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UMass Chan Medical School has three graduate schools: the T.H. Chan School of Medicine, the Morningside Graduate School of Biomedical Sciences and the Tan Chingfen Graduate School of Nursing. The collections in these communities archive and share research from UMass Chan students of all three schools, including doctoral dissertations, master's theses, published journal articles, research projects, capstone presentations and conference materials.

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Recently Published

  • Ethical Frameworks in Periviable Decision-Making: Patient Perspectives and Provider Patterns

    Delaney, Kathryn (2024-06-25)
    Background: Periviability counseling involves engaging in difficult ethical decisions. The ethical frameworks used by patients and their healthcare providers to discuss this topic have not been previously studied. Objectives: This study assessed the ethical frameworks used by patients and by providers during periviability counseling and subsequent decision making. Study Design: This mixed methods study included patients between gestation ages 21 weeks 0 days through 24 weeks 6 days who required periviability counseling, and the providers performing this counseling. Counseling sessions between providers and patients were recorded, as were semi-structured follow-up interviews with patients. These recordings were transcribed and analyzed using thematic analysis. Demographic surveys were given to providers, and patient demographic, obstetric, and delivery information was abstracted from medical records. Results: A total of 11 patients and 11 providers participated in either a recorded counseling session, a follow-up interview, or both. Qualitative analysis revealed the following themes: 1) decisions regarding periviable intervention are the patient’s to make, 2) desire to avoid pain or suffering of the fetus/newborn, especially as it related to CPR, 3) doing what is best for the family, including partners and other children at home, 4) “doing everything” as a good in itself, and 5) periviable complications disrupt expectations about parenthood or pregnancy which then need to be reimagined. These themes corresponded to four distinct ethical frameworks: principlism, care ethics, virtue ethics, and narrative ethics. All of the patients, and approximately three-quarters of providers used at least one of these ethical frameworks; most used a combination of frameworks. Conclusions: Patients and providers engaging in decision making surrounding periviable intervention use commonly accepted ethical frameworks to discuss and decide how to proceed with these pregnancies.
  • Calcineurin coordinates cell cycle progression with adaptation to environmental stress

    Flynn, Mackenzie J (2024-06-14)
    Sudden exposure to environmental stress threatens the viability of single-celled microbes and cells within complex tissues. In order to survive, cells must sense environmental changes and coordinate a transient cell cycle arrest with the appropriate adaptive response. Cells have several stress-responsive pathways that promote adaptation to distinct stressors, but how these pathways interact with one another is poorly understood. Here, we examined the response to calcium chloride stress, which activates the phosphatase calcineurin and the MAPK Hog1 in Saccharomyces cerevisiae. We discovered that calcineurin extends Hog1 activation, which causes prolonged downregulation of cell cycle-regulated genes and delays progression through multiple cell cycle phases. At the G1/S transition, crosstalk between calcineurin and Hog1 dramatically increases the duration of calcium-induced arrest. I found that Hog1 triggers arrest independent of calcineurin by decreasing G1 cyclin transcription and calcineurin maintains this arrest by extending Hog1-dependent activation of the G1 CDK inhibitor Cip1. These results suggest that stress-response pathway interactions tailor cell cycle arrest with adaptation to environmental stress. The immediate response to stress is well-characterized, but how cells maintain viability in challenging environments after recovering from a stress-induced arrest is unknown. I investigated the response to prolonged growth in calcium stress and found that calcineurin maintains fitness by promoting cell division and suppressing death. I determined that calcineurin helps cells proliferate and survive prolonged calcium exposure by two mechanisms, which differentially require a downstream transcription factor. Together, these findings highlight the importance of stress-response pathways during both acute and chronic environmental stress.
  • Kinetics of Pre-mRNA 3’ End Cleavage

