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Nanotherapeutics for Immune Modulation in SepsisDue to its complexity and heterogeneity, managing immune dysregulation in sepsis poses a significant clinical challenge. Thus, there is great demand to both improve our understanding of mediators of immune dysregulation in sepsis and develop nuanced therapeutic approaches to provide precise immune modulation for sepsis treatment. This thesis first investigates the novel phenomenon of cytokine charge disparity as a potential regulator of cytokine function. Then, two novel telodendrimer immune modulation approaches are presented as a personalized medicine strategy for sepsis. Through extensive database and literature review, we have established cytokine charge disparity as a potential mechanism for immune regulation. Using our versatile telodendrimers (TDs), we then optimized and validated our TD nanotrap approach for effective and selective targeting of plasma cytokines. Our lead selective TD nanotraps displayed charge selective cytokine targeting and our lead pan-affinitive TD nanotrap demonstrated superior cytokine removal efficacy compared to commercial resin control. Additionally, pan-affinitive TD nanotrap maintained efficacy across a wide range patient immune status, indicating promising therapeutic potential to reduce mortality risk associated with overwhelming cytokine profiles. To further expand our immune modulation tool set for sepsis treatment, we optimized our TD nanodrug for delivery of dimethyl itaconate (ITA) to control both hyperinflammation and pyroptosis. Encapsulating ITA into TD nanoparticles (ITA:TDNPs) resulted in a sustained-release profile and improved biocompatibility compared to free ITA. ITA:TDNPs more effectively inhibited both LPS- and LTA-induced inflammation and pyroptosis in macrophages compared to ITA or TDNP alone. Finally, ITA:TDNPs demonstrated superior therapeutic efficacy in both an IV LPS and polymicrobial cecal slurry sepsis model compared to individual therapies. Collectively, we have uncovered a novel phenomenon of cytokine charge disparity and validated it as a potential mechanism to regulate cytokine activity, as well as established it as targeting mechanism for effective immune modulation via charge selective TD nanotrap. We further developed an immune modulating TD nanodrug for ITA delivery to control both hyperinflammation and immune cell pyroptosis in sepsis. Through precise targeting of immune dysregulation in sepsis using a systematic multimodal TD therapeutic approach for personalized medicine, we may successfully improve patient outcomes in this devastating disease.
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Mechanism of Mitochondria-Induced Muscle Atrophy During AgingMitochondrial dysfunction is strongly associated with aging-related degenerative diseases including muscle atrophy. However, whether bioenergetic defects are the sole drivers of mitochondria-induced muscle atrophy remains unknown. The Chen lab discovered that various forms of mitochondrial damage can disrupt protein import, leading to the toxic accumulation of unimported mitochondrial precursors in the cytosol. This causes a stress termed mitochondrial Precursor Over-accumulation Stress (mPOS). A mouse model of mPOS was developed in which the mitochondrial inner membrane protein, ANT1, was overexpressed to saturate the protein import machinery. Ant1Tg/+ mice were found to have a severe muscle wasting phenotype. The overarching goal of this dissertation is to investigate the mechanism by which mPOS drives muscle wasting and its implications for normative muscle aging. The findings presented in this thesis led to three conclusions. First, we identified a novel mitochondria-to-lysosomal proteostatic axis through which mPOS induces lysosomal damage. Lysosomal damage subsequently causes the release proteolytic enzymes, which leads to excessive protein degradation and subsequent progressive muscle atrophy. Importantly, we found that this pathway operates independently of mitochondrial respiratory complex activity and reactive oxygen species (ROS) production. Second, we demonstrated the presence of mPOS in physiologically aged muscle. Sarcopenic muscle exhibited phenotypes similar to those found in Ant1Tg/+ mice, evidenced by overlapping transcriptional and proteomic profiles, and lysosomal damage. These findings indicate that mitochondria-induced changes to autophagic activity may play a central role in the pathogenesis of sarcopenia. However, considering the overall protein content of muscle is elevated during aging, we propose that reduced protein quality, rather than absolute protein content, drives sarcopenia. We therefore termed this phenomenon Muscle Atrophy Independent of Protein Content (MAIPC). Finally, we explored additional cellular factors that affect proteostasis and muscle mass maintenance. We found that the GCN2 kinase, a well-established activator of the Integrated Stress Response (ISR), plays a role in protecting myofibers from mPOS-induced stress and muscle wasting in Ant1Tg/+ muscle. Interestingly, we found that this effect is ISR-independent. The data presented in this dissertation provide valuable insights into the mechanistic role of mitochondrial dysfunction in both normative aging and chronic muscle wasting conditions. Our findings conclude that mitochondria-induced muscle atrophy is induced by mechanisms that extend beyond bioenergetic defects. By characterizing these alternative pathways, this work opens new avenues for therapeutic strategies targeting mitochondrial stress in chronic muscle wasting conditions.
