulates the physiology of dorsal striatum fast-spiking interneurons. To this end, we utilized ex vivo whole-cell patch clamp electrophysiology and found that 5-week ethanol vapor exposure specifically reduced GABAergic transmission onto dorsal striatum fast-spiking interneurons. This effect corresponded with a significant reduction in the number of GABAergic synapses that was predominantly seen on the soma and proximal processes of fast-spiking interneurons. Perineuronal nets, a subdivision of the extracellular matrix predominately expressed on parvalbumin-expressing interneurons, condense around the soma and proximal branches to regulate synaptic transmission. We found that 5-week ethanol vapor exposure degraded dorsal striatum perineuronal nets and that enzymatically degrading perineuronal nets reduces GABAergic transmission. Lastly, we investigated how GABAergic transmission responds to acute ethanol in slice and found that acute bath application of ethanol potentiates GABAergic transmission onto fast-spiking interneurons, but that 5-week ethanol vapor exposure prevents acute ethanol potentiation of GABAergic transmission, suggesting that GABAergic inputs onto fast-spiking interneurons undergo a homeostatic adaptation to repeated ethanol exposure. Collectively this work provides a novel insight into the pathophysiology of chronic ethanol exposure and identifies multiple therapeutic substrates for future interventions against compulsive drinking.
    • Trends in and Factors Associated With Maternal Autonomy and Its Role in Health Services Utilization and Neonatal/infant Deaths in Bangladesh

      Kibria, Gulam Muhammed Al; Hirshon, Jon Mark (2023)
      Introduction: The neonatal/infant mortality rate is high in most low- and middle-income countries, including Bangladesh. Maternal autonomy or decision-making ability improves health care utilization, and adequate health care utilization reduces neonatal/infant deaths. Previous studies reported determinants of neonatal/infant deaths in Bangladesh, but the role of sociocultural factors like maternal autonomy is unclear. Examining the trends (i.e., changes in prevalence) in and factors affecting maternal autonomy, and its role in neonatal/infant deaths could inform future research, programs, or policies to reduce neonatal/infant deaths in Bangladesh. The aims of the study are three-fold: (1) investigate trends in the prevalence of and factors affecting maternal autonomy; (2) identify the association of maternal autonomy with neonatal/infant deaths; and (3) investigate whether health care utilization mediates the association between maternal autonomy and neonatal/infant deaths. Methods: To investigate Specific Aim 1, data from Bangladesh Demographic and Health Survey (BDHS) 1999-00, 2004, 2007, 2011, 2014, and 2017-18 were analyzed. To examine Specific Aims 2 and 3, BDHS 2017-18 data were analyzed. Maternal autonomy (i.e., exposure variable) was defined as decision-making ability about own health care, large household purchases, and freedom of mobility. To describe prevalence, these three decisions were categorized as no, low, and high with decision-making ability about no, 1-2, and 3 decisions, respectively. Maternal autonomy was also dichotomized as the presence of at least 1 autonomy to investigate the association. Neonatal and infant deaths were defined as deaths within the first one month and one year, respectively. Three health care utilization variables were tested as mediators: at least four antenatal care (ANC) visits, hospital birth, and neonatal post-natal care (PNC). For all aims, the distributions of study samples were compared per exposure, mediators, and outcomes. The prevalence of maternal autonomy was reported, its trends were tested with Cochrane-Armitage test, and the factors associated with maternal autonomy were identified using multilevel logistic regression. Then, the as-sociations of maternal autonomy with neonatal and infant deaths were tested using multi-level logistic regression. Finally, to test mediation, ‘maternal autonomy’s relationships with health care utilization’ and ‘health care utilization’s relationship with neonatal and infant deaths were examined using multilevel logistic regression. Prevalence odds ratios (PORs) were reported with 95% confidence intervals (CIs). Results: The prevalence of maternal autonomy for all 3 decisions increased from 36.4% (95% CI: 34.8 to 38.1) in 1999-00 to 53.7% (95% CI: 52.1 to 55.2) in 2017-18. The prevalence of no autonomy declined from 27.9% (95% CI: 26.4 to 29.5) to 16.2% (95% CI: 15.1 to 17.3) during that period. These changes were observed regardless of decisions and most background characteristics of the participants. Overall, in the adjusted analysis, mothers with older age, higher education, and employment were more likely to have autonomy than their counterparts (POR > 1, p-value <0.05). Among mothers with no and any autonomy, the neonatal mortality rate was 30.4 (95% CI:19.0 to 48.1) and 15.0 (95% CI: 11.6 to 19.4) per 1000 live births; and infant mortality rate was 32.8 (95% CI: 20.8 to 51.3) and 19.3 (95% CI: 15.4 to 24.2) per 1000 live births, respectively. Compared to mothers with no autonomy, those with any autonomy (adjusted POR: 0.50, 95% CI: 0.28 to 0.88, p = 0.016) had lower odds of neonatal deaths. Similarly, the odds of infant mortality was lower among mothers with any autonomy (adjusted POR: 0.59, 95% CI: 0.35 to 0.99, p = 0.049). Maternal autonomy and neonatal/infant deaths did not have significant association with adequate health care utilization. Therefore, the mediational impact of healthcare utilization was not tested (p>0.05). Conclusion: During the past two decades, although the level of maternal autonomy has increased in Bangladesh, a significant proportion of mothers do not have autonomy. Expanding educational and earning opportunities may increase maternal autonomy in this country. Considering the association of maternal autonomy with neonatal/infant deaths, more research should be carried out to understand the association of other factors (e.g., socioeconomic factors) that may impact the association and to understand the perspectives of husbands, in-laws, and other family members on the role of autonomy.
    • The Neurobehavioral and Brain Morphometric Outcomes Associated with Bullying Victimization.