    Torres Ulloa, Leslie (2024-05-31)
    3’ end cleavage and polyadenylation are required steps in pre-mRNA maturation. The rate at which 3’ end cleavage occurs can determine the temporal availability of mRNA for subsequent function throughout the cell and is likely tightly regulated. While there are numerous high-throughput methods for global profiling of RNA maturation rates, the study of pre-mRNA 3’ end cleavage kinetics has remained limited to low-throughput approaches, and the temporal regulation of polyadenylation site choice that determines the composition of the 3’ UTRs of mRNAs remains poorly understood. This research project seeks to address this gap by introducing a novel genome-wide, site-specific methodology for estimating rates of pre-mRNA 3’ end cleavage, using metabolic labeling of nascent RNA, high-throughput sequencing, and mathematical modeling. Using in-silico simulations of nascent RNA-seq data, we show that our approach can accurately and precisely estimate cleavage half-lives for both constitutive and alternative sites. In Drosophila melanogaster S2 cells, we find that cleavage rates are fast but highly variable across sites, with alternative events being slowest. This variability in rates is underpinned by distinctive sequence elements, where an A-rich region upstream of the cleavage site, a U-rich element downstream of the cleavage site, and a higher density of polyadenylation signals, lead to faster cleavage reactions. Assessment of Polymerase II dynamics around cleavage sites reveals that cleavage rates are associated with the localization of RNA Polymerase II at the end of a gene and faster cleavage leads to quicker degradation of downstream read-through RNA. This approach for estimating pre-mRNA 3’ end cleavage kinetics opens new possibilities in the study of co-transcriptional regulation of mRNA expression and transcription termination across cellular states.
  • Variation in Depth of Sedation Targeted and Achieved among Mechanically Ventilated Patients and Associated Outcomes

    Rucci, Justin M (2024-05-30)
    Introduction: Sedative agents are commonly administered to patients receiving mechanical ventilation (MV). Practice guidelines recommend provision of light sedation within validated scoring systems (e.g., Richmond Agitation Sedation Scale [RASS]), but recognize some circumstances require deeper sedation. The real-world approaches to depth of sedation, and the impact of hospital sedation practices on patient outcomes, remain uncharacterized. Methods: We used the US based eICU collaborative research database to identify adult patients who received MV > 24 hours, who did not have a diagnosis that may require sedatives for indications other than facilitating MV, and who had recorded RASS goals and scores. We used mixed effects regression models to determine factors associated with initial RASS goals and rates of RASS score-goal concordance. We organized hospitals into quartiles of risk-adjusted RASS score-goal concordance, and used g-computation to evaluate differences in ventilator free days (VFD) at hospital day 28. Results: We identified a study sample of 1,650 adult patients (at 21 hospitals) who met inclusion/exclusion criteria. Hospital-level risk-adjusted initial RASS goals ranged from -1.4 to 0.2, and hospital-level risk-adjusted RASS score-goal concordance ranged from 27% to 64%. Patients admitted to hospitals in the highest quartile of score-goal concordance (quartile 4) were generally targeted for deeper sedation (median RASS goal -1.31) than patients admitted to hospitals in the lowest quartile (quartile 1) (median RASS goal -0.58). Compared to patients admitted to quartile 1 hospitals, patients at quartile 4 hospitals experienced fewer VFDs (adjusted incidence risk difference -2.4, 95% CI -4.26 to -0.36). Conclusion: US hospitals prescribe RASS goals in line with guideline recommendations for light sedation, but there is wide variation in achieving these RASS goals. Hospitals with higher RASS score-goal concordance typically prescribed deeper RASS goals, and patients admitted to these hospitals experienced fewer VFD.
  • Burnout Among Young Adults With Type 1 Diabetes

    Perez, Danielle; Sullivan-Bolyai, Susan L; Bova, Carol A; Fain, James A (2024-05-30)
    Purpose: The purpose of this qualitative descriptive study was to describe the experience of diabetes burnout in young adults with type 1 diabetes (T1DM). In addition, aims included participant perspectives of risk and protective factors associated with burnout and ways to balance everyday life with diabetes self-management (DSM). Methods: Young adults with T1DM (N = 11) were recruited through social media platforms and modified snowball sampling and interviewed. Informational redundancy was achieved. Qualitative thematic coding and analysis were conducted within and across transcripts. Results: Diabetes burnout was described as the willingness to put diabetes and DSM on the "back burner" and let things slide due to exhaustion, frustration, apathy, and the desire to be like everyone else for a while. Risk and protective factors were identified along with strategies to achieve balance of DSM in everyday life. Conclusions: This study identified a clear definition of diabetes burnout and acknowledges this concept as distinct and separate from other psychosocial conditions. Health care providers can utilize this information to identify individuals at risk for diabetes burnout and offer more effective support to lessen the overall burden associated with T1DM.
  • Dual engagement of the nucleosomal acidic patches is essential for deposition of histone H2A.Z by SWR1C