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Non-neutralizing Antibodies in the Complex Dance of Dengue Clearance and Immune AvoidanceDengue virus (DENV) is endemic in over 100 countries causing widespread morbidity and mortality. Approximately 400 million people are infected annually with one of the four immunologically and genetically distinct serotypes of DENV, resulting in 100 million symptomatic cases and at least 40,000 deaths. While the mechanisms behind the pathophysiology of severe DENV infection are complex and incompletely understood, it has been previously suggested that antibodies directed against the DENV envelope (E) protein can facilitate antibody dependent enhancement (ADE) of the virus during secondary DENV infections, increasing the number of infected cells and the clinical severity of infection in an exposed individual. However, there are other functional roles for antibodies outside neutralization of the virion. In this thesis, we describe the roles of non-neutralizing antibodies during DENV-infection. We show that IgG and IgA non-structural protein 1 (NS1)- and E-reactive antibodies are capable of mediating monocytic phagocytosis of DENV-infected cells. We show that secreted NS1 (sNS1) acts as immunological chaff and abrogates NS1-reactive antibody-mediated phagocytosis. We also begin to investigate the potential of phagocytosis of DENV-infected cells to lead to lead to infection of phagocytic monocytes. The findings described in these studies have implications in therapeutics and vaccinations targeting both NS1 and E protein.
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Unveiling cell-type-specific transcriptome and genetic regulation in postmortem brains of schizophrenia patientsSchizophrenia is a complex psychiatric disorder with a poorly understood etiology. This dissertation addresses three critical questions in schizophrenia research: identifying involved cell types, characterizing their transcriptomic changes, and elucidating how these changes mediate genetic risk. After rigorous evaluation, we conducted a comprehensive analysis of cell-type-specific gene expression in postmortem brains of schizophrenia patients and controls with single-cell RNA sequencing and cell deconvolution methods. Our findings provide compelling evidence for the involvement of upper-layer neurons and multiple non-neuronal cell types in schizophrenia. We observed significant alterations in synaptic function, neurodevelopment, immune response, and vascular transport within their respective cell types. Notably, we demonstrate that genetic risk for schizophrenia is predominantly enriched in neurons, particularly upper-layer neurons, with partial enrichment in oligodendrocyte precursor cells and vascular cells. This cell-type-specific approach offers novel insights into the molecular underpinnings of schizophrenia, potentially bridging the gap between genetic risk factors and clinical manifestations. By highlighting key genes and pathways, our study establishes a robust foundation for future research and opens avenues for innovative preventive and therapeutic approaches.
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Mucosal Innate Immunity of Human Surfactant Protein A Genetic Variants against SARS-CoV-2 InfectionMore than 7 million people have died of the coronavirus disease-2019 (COVID-19) since first reported in December of 2019. Infection in some patients manifests as life-threatening ALI/ARDS, multi-organ dysfunctions, and/or death characterized by active viral replication and profound inflammatory cell influx into tissues/organs. SARS coronavirus-2 (SARS-CoV-2) infects human angiotensin-converting enzyme 2 (hACE2)-expressing cells through its spike protein (S protein). The S protein is highly glycosylated and could be a target for lectins. Surfactant protein A (SP-A) is a collectin, expressed by lung alveolar type II cells and other mucosal epithelial cells; it plays a crucial role in innate immunity and inflammatory regulation. SP-A modulates pathogenic infection and disease severity by binding to microbial and host glycoproteins to alter infectivity and regulate host inflammation. The human SP-A gene is located on chromosome 10q22-23, which contains two functional genes SP-A1 and SP-A2 (gene names: SFTPA1 and SFTPA2), and a pseudogene. SP-A1 and SP-A2 are highly polymorphic and consist of several genetic variants, such as SP-A1 (variants 6A2, 6A4) and SP-A2 (variants 1A0, 1A3). It has been demonstrated that these variants have differential antiviral and immunoregulatory capacities in response to various viral infections. The goal of this study was to investigate the mechanistic role of human SP-A variants in response to SARS-CoV-2 infection and COVID-19 susceptibility and severity. The results from this study showed that native human SP-A can bind SARS-CoV-2 S protein, receptor-binding domain (RBD), and hACE2 in a dose-dependent manner. A decrease in S protein and RBD binding was observed in the presence of EDTA and sugars, indicating that the SP-A carbohydrate-recognition domain (CRD) mediates S protein binding in a calcium-dependent manner. We further showed that human SP-A can attenuate viral infectivity in susceptible host cells, evidenced by the dose-dependent reduction in viral load in infected cells. These results suggest that human SP-A can bind SARS-CoV-2 S protein, RBD, and hACE2 to attenuate SARS-CoV-2 infectivity in susceptible host cells. Next, we examined the variations in antiviral and immunoregulatory roles of human SP-A variants in response