Serine/threonine protein phosphatase-5 (PP5) is involved in tumor progression and survival, making it an attractive therapeutic target. Specific inhibition of protein phosphatases has remained challenging because of their conserved catalytic sites. PP5 contains its regulatory domains within a single polypeptide chain, making it a more desirable target. Here we used an in silico approach to screen and develop a selective inhibitor of PP5. Compound P053 is a competitive inhibitor of PP5 that binds to its catalytic-domain and causes apoptosis in renal cancer. We further demonstrated that PP5 interacts with FADD, RIPK1 and caspase 8, components of the extrinsic apoptotic pathway complex II. Specifically, PP5 dephosphorylates and inactivates the death effector protein FADD, preserving complex II integrity and regulating extrinsic apoptosis. Our data suggests that PP5 promotes renal cancer survival by suppressing the extrinsic apoptotic pathway. Pharmacological inhibition of PP5 activates this pathway, presenting a viable therapeutic strategy for renal cancer.

Chromatin structure and organization controls DNA's accessibility to regulatory factors and influences gene regulation. Heterochromatin, or condensed chromatin containing mostly silenced genes, self-assembles through weak, multivalent interactions with its associated proteins that contain intrinsically disordered regions (IDRs) and undergoes liquid-liquid phase separation (LLPS). However, the details of the intricate molecular interactions that drive heterochromatin LLPS are not fully understood. It is crucial that we uncover the molecular mechanisms involved as it regulates vital nuclear functions, and dysregulation is implicated in neurological disorders and cancer. Here, we focus on two members of the methyl-CpG-binding domain (MBD) family of proteins, MBD2 and MBD3, that recognize and interpret methylated residues on heterochromatin's underlying DNA. We use an integrated approach to explore the driving forces that allow them to undergo LLPS and how known interactors influence this process. Using computational approaches that assess amino acid sequence features, we found that MBD2 and MBD3 are highly disordered proteins predicted to undergo LLPS. Although they are highly similar in sequence, they have distinct clustering patterns of certain residue types that suggest the molecular basis of how they phase separate differs between them. We have tested these predictions in vitro and in cellulo and have demonstrated their ability to phase separate individually, together and with methylated DNA using UV-Vis spectroscopy and microscopy. Through truncations of MBD2 and MBD3, we have found that their ability to undergo LLPS is dictated by a balance between hydrophobic interactions, likely arising from their associative domains, and electrostatic interactions, arising from their highly charged termini, occurring within or between the proteins and DNA. Finally, using scattering techniques such as small-angle X-ray scattering (SAXS) and dynamic light scattering (DLS), we have demonstrated that MBD2 and MBD3 are self-interacting proteins that form large assemblies. We propose that MBD2 and MBD3, through their ability to self-interact via hydrophobic and electrostatic forces, undergo LLPS and foster a biochemically unique environment to sequester binding partners and perform their functions as transcriptional repressors and heterochromatin organizers. Uncovering the driving forces that assemble MBD protein-based droplets will give us insight into the higher-order, LLPS-mediated organization of heterochromatin and how it functions within this structure. Additionally, understanding how disease-related aberrations influence biomolecular condensate dynamics will provide novel therapeutic targets.

Myosin 1e (myo1e) is a long-tailed class I myosin implicated in breast cancer progression and the development of focal segmental glomerulosclerosis (FSGS). This dissertation characterizes how myosin functions in these distinct pathologies. In chapter 2, I dissect the role that myo1e plays in the metastasis of breast cancer cells. Using the highly invasive 4T1 cell line, I demonstrate that cells deficient in myo1e exhibit altered morphologies and slower migration rates. Dissection of the migration defects in myo1e KO cells led us to investigate the role of myo1e in organelle trafficking, integrin endocytosis and the assembly and disassembly of focal adhesions. Our preliminary results suggest that cells deficient in myo1e exhibit reduced rate of focal adhesion disassembly. In chapter 3, I characterize three novel mutations in myosin 1e that were isolated from patients with FSGS: A92E, H506D, and G562R. Using in silico and comparative sequence analyses, I demonstrate that these mutations are likely pathogenic and highly evolutionarily conserved. Expressing these mutants in Madin-Darby Canine Kidney (MDCK) cells, I demonstrate that mutants myo1eH506D and myo1eG562R exhibit proper membrane enrichment, while the myo1eA92E mutant mislocalizes to the cytoplasm. Additional characterization of the properly localizing myo1eG562R mutant demonstrated that its junctional dynamics were not different from the junctional dynamics of myo1eWT. Taken together, our findings in chapter 3 demonstrate functional differences among myo1eA92E and myo1eH506D and myo1eG562R mutants and how these differences may contribute to their pathogenicity.