    Baier, Alexander S; Gioacchini, Nathan; Eek, Priit; Leith, Erik M; Tan, Song; Peterson, Craig L (2024-05-29)
    The yeast SWR1C chromatin remodeling enzyme catalyzes the ATP-dependent exchange of nucleosomal histone H2A for the histone variant H2A.Z, a key variant involved in a multitude of nuclear functions. How the 14-subunit SWR1C engages the nucleosomal substrate remains largely unknown. Studies on the ISWI, CHD1, and SWI/SNF families of chromatin remodeling enzymes have demonstrated key roles for the nucleosomal acidic patch for remodeling activity, however a role for this nucleosomal epitope in nucleosome editing by SWR1C has not been tested. Here, we employ a variety of biochemical assays to demonstrate an essential role for the acidic patch in the H2A.Z exchange reaction. Utilizing asymmetrically assembled nucleosomes, we demonstrate that the acidic patches on each face of the nucleosome are required for SWR1C-mediated dimer exchange, suggesting SWR1C engages the nucleosome in a 'pincer-like' conformation, engaging both patches simultaneously. Loss of a single acidic patch results in loss of high affinity nucleosome binding and nucleosomal stimulation of ATPase activity. We identify a conserved arginine-rich motif within the Swc5 subunit that binds the acidic patch and is key for dimer exchange activity. In addition, our cryoEM structure of a Swc5-nucleosome complex suggests that promoter proximal, histone H2B ubiquitylation may regulate H2A.Z deposition. Together these findings provide new insights into how SWR1C engages its nucleosomal substrate to promote efficient H2A.Z deposition.
  • Single-cell genomics and regulatory networks for 388 human brains

    Emani, Prashant S; Liu, Jason J; Clarke, Declan; Jensen, Matthew; Warrell, Jonathan; Gupta, Chirag; Meng, Ran; Lee, Che Yu; Xu, Siwei; Dursun, Cagatay; et al. (2024-05-24)
    Single-cell genomics is a powerful tool for studying heterogeneous tissues such as the brain. Yet little is understood about how genetic variants influence cell-level gene expression. Addressing this, we uniformly processed single-nuclei, multiomics datasets into a resource comprising >2.8 million nuclei from the prefrontal cortex across 388 individuals. For 28 cell types, we assessed population-level variation in expression and chromatin across gene families and drug targets. We identified >550,000 cell type-specific regulatory elements and >1.4 million single-cell expression quantitative trait loci, which we used to build cell-type regulatory and cell-to-cell communication networks. These networks manifest cellular changes in aging and neuropsychiatric disorders. We further constructed an integrative model accurately imputing single-cell expression and simulating perturbations; the model prioritized ~250 disease-risk genes and drug targets with associated cell types.
  • Using a comprehensive atlas and predictive models to reveal the complexity and evolution of brain-active regulatory elements

    Pratt, Henry E; Andrews, Gregory; Shedd, Nicole; Phalke, Nishigandha; Li, Tongxin; Pampari, Anusri; Jensen, Matthew; Wen, Cindy; Gandal, Michael J; Geschwind, Daniel H; et al. (2024-05-23)
    Most genetic variants associated with psychiatric disorders are located in noncoding regions of the genome. To investigate their functional implications, we integrate epigenetic data from the PsychENCODE Consortium and other published sources to construct a comprehensive atlas of candidate brain cis-regulatory elements. Using deep learning, we model these elements' sequence syntax and predict how binding sites for lineage-specific transcription factors contribute to cell type-specific gene regulation in various types of glia and neurons. The elements' evolutionary history suggests that new regulatory information in the brain emerges primarily via smaller sequence mutations within conserved mammalian elements rather than entirely new human- or primate-specific sequences. However, primate-specific candidate elements, particularly those active during fetal brain development and in excitatory neurons and astrocytes, are implicated in the heritability of brain-related human traits. Additionally, we introduce PsychSCREEN, a web-based platform offering interactive visualization of PsychENCODE-generated genetic and epigenetic data from diverse brain cell types in individuals with psychiatric disorders and healthy controls.
  • Spatial Transcriptomics Reconstruction of Mouse Olfactory System