to SARS-CoV-2 infection. The binding studies showed that in vitro-expressed SP-A variants differentially interact with S protein. Moreover, cells inoculated with SARS-CoV-2 pretreated with the 1A0 variant had a more reduced virus titer than those pretreated with the 6A2 variant, indicative of their differential antiviral capacities. These findings from in vitro studies demonstrated that human SP-A and their genetic variants directly interact with viral S protein to differentially modulate SARS-CoV-2 infectivity. To perform in vivo study, six genetically modified double-hTG mouse lines, expressing both hACE2 and the respective SP-A variants: (hACE2/6A2 (6A2), hACE2/6A4 (6A4), hACE2/1A0 (1A0), and hACE2/1A3 (1A3), one SP-A knockout (hACE2/SP-A KO (KO) and one hACE2/mouse SP-A (K18) mice, were generated and challenged intranasally with 103 PFU SARS-CoV-2 (Delta) or saline (Sham). We observed that these infected mice had differential COVID-19 severity. Infected KO and 1A0 mice had more mortality and lung injury compared to other mouse lines, and disease severity correlated with enhanced upregulations of inflammatory genes that play vital roles in host immunity such as MyD88 and Stat3 in the lungs of KO and 1A0 mice. Furthermore, pathway analysis identified several important signaling pathways involved in lung defense, including pathogen-induced cytokine storm, NOD1/2, toll-like receptor, neuroinflammation, and Trem1 signaling pathways. Consistent with the transcriptomic data, expressions of inflammatory mediators such as G-CSF, IL-6, and IL-1β were comparatively higher in the lungs and sera of KO and 1A0 mice with the highest mortality rate. We further examined other organ injuries (kidney, intestine, and brain) in the infected mice; we found a more severe acute kidney injury (AKI) and intestinal damage in KO and 6A4 mice compared to other double-hTG mice. Viral titers were generally lower in the kidneys and brains of infected double-hTG mice relative to KO mice. Inflammatory mediators like TNF-α, IL-6, IL-1β, and MCP-1 were comparatively higher in KO and 6A4 mice with the most severe AKI. High virus presence and inflammatory markers were also observed in the brain and hippocampus of all infected mice. The results from in vivo studies suggest that SP-A variants differentially protect against severe COVID-19. Furthermore, the human COVID-19 patient studies revealed increased SP-A levels in the saliva of COVID-19 patients compared to healthy controls and highlighted the potential use of SP-A levels as a biomarker for COVID-19 severity. Collectively, these findings underscore the importance of host innate immune collectins and contribute to our understanding of the roles of host genetic variations in the observed population-level differences in COVID-19 susceptibility and severity.
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Deciphering cellular dynamics and crosstalk of trabecular meshwork and Schlemm's canal cells in a bioengineered 3D extracellular matrix hydrogel microenvironmentIn the conventional outflow pathway, Schlemm's canal (SC) inner wall endothelium interfaces with the trabecular meshwork (TM). Biomechanical changes in this microenvironment contribute to increased resistance to aqueous outflow, a characteristic of ocular hypertensive glaucoma. Notably, TM undergoes fibrotic-like remodeling and stiffening. Existing in vitro TM/SC models fail to accurately replicate native cell-cell and cell-extracellular matrix (ECM) interactions, limiting their use for studying glaucomatous outflow pathobiology. In this dissertation, we utilized a biomimetic ECM hydrogel system made from natural polymers resembling native tissue proteins. This ECM hydrogel can be (i) used to encapsulate donor-derived primary human TM cells or (ii) employed as a substrate for culturing donor-derived primary human SC cells on top. As ECM hydrogels gradually emerge as a preferred model in diverse research laboratories, a standardized fabrication method is essential to improve accessibility and consistency across experimental protocols. Thus, a detailed methodology for producing these ECM hydrogels is provided in Chapter 2. In Chapter 3, using the 3D TM hydrogel system, we demonstrated that simvastatin-mediated inactivation of Yes-associated protein (YAP) and transcriptional coactivator with PDZ binding motif (TAZ) attenuates pathological changes in TM cells. YAP/TAZ are key mechanotransducers involved in glaucoma pathogenesis and are shown to be regulated by the mevalonate pathway. By inhibiting this pathway, we hypothesized that statins could potentially improve TM cell pathobiology by modulating YAP/TAZ activity. Thus, targeting the mevalonate pathway with statins may offer therapeutic potential for glaucoma. Despite significant progress in understanding TM and SC cells individually, the dynamic interactions between them and their role in glaucoma pathogenesis remain poorly understood. These interactions are crucial in the pathogenesis of glaucoma, yet no effective model exists to study them. Therefore, in Chapter 4, we developed a novel co-culture hydrogel system to explore TM-SC interactions and assess how glaucomatous TM cells affect SC behavior. Our findings show that glaucomatous TM cells alone can induce pathological changes in SC cells, underscoring the critical role of cell-cell and cell-ECM interactions in glaucoma progression. Collectively, these biomimetic ECM hydrogels provide a unique platform for investigating glaucomatous outflow mechanisms and offering insights into disease pathogenesis.