Background The COVID-19 pandemic abruptly reversed the long-standing practice of open visitation in children’s hospitals, due to the concern that hospital visitors might contribute to the spread of disease. However, little is known about the unintended consequences of such policies, including the potential that they may disproportionately impact children and families of color and those from low-income communities. Methods We reviewed requests for an exception to a pediatric visitation policy made between August and November 2020 at a midsize American children’s hospital and collected data regarding details of the requests, demographics, family characteristics, and the patients’ medical histories. We compared the sample to the general patient population using bivariate tests and developed a logistic regression model to explore factors associated with the receipt of requests for an exception to a visitation policy. Results Regression models indicated that Black families were less likely to have their request for an exception to the visitation policy granted, compared to White families (odds ratio, OR = 0.06; 95 percent confidence interval, CI 0.01-0.84; p < .05). The families of children who were admitted to critical care were more likely to have their request for an exception granted (OR = 28.35; 95 percent CI 1.43-562.37, p < .05). Two of the three reviewers of requests for exceptions were found to be less likely to grant a request for an exception (OR = 0.05; 95 percent CI 0.00-0.84; p < .05; OR = 0.03; 95 percent CI 0.00-0.67; p < .05). Conclusions Our findings highlight the need to reconsider the risks and benefits of highly restrictive visitation policies that disproportionately impact vulnerable and marginalized children and their families. This study also provides a model for the broader, prospective analysis of the potential for disparities in the impact of any institutional policy.

Context: The first imported U.S. Ebola Hemorrhagic Fever case during the 2014 West Africa Ebola outbreak triggered an increase in online activity through various social media platforms, including Twitter. Objectives: The purpose of our study was to examine characteristics of local health departments (LHDs) tweeting about Ebola, in addition to how and when LHDs were communicating Ebola-related messages. Design: All tweets sent by 287 LHDs known to be using Twitter were collected from September 3 to November 2, 2014. Twitter data were merged with the 2013 National Association of County and City Health Officials (NACCHO) Profile study to assess LHD characteristics associated with sending Ebola-related tweets. To examine the content of Ebola tweets, we reviewed all such tweets and developed a codebook including four major message categories: information-giving, news update, event promotion, and preparedness. A timeline tracking the trends in Ebola tweets was created by aligning daily tweets with major Ebola news events posted on the Centers for Disease Control and Prevention (CDC) Ebola website. Results: Approximately 60% (n=174) of all LHDs using Twitter sent a total of 1 648 Ebola-related tweets during the study period. Sending more tweets in general (OR: 2.42; 95% CI: 1.00-5.84) and employing at least one Public Information Specialist (OR: 2.61; 95% CI: 1.14-5.95) significantly increased the odds that an LHD tweeted about Ebola. Of all the Ebola tweets collected, 78.6% were information-giving, 22.5% were on preparedness, 20.8% were news updates, and 10.3% were event promotion tweets. A temporal analysis of Ebola tweets indicated five distinct waves, each corresponding with major Ebola news events. Conclusions: Twitter has become a communication tool frequently used by many LHDs to respond to novel outbreaks, but messaging strategies vary widely across LHDs. We present several recommendations for LHDs using this novel communication channel during outbreaks and other emergent events.