    Wang, I-Hao (2024-05-20)
    The olfactory system is crucial for animals in tasks such as foraging, mate selection, and predator avoidance due to its ability to detect and distinguish a vast array of environmental chemicals. Mice detect these chemicals via olfactory receptor (OR) proteins, which are uniquely expressed by olfactory sensory neurons (OSNs); each OSN expresses only one OR type. OSNs with the same OR converge their axons to a specific location in the olfactory bulb (OB), forming a structure known as a glomerulus. This precise organization ensures a consistent, spatially invariant pattern of glomerular activation for each odorant, playing a likely role in the brain's decoding of odor identities. Nevertheless, the exact locations of most glomeruli are unknown, and the mechanisms that create consistent glomerular maps across different animals are not fully understood. In this study, we leveraged spatial transcriptomics and machine learning to map the majority of glomerular positions within the mouse OB. Furthermore, single-cell RNA sequencing revealed distinct transcriptional profiles for each OSN type, characterized not only by their OR gene but also by a unique set of axon guidance genes. These profiles can predict the eventual location of each OSN's glomerulus within the olfactory bulb. We also identified a correlation between the spatial distribution of glomeruli and the characteristics of their corresponding ORs, suggesting a chemotopic arrangement in the mouse olfactory system. Additionally, we probed the complexity of the OB by creating a spatially resolved cell atlas through spatial single-cell transcriptomics, revealing the identity and distribution of neuron subtypes that contribute to odor perception.
  • Developing an Adeno-Associated Viral Gene Therapy for Sialidosis Using Small and Large Animal Models

    Gallagher, Jillian (2024-05-17)
    Sialidosis is a rare, fatal lysosomal storage disease caused by mutations in NEU1, encoding neuraminidase 1 (NEU1) resulting in toxic accumulation of sialylated glycoproteins. Type I patients have adolescent onset with myoclonus, ataxia, seizures, cherry-red spot, and vision loss, with death in midlife. Type II patients are more severe with the addition of hepatosplenomegaly, coarse facial features, dysostosis multiplex, developmental delay, and death in childhood. There are no approved therapies for sialidosis. We developed three adeno-associated viral (AAV) gene therapy vector strategies to treat sialidosis encoding: Neu1 alone (AAV9-Neu1), Neu1 modified to enhance secretion (AAV9-Idua-Neu1) or Neu1 in combination with its chaperone protein cathepsin A (AAV9-Ctsa-Bici-Neu1). Neonatal Neu1-KO mice were injected by intracerebroventricular injection. Increased survival and significant improvement in motor ability was noted for all vectors, however seizures were observed in AAV9-Neu1 treated mice at 10 months of age. Reduced sialic acid and PPCA were found in peripheral organs and brains of treated mice. NEU1 was increased in peripheral organs and the brain, however AAV9-Ctsa-Bici-Neu1 treated mice had NEU1 levels similar to WT mice. Pathology of peripheral organs were markedly ameliorated with AAV gene therapy. We generated a sialidosis type II sheep using CRISPR-SpCas9 (A320del), which is in the same location as the amino acid change in patients (A319V). Importantly, the A320del sheep exhibits a neurological phenotype unlike the Neu1-KO mouse. Overall, the most efficacious vector is AAV9-Ctsa-Bici-Neu1, which will be further tested in the sialidosis sheep model. These studies will inform future clinical trials for this devastating disease.
  • Exploring the relationship between school-supervised asthma therapy and social determinants of health in pediatric asthma care