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HIV-1 has a sweet tooth: glucose metabolism drives the multistep process of HIV-1 latency reversalThe major barrier to a cure for HIV-1 is the establishment of latency in long-lived CD4+ T cells within lymphoid tissues which readily fuel viral rebound upon antiretroviral therapy (ART) interruption. Therapeutic approaches aimed at eliminating these HIV reservoirs with latency reversal agents (LRAs) have hitherto yielded underwhelming results in clinical trials owing to our incomplete understanding of the exact determinants of meaningful latency reversal in vivo. While previous studies have associated glycolysis with HIV productive replication and latency reversal, the exact role and mechanistic link of glycolysis to HIV latency reversal remains undefined. Furthermore, few studies have investigated HIV latency under physiologically relevant metabolic conditions found in the anatomical reservoirs of HIV in vivo. The studies in this thesis reveal that glycolysis is a metabolic determinant of HIV latency reversal, particularly during physiological hypoxia. We show that the capacity of LRAs to modulate glycolysis determines their efficacies over a physiological range of glucose and oxygen availabilities as found across tissues in vivo. Mechanistically, glycolysis fuels histone lactylation, a novel post-translational modification (PTM) which we show is a stronger predictor xviii of latency reversal than the canonically recognized acetylation marks, and promotes chromatin accessibility at the HIV LTR. Beyond histone PTM modulation, glycolysis also modulates HIV RNA splicing, a critical post-transcriptional step in HIV latency reversal. Specifically, multiple splicing of rev, an HIV regulatory gene, is significantly downmodulated by glycolytic restriction in a hypoxia-dependent fashion. Finally, we show that glucose and oxygen availability impact the phosphorylation and lactylation of splicing factor 3B subunit 1 (SF3B1), a core component of the U2 spliceosome complex and HIV dependency factor which provides preliminary mechanistic insight to how glycolysis and hypoxia modulate HIV RNA splicing. Collectively, our findings uncover glucose and oxygen availability as critical metabolic determinants of HIV-1 latency reversal and support the rationale that physiologically relevant experimental conditions should be utilized in studies aimed at identifying therapeutic agents that effectively target the latent reservoir in vivo.