      Menken, Miriam; Chang, Linda, Ph.D. (2023)
      Childhood bullying victimization was linked to several debilitating long-term outcomes, including a doubled risk for suicide attempts in adulthood and an increased risk for anxiety disorders in adolescence. While several studies focused on self-harm or suicidal outcomes associated with bullying in adolescents, few identified the sex-specific neurological, cognitive, and suicidal ideation outcomes associated with bullying victimization in preadolescent children. Additionally, no studies examined whether changes in cortical morphometry might mediate the impact of victimization on cognition. Furthermore, most previous studies that evaluated bullying victimization and neurological or cognitive outcomes were limited by relatively small sample sizes. We used data from the ongoing, longitudinal Adolescent Brain Cognitive Development (ABCD) Study dataset of 11,875 children to determine the cross-sectional (baseline) and longitudinal (matched sample from baseline and second annual follow-up visits) sex-specific neurobiological outcomes associated with bullying victimization. We also performed a methodological study to examine the importance of including socioeconomic factors in all analyses, while replicating the results from a prior study on the impact of initiation of tobacco use on brain morphometry and cognition. This dissertation reports on several novel findings. First, we found that bullying victimization was associated with persistently poorer cognition at baseline and at the second annual follow-up. Second, we identified several novel sex-specific relationships between bullying, self-harm, and behavioral problems in preadolescent children. Third, bullying victimization was associated with larger cortical and subcortical volumes, larger cortical surface area, but thinner cortices. Fourth, larger surface area in the fusiform gyrus partially suppressed (compensated for) and thinner precentral gyrus cortices partially mediated (contributed to) the effect of bullying on cognition. Additionally, our methodological study emphasized the need to adjust for socioeconomic factors when addressing complex human behaviors and brain development. Together, these findings support our overall hypothesis that bullying victimization in preadolescent children is linked to several major neurobehavioral and brain morphometric outcomes. Our findings can better inform schools and communities of the negative consequences of bullying victimization and help them design interventions and treatments that address potential contributory factors based on sex, socioeconomic level, and race/ethnicity.
    • Cell Subtype Specific Investigation of Cocaine-induced Transcriptomic Adaptations in the Nucleus Accumbens

      Choi, Eric; Lobo, Mary Kay (2023)
      Substance use disorder is a chronic disease and a leading cause of disability around the world. The nucleus accumbens (NAc) is a major brain hub mediating reward behavior. Studies demonstrate exposure to cocaine is associated with molecular imbalance in NAc medium spiny neurons (MSNs), dopamine receptor 1 and 2 enriched D1-MSNs and D2-MSNs. We previously reported repeated cocaine exposure induced transcription factor early growth response 3 (Egr3) mRNA in NAc D1-MSNs, and reduced it in D2-MSNs. Here, we report that repeated cocaine exposure in mice induces MSN subtype specific bidirectional expression of the Egr3 corepressor NGFI-A-binding protein 2 (Nab2). Using CRISPR activation and interference tools combined with Nab2 or Egr3 sgRNAs, we mimicked these bidirectional changes in Neuro2a cells. Furthermore, we investigated MSN subtype specific bidirectional expressional changes of histone lysine demethylases Kdm1a, Kdm6a and Kdm5c in mouse NAc after repeated cocaine exposure. Employing a light inducible Opto-CRISPR-KDM1a system, we were able to downregulate Egr3 and Nab2 transcripts in Neuro2A cells and cause similar bidirectional expression changes we observed in D1-MSNs and D2-MSNs of mouse repeated cocaine exposure model. Contrastingly, our Opto-CRISPR-p300 activation system induced the Egr3 and Nab2 transcripts and caused opposite bidirectional transcription regulations. We further investigated the role of Nab2 in mouse cocaine self-administration. Specifically, we report that NAc D2-MSN specific silencing of Nab2 attenuates cocaine seeking after 10 days of cocaine self-administration in mice. Furthermore, we observed that Nab2 KD in D2-MSNs caused lower cocaine intake in female mice. Notably, lower intake was not observed in male mice. Next, using single-nucleus RNA-sequencing (snRNA-seq), we investigated cocaine and D2-MSN specific Nab2 KD induced alterations in transcriptomic landscape in mouse NAc with single-cell resolution. We then performed cell population specific gene co-expression network analysis and unbiasedly identified modules of differentially expressed genes (DEGs). Notably, we observed that several D2-MSN specific modules of DEGs were up-regulated after cocaine self-administration, and this effect was blocked with Nab2 silencing. Together, the findings in this dissertation provide mechanistic insights into cocaine-induced MSN subtype specific adaptations in the NAc with a particular focus on the D2-MSN specific role of Nab2.
    • The Impact of SARS-CoV-2 Accessory Proteins and Non-Spike Mutations on Viral Pathogenesis