    Al-Halbouni, Layana; Ryan, Grace W; Radu, Sonia; Spano, Michelle; Sabnani, Reshma; Phipatanakul, Wanda; Gerald, Lynn B; Garg, Arvin; Pbert, Lori; Trivedi, Michelle (2024-05-16)
    Background: Social determinants of health (SDoH), including access to care, economic stability, neighborhood factors, and social context, strongly influence pediatric asthma outcomes. School-supervised asthma therapy (SST) is an evidence-based strategy that improves asthma outcomes, particularly for historically marginalized children, by providing support for daily medication adherence in school. However, little is known about the relationship between these programs and the adverse SDoH commonly affecting underrepresented minority and marginalized children with asthma. Methods: We examined qualitative data from interviews (n = 52) conducted between 2017 and 2020 with diverse multi-level partners involved in Asthma Link, a SST intervention. Participants included end-users (children and their parents), deliverers (school nurses and pediatric providers), and systems-level partners (e.g., insurers, legislators, and state officials). We used inductive coding to determine themes and subthemes and deductive coding using the Healthy People 2030 SDoH framework. Results: Three themes emerged: (1) SST mitigates adverse SDoH (improves access to preventive healthcare and asthma health literacy), (2) SST benefits children experiencing specific adverse SDoH (provides a consistent medication routine to children with unstable family/housing situations) and (3) specific adverse SDoH impede SST implementation (economic instability, culture and language barriers). Conclusion: This study suggests an important relationship between SDoH and SST that warrants further evaluation in our future work on this community-based asthma intervention. Moreover, our findings underscore the importance of measuring SDoH in the implementation and evaluation of pediatric asthma interventions, particularly given the strong influence of these social factors on child health outcomes.
  • Progressive CD4 T-Cell Dysfunction During Chronic Tuberculosis is Associated with Bacterial Recrudescence

    Chang, Evelyn (2024-05-13)
    Tuberculosis remains a leading cause of death globally, and approximately 5-10% of those infected develop pulmonary disease, often after initially controlling infection. Why immunity fails is unknown. CD4 T cells are crucial for immunity against Mtb, yet little is known about how Mtb infection might modulate CD4 T cell function, particularly late during infection. We hypothesize that failure of CD4 T cell function permits bacterial recrudescence and the development of active disease. Here, we develop a minimalistic adoptive transfer model to study antigen-specific T cells in the lungs of Mtb-infected mice. Late during infection, a decrease in T cell polyfunctionality and production of IL-2, TNF, and IFNγ is accompanied by the accumulation of PD-1 and TIM-3 expressing T cells. Transcriptional profiling identifies signatures of senescence and exhaustion in these cells. In C57Bl/6 mice, a similar co-inhibitory receptor expression and loss of function is observed on antigen-specific CD4s. Single-cell sequencing reveals that most expanded parenchymal T cells are hypofunctional, even early post-infection. However, no evidence of exhaustion or senescence was found. As TCR affinity and signaling strength affects T cell differentiation, we next examined whether TCR affinity impacts T cell function. Higher affinity CD4 T cells differentiate into polyfunctional Th1 cells while lower affinity clones skew towards a Th17 phenotype. The distinct clusters formed by high and low affinity clones on a UMAP projection show how TCR signaling strength affects T cell differentiation during chronic infection. These data provide insight into mechanisms modulating CD4 T cell dysfunction during chronic infection. Further study into these mechanisms will inform therapeutic options.
  • I saw the 'puff of smoke' sign before it vanished into thin air

    Suh, Lyle; Puri, Ajit S; Singh, Jasmeet; Kuhn, Anna Luisa (2024-05-10)
    Moyamoya is characterized as a non-atherosclerotic and non-inflammatory vasculopathy that leads to progressive stenosis of the intracranial internal carotid arteries as well as the Circle of Willis. While it can be idiopathic (Moyamoya disease) or associated with another condition (Moyamoya syndrome), there is a characteristic 'puff of smoke' sign that can be appreciated on cerebral angiography.
  • Investigating the function and regulation of SARM1