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Kohlschütter-Tönz protein ROGDI is the homolog of yeast Rav2 and a novel Rabconnectin-3 subunitV-ATPases are rotary proton pumps that are extraordinarily well-conserved among eukaryotes. V-ATPases function primarily to acidify intracellular compartments, critical to maintaining cellular homeostasis. The V-ATPase-generated proton gradient provides the optimal environment for lysosomal catabolism and drives intracellular protein trafficking. V-ATPases serve important functions throughout the human body. For example, V-ATPase activity energizes the active transport of neurotransmitters into synaptic vesicles, regulates the acid/base balance in the kidney, and helps the immune system recognize invading pathogens. However, when V-ATPase activity is inappropriately increased or decreased, these processes are affected, and disease can result. V-ATPases are composed of peripheral V₁ and integral membrane V₀ subcomplexes; V₁ hydrolyzes ATP and transmits rotation to V₀, which moves protons across a membrane. V-ATPase activity is regulated in part through the reversible association of the V₁ subcomplex and V₁C subunit from V₀. Upon disassembly, both V₁ and V₀ are catalytically inactivated. In yeast, the RAVE complex catalyzes the efficient reassembly of V-ATPases. Rabconnectin-3 is the human homolog of the RAVE complex and functions similarly. Mutations in the Rabconnectin-3 complex can reduce V-ATPase activity through decreased assembly, which leads to disease. Both Rabconnectin-3 subunits share substantial homology with the RAVE subunit Rav1. We have identified the poorly characterized protein ROGDI as the mammalian homolog of the yeast RAVE subunit, Rav2. ROGDI shares strong functional and structural homology with yeast Rav2. Expression of ROGDI in a rav2Δ yeast strain partially rescues the growth phenotype characteristic of RAVE mutants. ROGDI binds to the structurally conserved N-terminal β-sheet rich domain. AlphaFold3 modeling predicts that ROGDI binds between the Rabconnectin-3 subunits. ROGDI coimmunoprecipitates with Rabconnectin-3 and V-ATPase subunits. Additionally, ROGDI is present alongside V-ATPase and Rabconnectin-3 subunits on lysosomal membranes. This indicates that, like RAVE and Rav2, Rabconnectin-3 and ROGDI localize intracellular regions rich in V-ATPases. Identifying ROGDI as a novel Rabconnectin-3 subunit is a substantial step forward in our understanding of Rabconnectin-3 and how it influences V-ATPase activity.
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Human cytomegalovirus (HCMV) exploits heat-shock transcription factor 1 (HSF1) to promote viral replication: a potential novel antiviral target to combat HCMV infectionHuman cytomegalovirus (HCMV) is a highly prevalent beta-Herpesviridae virus infecting almost 80-90 % of the world population. Though HCMV infection is typically asymptomatic, it can cause significant morbidity and mortality among immunocompromised individuals. Because of its obligate intracellular nature, HCMV modulates the cellular environment to promote infection. HCMV activates different cellular responses and signaling pathways to facilitate a favorable state for viral replication. During the lytic cycle of HCMV infection, viral entry, and replication inside the cell initiate stress response due to nutrient deficiency, energy depletion, hypoxia, and proteotoxic stress. Stress responses are designed to sense the damage, initiating a cascade of events to survive the stress. Several studies showed that HCMV usurps components of heat shock-stress response (HSR) to mitigate stress-associated damage and promote viral gene expression and replication. In this study, we found that HCMV infection in fibroblast cells induces a unique biphasic activation of heat shock transcription factor 1 (HSF1), a master transcription factor that is activated in response to heat-induced proteotoxic stress. HCMV binding to the integrin-ᵝ receptor activates HSF1 through Src- kinases. Importantly, HCMV infection drives the translocation of HSF1 into the infected cell nucleus. During canonical activation of HSF1, nuclear HSF1 binds to the specific sequence on the genome called heat shock element (HSE) and initiates transcription of a wide variety of stress-related genes. Interestingly, HCMV also utilizes this master transcription factor by harboring HSEs on major immediate early promoter (MIEP) to regulate viral immediate early (IE) gene expression. We found inhibition of HSF1 with a novel anti-HSF1 targeting drug SISU102 (Direct Targeted HSF1 InhiBitor) attenuated IE protein expression, indicating that the HSF1 regulates HCMV lytic replication. Additionally, inhibition of HSF1 reduced late (L) gene expression and subsequent viral progeny production. To explore HSF1 as a potential in vivo anti-HCMV target, we employed a murine model involving the subcutaneous transplantation of human skin into athymic nude mice. Treatment with SISU102 significantly diminishes viral replication in skin xenografts compared to the vehicle-treated group, indicating HSF1 as a possible cellular protein target for HCMV antiviral therapy. Overall, our data suggest that HCMV infection rapidly activates HSF1 during viral binding and entry, driving nuclear localization to promote lytic replication, which can be exploited as an antiviral strategy.
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Microbiota Colonization Dynamics Dictate Systemic IgAEvolution of the mammalian gut is intimately linked with the microbes that inhabit this space. Immunological development of gastrointestinal and systemic tissues is fundamentally dependent on stimulation by symbiotic microorganisms. In some cases, the same species that are critical for host immunity display pathogenic qualities when homeostasis is disrupted. Bacteroides fragilis is one such species with numerous symbiotic and pathogenic characteristics. This thesis explores the generation of B. fragilis-specific systemic IgA and the role of this response in protecting the host from B. fragilis pathogenicity. Induction of systemic IgA specific to B. fragilis requires exposure of this bacterium to small intestinal Peyer's patches and results in migration of newly generated IgA plasma cells to systemic tissues. Colonization dynamics of B. fragilis in mouse models with endogenous gut microbiota revealed that the magnitude of systemic IgA responses occurs in a dose-dependent fashion. Finally, a framework for establishing B. fragilis colonization and subsequent immune modulation within a highly diverse intestinal ecosystem was developed.