Objectives: To examine how dementia is associated with COVID-19 risk and adherence to COVID-19 mitigation behaviors, and whether mitigation behaviors mediate the relationship between dementia and COVID-19 risk. Methods/Design: We analyzed 2019 and 2020 data from the National Health and Aging Trends Study, a national prospective cohort study of United States older adults age 65+. Outcomes were COVID-19 diagnosis and adherence to COVID-19 mitigation behaviors (handwashing, mask-wearing, and social distancing). Results: Among the 3257 older adults in this study, 485 (14.9%) had dementia in 2019 and 98 (3.1%) were COVID-19 positive in 2020. Dementia significantly increased the odds of COVID-19 by 129% (odds ratio [OR] = 2.29, 95% confidence interval [CI] 1.32 to 3.97), and remained elevated after adjusting for sociodemographics and health (OR = 1.67, 95% CI 0.90 to 3.11). Dementia significantly decreased the odds of handwashing by 72% (OR = 0.28, 95% CI 0.17 to 0.44), which remained lower after adjusting for sociodemographics and health (OR = 0.53, 95% CI 0.23 to 1.21). Dementia was not significantly associated with mask-wearing and social distancing. The relationship between dementia and COVID-19 was primarily mediated by functional impairment, income, and residential setting. Conclusions: Dementia was associated with an increased COVID-19 risk and lower adherence to handwashing among U.S. older adults. Adherence to COVID-19 mitigation behaviors did not mediate COVID-19 risk by dementia status. For older adults with dementia, COVID-19 risk could be decreased by prioritizing health interventions.

Objective This study compared the geospatial distribution of Ebola tweets from local health departments (LHDs) to online searches about Ebola across the United States during the 2014 Ebola outbreak. Methods Between September and November 2014, we collected all tweets sent by 287 LHDs known to be using Twitter. Coordinates for each Ebola tweet were imported into ArcGIS 10.2.2 to display the distribution of tweets. Online searches with the search term “Ebola” were obtained from Google Trends. A Pearson correlation was conducted to access the relationship between online search activity and per capita number of LHD Ebola tweets by state. Results Ebola tweets from LHDs were concentrated in cities across the northeast states, including Philadelphia and New York City. In contrast, states with the highest online search queries for Ebola were primarily in the south, particularly Oklahoma and Texas. A weak, negative, non-significant correlation (r=-.03, p=.83, 95% CI -.30-.25) was observed between online search activity and per capita number of LHD Ebola tweets by state. Conclusions We recommend LHDs consider using social media to communicate possible disease outbreaks in a timely manner, and consider using online search data to tailor their messages to align with the public health interests of their constituents.

Introduction: Prior research has identified a link between sleep disturbances and cognitive impairment, however, no study has examined this relationship using a national U.S. sample. This study examines how multiple longitudinal measures of sleep disturbances (sleep-initiation insomnia, sleep-maintenance insomnia, sleep medication usage) are associated with dementia risk. Methods: Ten annual waves (2011–2020) of prospective cohort data from a nationally representative U.S. sample of older adults age 65 and older were analyzed from the National Health and Aging Trends Study (NHATS). Sleep disturbances were converted into a longitudinal score and measured as sleep-initiation insomnia (trouble falling asleep in 30 minutes), sleep-maintenance insomnia (trouble falling asleep after waking up early), and sleep medication usage (taking medication to help sleep). Cox regression models analyzed time to dementia diagnosis for a sample of 6,284 respondents. Results: In the unadjusted model, sleep-initiation insomnia was significantly associated with a 51% increased dementia risk (hazard ratio [HR]=1.51, 95% confidence interval [CI]=1.19–1.90). Adjusted for sociodemographics, sleep medication usage was significantly associated with a 30% increased dementia risk (aHR=1.30, 95% CI=1.08–1.56). Adjusted for sociodemographics and health, sleep-maintenance insomnia was significantly associated with a 40% decreased dementia risk (aHR=0.60, 95% CI=0.46–0.77). Conclusions: These findings suggest sleep-initiation insomnia and sleep medication usage may elevate dementia risk. Based on the current evidence, sleep disturbances should be considered when assessing the risk profile for dementia. Future research is needed to examine other sleep disturbance measures and to explore mechanisms for decreased dementia risk among older adults with sleep-maintenance insomnia.