    Icso, Janneke D (2024-05-07)
    Wallerian degeneration is a process characterized by axonal fragmentation and myelin disintegration, and is the hallmark of neurodegenerative diseases, traumatic axonal injuries, and peripheral neuropathies. SARM1 is a key promoter of Wallerian degeneration. In injured axons, SARM1 cleaves NAD+ to nicotinamide and a mixture of ADPR and cADPR, which ultimately leads to the axonal degradation that characterizes Wallerian degeneration. SARM1 deletion in animal models of degenerative diseases prevents axonal degeneration, indicating that SARM1 is an attractive therapeutic target. Herein, we describe the investigation of the function and regulation of SARM1. In the initial kinetic characterization of TIR-1, the nematode ortholog of SARM1, we show that these enzymes are regulated by a phase transition to a gel/solid-like state that increases the catalytic activity of the enzyme 27-fold. Moreover, we show that the phase transition of SARM1/TIR-1 occurs in vivo in multiple biological contexts: axon degeneration and intestinal immunity. The phase transition also reduces the threshold for NMN-induced SARM1 activation physiologically relevant levels (140-fold). Furthermore, we delineate the catalytic mechanism of SARM1/TIR-1 and demonstrate that these enzymes use an oxocarbenium-like intermediate to catalyze myriad chemical reactions. This intermediate is common to NAD+ hydrolysis and cyclization as well as the base exchange reaction with NADP+ and nicotinic acid. Finally, we investigate mono-ADP-ribosylation as a novel SARM1 function. Given the critical role of SARM1 in axon degeneration, SARM1 inhibition is likely to attenuate the pathophysiology of Wallerian-like diseases. We expect these findings to inform the further development of inhibitors targeting SARM1.
  • Identifying Therapeutic Oligonucleotide-Induced Neurotoxicity and Methods for Safe Delivery to the Central Nervous System

    Miller, Rachael (2024-04-29)
    Huntington’s disease (HD) is a hereditary neurodegenerative disorder caused by an autosomal dominant mutation in Exon 1 of the Huntingtin gene (Htt). There are no approved treatments for HD. Oligonucleotide therapeutics (ASOs and siRNAs) offer a new strategy to treat genetically defined CNS diseases. These therapeutics aim to attenuate disease pathogenesis by targeting Htt mRNA to reduce the toxic mutant protein. Recent technological advancements now enable robust distribution and efficacy throughout mouse, sheep, and NHP brains. However, oligonucleotides can cause acute neurotoxicity when injected directly into the CSF. This dissertation aims to optimize oligonucleotide delivery for the treatment of HD by addressing safety issues across species. We used electroencephalography (EEG) and electromyography (EMG) in awake animals to confirm that direct CSF injection of oligonucleotides induces seizures. We hypothesized that this was due to the negatively charged oligonucleotides changing the delicate balance of divalent cations in the CSF. To address this issue, we developed an artificial CSF (aCSF) buffer supplemented with Ca2+ alone, Mg2+ alone, or Ca2+ and Mg2+ in the injected solution to prevent the imbalance. Real-time EEG monitoring in awake mice and lambs confirmed the absence of seizures when oligonucleotides were delivered in the new aCSF buffer. In summary, this dissertation identified a potential cause of oligonucleotide-induced acute neurotoxicity, developed a method to safely deliver oligonucleotides to the CNS with Ca2+/Mg2+-enriched buffers, and demonstrated the viability of this formulation in a large animal model. These findings support a new method for safely delivering oligonucleotides to the CNS to treat neurological diseases.
  • Epigenetic Enablers of Meningioma Growth

    Berry, Bethany C (2024-04-26)
    Meningiomas are the most common primary intracranial brain tumor, often causing significant disability and sometimes even death. The most aggressive meningiomas commonly exhibit extensive genomic disruption that can lead to genotoxic and proteotoxic stress, but the mechanisms that enable these tumors to thrive are unknown. To date, meningiomas have no effective chemotherapy. This study used a high-throughput bioactive small molecule screen of established meningioma cell lines and RNA-sequencing of patient meningiomas to identify EHMT2/G9a inhibitors as potent cytotoxic agents in meningioma in vitro. Further, studies using the small molecule EHMT2/G9a inhibitor, UNC0631 demonstrated reduced tumor growth in an orthotopic xenograft mouse model of meningioma in vivo. We used CUT&Tag and transcriptomic analyses of established meningioma cell lines after EHMT2/G9a inhibition to identify the unfolded protein response (UPR) and endoplasmic reticulum (ER) stress apoptotic signaling pathway as key factors in EHMT2/G9a-mediated meningioma cell death. After EHMT2/G9a inhibition, we observed a collapse of heat shock protein expression and hypothesized that downregulation of a deacetylase, Sirtuin 1 (SirT1), may be responsible. Consistent with decreased SirT1 activity, Heat Shock Factor 1 exhibited increased lysine acetylation and decreased DNA binding at the promoters of downregulated heat shock proteins. Overexpression of SirT1 or shRNA-mediated knockdown of the ER stress response mediators, ATF4 or CHOP/DDIT3, decreased meningioma cell death caused by EHMT2/G9a inhibition. The molecular chaperone and ER stress inhibitor, 4-phenylbutyric acid, abrogated meningioma cell death occurring after EHMT2/G9a inhibition. In conclusion, epigenetic maintenance of heat shock protein activity and suppression of the UPR/ER stress apoptotic signaling pathway by EHMT2/G9a and SirT1 are essential for aggressive meningioma growth.
  • Advancing Oligonucleotide Technologies for Malignant Brain Tumors and Other Central Nervous System Diseases