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Investigating the role of formin FHOD3 during myofibrillogenesis in embryonic chick cardiomyocytesFormins are major actin polymerizing proteins which act via the FH2 domain to promote actin nucleation and polymerization, as well as the FH1 domain to accelerate FH2 mediated actin elongation. FHOD3 is a formin that has been shown to be expressed predominantly in the heart and is critical for myofibril maturation during development in mice. FHOD3 has been shown to localize where actin filaments overlap myosin filaments within the sarcomeres of mice, rat, and human induced pluripotent stem-cell derived cardiomyocytes, flanking both sides of the M-line in the sarcomere. However, the role of FHOD3 in the myofibrillogenesis and the timing of FHOD3's activity in myofibrils has yet to be determined. Using RT-PCR, I successfully identified expression of at least two different isoforms of FHOD3 within heart tissue, matching to predicted isoforms X5 and X6. I also identified two chemically conserved regions within the FHOD3 amino acid sequence that are related to the cardiac FHOD3 isoform's localization to myofibrils. Using immunofluorescence microscopy and western blotting I found that FHOD3 is present within embryonic chick cardiomyocytes and that the localization of FHOD3 matches prior reports. FHOD3 was determined to be transiently expressed at significantly higher rates on Days 3 and 4 of culture in cardiomyocyte myofibrils. 90% of measured sarcomeres containing FHOD3 had a Z-line to Z-line length ranging from 1.4-1.9 µm, suggesting not only a length-dependent role of FHOD3, but a myofibril maturity dependent localization of FHOD3. These observations illustrate that FHOD3 likely does not have a function in the initiation of myofibrillogenesis but may instead have a role in the maturation and elongation of sarcomeres. The transient nature observed also suggests that FHOD3 may be localized within the sarcomere only as needed. Knockdowns of FHOD3 performed with shRNAs showed no indication of knockdown causing myofibrillar disruption. Knockdowns of FHOD3 using DsiRNAs were statistically inconclusive for knockdown occurring but did have an upwards nonsignificant trend in the percentage of myofibril disruption in cardiomyocytes.
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HSV-1 targets a novel antiviral response of the STING pathwayIn order to establish a successful infection, herpes simplex virus-1 (HSV-1), a ubiquitous virus with high seropositivity in the human population, must undermine a multitude of host innate and intrinsic immune defense mechanisms, including key players of the stimulator of interferon genes (STING) pathway. Recently it was discovered that not only de novo produced intracellular 2'-3'cGAMP, but also extracellular 2'-3'cGAMP can activate the STING pathway by being transported across the cell membrane via the folate transporter, SLC19A1, the first identified extracellular antiporter of this critical signaling molecule in cancer cells. We hypothesized that the import of exogenous 2'-3'cGAMP would function to establish an antiviral state similar to that seen with the paracrine antiviral activities of interferon. Further, to establish a successful infection, viruses, such as HSV-1, must undermine this induction of the STING pathway by inhibiting the biological functions of SLC19A1. Herein, we report that treatment of the monocytic cell line, THP-1 cells and SH-SY5Y neuronal cell line with exogenous 2'-3'cGAMP induces interferon production and establishes an antiviral state. Using either pharmaceutical inhibition or genetic knockout of SLC19A1 blocks the 2'-3'cGAMP-induced inhibition of viral replication. Additionally, HSV-1 infection results in the reduction of SLC19A1 transcription, translation, and importantly, the rapid removal of SLC19A1 from the cell surface of infected cells. Our data indicate SLC19A1 functions as a newly identified antiviral mediator for extracellular 2'-3'cGAMP which is undermined by HSV-1 protein ICP27. This work presents novel and important findings about how HSV-1 manipulates the host's immune environment for viral replication and discovers details about an antiviral mechanism which information could aid in the development of better antiviral drugs in the future.