      McGrath, Marisa; Frieman, Matthew B. (2023)
      In December of 2019, a viral pneumonia emerged in Wuhan, Hubei Province, China. This disease, known as COVID-19, would continue to spread rapidly. The causative agent of this pneumonia was later found to be a novel coronavirus designated SARS-CoV-2. To date, this virus is responsible for over 750 million cases and nearly 7 million deaths. Due to the massive impact this virus had on the population, efforts to manufacture a vaccine based on the immunodominant spike antigen were both swift and successful. Despite this development, the virus continues to spread, and additional characterization of SARS-CoV-2 is needed to assist in the development of therapeutics and preparedness for significant resurgence. We were specifically interested in the accessory proteins located at the 3′ end of the viral genome. Coronaviral accessory proteins have previously been shown to impact viral replication and pathogenesis, although they are not necessary for the virus to cause disease. To assess the impact of the accessory proteins of SARS-CoV-2 on viral replication and pathogenesis, we synthesized novel infectious clones of this virus that lacked one or more of these accessory proteins and examined their replicative fitness and impact on pathogenesis in a mouse model. Soon after we began our work with these accessory proteins, viral variants of SARS-CoV-2 emerged and began to dominate over the wildtype strain. We noticed a significant number of mutations in the accessory protein region of these genomes and aimed to characterize their impact on pathogenesis through the synthesis of novel infectious clones possessing the variant spike protein in the wildtype background to allow for determination of the effect of non-spike mutations on variant pathogenesis. Our work with these deletion viruses and variant spike viruses led us to determine that the accessory protein ORF8 of SARS-CoV-2, which differs significantly from that of SARS-CoV-1, contributes significantly to the modulation of inflammation in the lungs during infection and the variants of SARS-CoV-2 have evolved to lose this function.
    • Myofibrillar Malformations are Driven by Aberrant Microtubule Detyrosination in Aging and Dystrophy

      Harriot, Anicca; Ward, Christopher, Ph.D. (2023)
      The highly ordered structure of the myofibrillar matrix is essential for optimal muscle function. However, in dystrophy and aging myofibrils become misaligned resulting in misorientation of force vectors and dyssynchronous activation of sarcomeres. Alterations in muscle fiber structure have been linked to the decreased contractile force, increased susceptibility to injury and the activation of signaling pathways that predispose muscle atrophy. Remodeling of the microtubule (MT) network, marked by increased MT density and post-translational modification by detyrosination, are also pathognomonic of skeletal muscle disease/dysfunction. Our work in Duchenne muscular dystrophy, and new work in aging, align these MT changes to increased cytoskeletal stiffness and dysregulated excess in mechano-activated reactive oxygen species (ROS) and calcium (Ca2+) signals that contribute to contraction-induced injury. Given the essential role of deTyr-enriched MT arrays in myofibrillar growth, maintenance, and repair, we hypothesize that in healthy aging and in disease, alterations in cytoarchitecture, including increased densification, detyrosination, and disorganization of the MT network, underscore myofibrillar malformations and deleterious changes in sarcomere alignment. To this end, our recent work has focused on characterizing morphological changes to myofiber structure in relation to the structure and properties of the MT network across the trajectory of dystrophy and healthy aging. Evidence that myofibers with structural malformations also arise in these conditions to promote muscle damage, led us to question whether these myofibrillar malformations co-segregate with MT alterations. Using an automated imaging approach, we profiled myofiber, sarcomere, and cytoskeletal structure to quantify the coincidence of altered myofibrillar structure, altered MT density, and shifts in MT mediated mechanotransduction. At areas of myofibrillar malformation, we find increased MT density and concomitant increase in MT post-translational modification. Through genetic increase in the detyrosinating enzyme complex VASH2/SVBP expression in otherwise health mice, we find densified deTyr-enriched MT arrays that co-segregate with the onset of myofibrillar malformations. We further show that the suppression of contraction injury, by targeting MTs, may have been due to their effect at these structures. We conclude that disease-dependent densification of deTyr-enriched MT arrays underscores the altered myofibrillar structure in dystrophic skeletal muscle fibers. Ultimately, this work implicates a relationship between disease-dependent MT alterations and the genesis of disordered myofiber and sarcomere structure.