    Sarli, Samantha L (2024-04-26)
    Oligonucleotides are a class of synthetic, nucleic acid-based drugs that modulate the expression of disease-causing genes. These drugs are chemically modified to ensure safe and effective activity in relevant tissues in vivo. However, in complex tissues such as the brain, oligonucleotides can show striking differences in activity across cell types. Determining the activity profile of an oligonucleotide in distinct cell types can inform on mechanism of action and is key in moving these drugs towards the clinic. In this thesis, I broadly focused on developing tools to measure efficacy and improve the safety of gene-silencing oligonucleotides in the central nervous system (CNS). Much of this work was dedicated to glioblastoma multiforme (GBM), a universally lethal brain tumor that is largely resistant to current surgical and drug interventions. To this end, I designed a method to measure gene silencing by oligonucleotides in GBM xenografts versus normal brain tissue in vivo. I assessed the impact of conjugates on oligonucleotide activity patterns in tumor and normal brain cells and identified conjugates with potential GBM applications. In addition, I studied formulation as a strategy to mitigate acute neurotoxicity induced by antisense oligonucleotides (ASOs) delivery to the CNS. I also optimized immunostaining methods for a phosphorothioate-specific antibody to characterize biodistribution of monovalent and multivalent ASOs. In summary, this thesis expands the frameworks used in the rational design of these drugs and further
  • Deciphering Splicing Anomalies in Pancreatic Islets and Cancer Cells

    MacMillan, Hannah J (2024-04-25)
    Efficient splicing hinges upon an intricate balance between splice site choice accuracy and temporal coordination. It is well known that splicing programs are widely dysregulated in disease contexts such as diabetes and cancer, but the mechanism and consequence is not always clear. Here, we investigate splicing outcomes in the context of SNPs and splicing factor mutations. Specifically, we examine back-splicing of the antisense non-coding RNA in the INK locus (ANRIL) in cardiometabolic disease and then query the consequences of recurrent splicing factor mutations SF3B1K700E and U2AF1S34F in cancer. We characterize consistent circular ANRIL isoforms across dozens of pancreatic islet donors and find that individuals with a Type II Diabetes T2D risk-SNP in exon 2 of ANRIL produce more circANRIL. We also find that a higher circular:linear ANRIL isoform ratio is associated with decreased beta cell proliferation, drawing an association between back-splicing and disease phenotype. Outside of transcriptomic variation due to individual SNPs relating to cardiometabolic disease, we investigate the consequences of missense mutations within core splicing factors in cancer. Through a combination of nascent and steady state RNA sequencing and mathematical modeling, we find that both highly prevalent mutations in MDS and leukemias, SF3B1K700E and U2AF1S34F, cause a significant global alteration in splicing rate. Our result suggests that prevalent spliceosomal mutation not only contributes to disease progression in the form of select mis-spliced transcripts as previously reported, but also through a global disruption in the temporal coordination of mRNA processing. Together, our research sheds further light on the interplay between aberrant splicing mechanisms and the progression of disease phenotypes.
  • Mechanisms of Interferon-α/β Receptor (IFNAR) Dependent and Independent Autoimmune Diabetes in LEW.1WR1 Rats