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Structural insights of the histone H3 tail and its role in the mechanism of histone H3 lysine-4 methylationStructural insights of the histone H3 tail and its role in the mechanism of H3 lysine-4 methylation Gene expression relies on the proper chromatin structure to provide the necessary access to the DNA for the large transcription complexes to carry out their tasks. If the chromatin is tightly condensed, transcription is unable to occur. To regulate and initiate access to the DNA, an elaborate network of histone modifying enzymes, chromatin remodeling complexes, and other supporting proteins must coordinate the writing, reading, and erasing of histone post-translational modifications (PTMs). One such PTM, methylation of histone H3 on the lysine-4 (H3K4) residue, is critically important for maintenance of gene expression states. This is done in a spatiotemporal manner, which is influenced by the number of methyl groups that are present. However, an understanding of how the degree of H3K4 methylation is regulated remains elusive. In this dissertation, we demonstrate the remarkable conservation of length and composition in the flexible N-terminal tails of histone proteins across evolution. Recent structural studies indicate several methyltransferase complexes bind to the nucleosome core, often leaving the N-terminal tails unbound. Research from our lab has also demonstrated that non-processive buildup of lysine-4 methyl groups takes place at multiple active sites. Based on these observations, we propose a hypothesis whereby the histone H3 tail acts as a swinging arm substrate, delivering residue side chains to different active sites to facilitate the progressive establishment of these epigenetic states. To investigate this hypothesis, we employed the CRISPR/Cas9 system in Saccharomyces cerevisiae to systematically modify the length of the H3 tail. We monitored histone H3 lysine 4 (H3K4) methylation, mediated by SET1, the primary H3K4 methyltransferase in budding yeast. Our findings demonstrate that altering the length of the H3 tail has varying effects on the extent of H3K4 methylation, in accordance with the swinging arm model. We also demonstrate that three proline residues are responsible for providing a segmented, tripartite structure with hinge-like joints that likely influence the tail's range of motion. Furthermore, the results support the proposed multiple active-site model, where mono-, di-, and trimethylation occur at distinct active sites within the COMPASS or MLL Core Complexes.
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Role of TLDc Proteins Oxr1 and Rtc5 in Yeast V-ATPase Reversible DisassemblyThe vacuolar H+-ATPase (V-ATPase; V1Vo-ATPase) is a highly conserved, ATP hydrolysis-driven dedicated proton pump found on the membranes of intracellular organelles in virtually all eukaryotic cells and on the plasma membrane of specialized cell types. Regulation of V-ATPase activity is key to maintaining normal physiological functions, as aberrations in its activity are associated with several pathophysiological conditions. V-ATPase activity is mainly regulated by a mechanism called reversible disassembly, in which the assembly state - and hence the activity - of the enzyme is controlled by nutrient availability and extracellular cues. During the process, V-ATPase activity becomes either turned off by dissociation of the V1-ATPase from the Vo proton channel, or turned on by reassembling the two subcomplexes into an active enzyme. While the process is well-characterized at the cellular level, the molecular mechanism at the level of the enzyme remains elusive. Here, we show that two TLDc proteins, Oxr1p and Rtc5p, control the assembly state of yeast V-ATPase, with the former promoting disassembly, and the latter (re)assembly of the enzyme. Based on cryoEM analysis and in vitro and in vivo approaches, we discovered that Oxr1p is a V-ATPase disassembly factor. Oxr1p binding to V-ATPase results in autoinhibited V1 in two steps - first producing a disassembly intermediate, which, upon ATP hydrolysis, gets converted into autoinhibited V1. From in vitro experiments, we find that the second TLDc protein, Rtc5p, primes autoinhibited V1 for (re)assembly with Vo. CryoEM structures of Rtc5p bound V1 show Rtc5p's C-terminal ⍺ helix inserted into the catalytic core of the enzyme, thereby opening a second catalytic site, a conformational change that may facilitate (re)assembly of V1Vo. In vivo experiments, however, suggest that Rtc5p is not essential for V-ATPase reassembly in the cell, suggesting redundancy and/or alternative pathways. Overall, this study enhances our understanding of the molecular basis for the regulation of V-ATPase activity by reversible disassembly.