Cell division, migration, and maintaining cell morphology are all essential and dynamic cell processes that require precise coordination of the actin cytoskeleton. Actin monomers assemble into polar actin filaments that have a faster-growing (barbed) end and a slower-growing (minus) end. Here we examine the role of IQ-motif containing GTPase Activating Protein 1 (IQGAP1) in regulating actin filament assembly. This 189 kDa actin- binding protein slows actin filament assembly by interacting with the barbed end. Using extensive truncation analysis and single molecule microscopy techniques, we determined that IQGAP1 interacts with actin filament ends via residues within its IQ motifs. The barbed ends of actin filaments are intricately and competitively regulated by specific proteins and complexes of proteins that promote (formins) or inhibit (capping proteins) actin filament growth. We next examined the role of IQGAP1 in competitive interactions with the prominent barbed end regulators including formin and capping protein. Using fluorescently tagged proteins, IQGAP1 can be directly visualized on filament ends with individual formins and capping proteins and with formin-capping protein complexes. Interactions between IQGAP1 and formin on the ends of filaments slows formin-mediated actin assembly from 22.68 ± 2.9 subunits s-1μM-1 to 6.13 ± 0.7 subunits s-1μM-1. Further, IQGAP1 interacts with decision complexes on filament ends, creating a more complex decision complex, which decreases the dwell time on the end by 18-fold. We next examined the relevance of IQGAP1-mediated capping in cells using readouts of actin assembly: cell morphology, actin filament structure, and cell migration. Cells lacking IQGAP1 displayed significant changes to cell morphology and actin filament structures Cells expressing IQGAP1 or a capping deficient IQGAP1(CD), unable to bind filament ends, on a plasmid did not display significant changes to cell morphology or actin filament structure compared to wildtype cells. However, cells expressing IQGAP1(CD) displayed significantly slower wound closure compared to cells with endogenous IQGAP1. These results suggest that IQGAP1-mediated capping is a physiologically relevant mechanism of regulating actin filament assembly. This study reveals a role for IQGAP1 as a transient capper that promotes protein exchange on filament ends, which may have implications in the regulation of actin filament lengths in cells.

Psychiatric disorders are a leading cause of disability, premature mortality, and economic burden globally, with a 48.1% increase in cases between 1990 and 2019. Stress in early life has been linked to hippocampal damage and the development of psychiatric disorders later in life. Personal stressors have also been found to be significantly associated with psychological distress, anxiety, and depression. Prenatal stress, which occurs when the fetus is exposed to excess glucocorticoids from maternal stress or synthetic glucocorticoids, has been linked to cognitive and behavioral outcomes later in life, possibly due to its impact on fetal development. In this project, I aimed to study the effect of cortisol on neural progenitor cells (NPCs) differentiated from human induced pluripotent stem cells (iPSCs). Cell type was confirmed using iPSC and NPC marker gene and protein expression by qPCR and immunofluorescence. The loss of undifferentiation marker SSEA4 in NPCs suggests that they are differentiated. The NPCs expressed SOX2, Nestin, SOX1, and PAX6, as confirmed by qPCR and immunofluorescence analyses. The percentage of SOX2, SOX1, PAX6, and Nestin positive cells was quantified based on colocalized signals with the nuclear marker DAPI. Novel Nestin isoforms that may be present in NPCs but not in iPSCs were identified. Currently, only one Nestin isoform is known to us. Novel, unpublished data from collaborators was used to confirm that there are Nestin isoforms present in fetal and adult human isoform sequencing study. Glucocorticoid (GC) response genes were upregulated after cortisol treatment in NPCs, as confirmed by qPCR. I measured NPC proliferation in response to cortisol and identified that cortisol did not significantly increase NPC proliferation. However, RNA sequencing showed differential expression of proliferation-related genes in cortisol-treated NPCs. RNA sequencing analysis revealed that 59 genes were differentially expressed in cortisol treated NPCs (including ZBTB16 and TSC22D3 measured previously using qPCR) compared to controls, out of which nine genes are associated with psychiatric traits based on GWAS studies. The study showed that cortisol can alter gene expression in NPCs, which may have implications for psychiatric disorders. Finally, the study emphasizes the novelty of exploring the role of cortisol in iPSC derived NPCs and highlights the need for further research in this area.