    Arowosegbe, Adediwura (2024-04-22)
    Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing pancreatic β cells by immune cells, leading to insulin deficiency. T1D is driven by intricate interactions between the innate and adaptive immune mechanism, and autoreactive T-cells perpetuate destruction of islets cells following priming by proinflammatory cytokines and chemokines. Although the primary risk factor for T1D is genetic, environmental factors have been implicated as possible triggers or accelerators in the pathogenesis of T1D. Viral infections, especially enteroviruses have been implicated in the pathogenesis of T1D. Mechanisms proposed for such association include virus-induced innate immune responses including type I interferon (IFN) that unmask β-cells for recognition by autoreactive T-cells. Type I IFN has been implicated in the early stages of T1D autoimmunity and previous studies in our rat model highlight the essential role of virus-induced, type I IFN responses as rats lacking type I IFN signaling (Ifnar1-/- LEW.1WR1 rats) have delayed onset and up to 50% reduction in the incidence of autoimmune diabetes. The goal of my thesis research is to delineate type I IFN dependent and independent mechanisms that drive autoimmune diabetes using LEW.1WR1 rats, models in which autoimmune diabetes can be induced with combined poly I:C and virus infection. I hypothesize that in Ifnar1-/- LEW.1WR1 rats, type II IFN and non-interferon innate immune responses compensate for type I IFN responses and drive the adaptive immune cells to mediate autoimmune diabetes. Transcriptome profiles of wild type (WT) and Ifnar1-/- LEW.1WR1 rat islets over a time course were analyzed to define temporal transcriptional events that lead to autoimmune diabetes in prediabetic LEW.1WR1 rats following poly I:C and KRV treatment. Pancreatic sections of treated rats were also analyzed using RNA-in situ hybridization (RNA-ISH) to spatially map islet cells inflammation and to correlate with local islet T cell recruitment. In WT LEW.1WR1 rats, a transcriptional signature characterized by interferon-stimulated genes, chemokines, major histocompatibility class I, and genes for the ubiquitin-proteasome system was identified in subsets of β and α cells. These signature cells increased in frequency over time and correlated with local islet T cell recruitment. Type I IFN genes as well as genes for the ubiquitin-proteasome system are largely suppressed in poly I:C + KRV treated Ifnar1-/- rats compared to control Ifnar1-/- rats, while MHC class I genes were upregulated in poly I:C + KRV treated Ifnar1-/- rats compared to control Ifnar1-/- rats. Cytokine analysis reveal an increase in levels of IFNγ in poly I:C + KRV treated Ifnar1-/- rats compared to WT rats. Blocking IL-1R does not protect from diabetes in WT and Ifnar1-/- treated rats although both are protected with depletion of CD8+ T cells. Infiltration of immune cells in WT rats was accompanied with extensive islet damage while infiltration in Ifnar1-/- rats was less destructive. Inhibition of the JAK signaling pathway confirmed the requirement of the JAK-STAT signaling pathway and CD8+ T cells in progression to diabetes in the WT rats and further testing to confirm this requirement in Ifnar1-/- rats is ongoing. Although CCL proteins were expressed on immune cells, blockade of CCR5 had no effect on diabetes incidence. Collectively, the results from these studies provided mechanistic insights into the essential role of virus-induced, innate immune responses in the early phase of autoimmune diabetes pathogenesis.
  • CD20/MS4A1 is a mammalian olfactory receptor expressed in a subset of olfactory sensory neurons that mediates innate avoidance of predators

    Jiang, Hao-Ching; Park, Sung Jin; Wang, I-Hao; Bear, Daniel M; Nowlan, Alexandra; Greer, Paul L (2024-04-18)
    The mammalian olfactory system detects and discriminates between millions of odorants to elicit appropriate behavioral responses. While much has been learned about how olfactory sensory neurons detect odorants and signal their presence, how specific innate, unlearned behaviors are initiated in response to ethologically relevant odors remains poorly understood. Here, we show that the 4-transmembrane protein CD20, also known as MS4A1, is expressed in a previously uncharacterized subpopulation of olfactory sensory neurons in the main olfactory epithelium of the murine nasal cavity and functions as a mammalian olfactory receptor that recognizes compounds produced by mouse predators. While wildtype mice avoid these predator odorants, mice genetically deleted of CD20 do not appropriately respond. Together, this work reveals a CD20-mediated odor-sensing mechanism in the mammalian olfactory system that triggers innate behaviors critical for organismal survival.

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