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Exploring the role of single nucleotide polymorphisms in varicella zoster virus vaccine attenuation in skinVaricella zoster virus (VZV) is a disease that can be detrimental to the health of children in its primary form, chicken pox, and later in the elderly as its reactivated form, shingles. Before the advent of the vaccine, Varivax, VZV was endemic in the United States as it is highly contagious and can be spread through both direct contact and aerosol particles. Varivax, or vOka, is a live attenuated vaccine, and while effective, has side effects ranging from rashes to possible VZV reactivation. While the vaccine has reduced the incidence and severity of VZV, there is still little known about the mechanism of its attenuation in skin. vOka is genetically heterogeneous with hundreds of single nucleotide polymorphisms (SNPs) that are a mixture of wild-type and vOka nucleotides. Previous studies have demonstrated the key to attenuation may be through five SNPs in the open reading frame (ORF) 62 region found to be fixed and stable across different licensed vOka preparations around the world. ORF62 contains the gene for IE62, a transactivator protein responsible for regulating the expression of viral genes and the host gene for KRT15, a cytokeratin protein. This project focused on if two SNPs, located in the loci positions 106262 and 107252, that are found to be almost 100% conserved across all variations of vOka are responsible for the attenuation in human skin and induction of KRT15. We evaluated four mutant viruses with SNPs found in vOka and discovered that a double SNP mutation stunted virus growth in HFF cells. In addition, we found no significant difference in the growth of our viruses in skin but variability in successful infection. Furthermore, in infected skin, we found that VZV-ORF57-Luc and single mutant virus, 68-958, upregulate KRT15 expression with VZV infection while single mutant virus, 68-62S-A, may downregulate KRT15 expression with VZV infection. This project is important because it may reveal the molecular basis of attenuation of the licensed varicella vaccine. This information could be used to make a vaccine that contains only the attenuated genotype.
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From bud scars to molecular insights: investigating V-ATPase function and assembly in yeast replicative agingAging is a complex process that involves the progressive decline of physiological functions over time. As the global population continues to age, understanding the mechanisms underlying aging has become an important area of study. Lysosomes play a crucial role in maintaining protein quality control and degrading unneeded or damaged proteins through proteolysis. Therefore, lysosomes play a prominent role in theories of aging due to their significant role in cellular homeostasis. Interestingly, many of the hallmarks of aging are conserved between yeast and humans, highlighting the relevance of yeast as a model organism to study aging processes. One key enzyme responsible for acidifying lysosomes and lysosome-like vacuoles in yeast is the vacuolar-type H+-ATPase (V-ATPase). Despite evidence showing that lysosomes alkalinize with age, compromising their proteolytic function, little is known about the regulation of V-ATPase in aging cells. Yeast cells divide asymmetrically with each division leaving a "bud scar" that can be stained to determine replicative age. In comparing cells of distinct replicative age, we find significant decreases in V-ATPase assembly, accompanied by poor vacuolar acidification, in older cells. Remarkably, partial disassembly of the V-ATPase occurs at a relatively early age, indicating its potential as a phenotypic driver in the aging process. Reversible disassembly is controlled in part by the activity of two opposing and conserved factors, the RAVE complex and Oxr1. The RAVE complex promotes V-ATPase assembly and a rav1∆ mutant has a significantly shorter lifespan than wild-type cells; Oxr1 promotes disassembly and an oxr1∆ mutation significantly extends lifespan. These data indicate that reduced V-ATPase assembly may drive the loss of lysosome acidification with age and place the balance of V-ATPase assembly factors at the center of this process. Caloric restriction, defined as reduced calories with adequate nutrition, has been shown to extend lifespan in multiple organisms including yeast. We find that caloric restriction reverses the age-related decreases in V-ATPase assembly and vacuolar acidification in yeast as well as restoring balance of assembly factors. We investigated three conserved metabolic signaling pathways that have been linked to acidification, caloric restriction, and aging: PKA, mTORC1/S6K (TORC1/Sch9 in yeast), and AMPK (Snf1 in yeast). By utilizing non-essential nutrient mutations in these signaling pathways, we determined the impact on V-ATPase assembly during replicative aging. Mutations compromising TORC1 function were known to extend lifespan and preserved V-ATPase assembly even in older cells. In contrast, a mutation that prevents recruitment of Snf1/AMPK to vacuoles prevented V-ATPase assembly even in young cells and shortened lifespan. This study provides novel insights into the importance of V-ATPase assembly and function in the aging process and suggests novel interventions to promote health aging.
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Kcnh2 Expression Profile and Continuous EEG/ECG Monitoring in a Rabbit Model of Long QT Syndrome Type 2Long QT Syndrome (LQTS) is a classically studied cardiac condition characterized by a prolonged ventricular excitation-repolarization interval (QT interval) on an electrocardiogram (ECG). LQTS is associated with an increased risk of arrhythmias and sudden cardiac death. People with LQTS, particularly those with Long QT Syndrome Type 2 (LQT2), are also at an increased risk of seizures/epilepsy. LQT2 is caused by loss of function variants in the KCNH2 gene. The dual neuro-cardiac phenotype of LQT2 can likely be explained by expression of KCNH2 in both the brain and heart. Using a rabbit model that harbors an endogenous knock-in mutation in one allele of the pore domain of the Kcnh2 gene, I characterized the molecular expression of WT vs. mutant Kcnh2 and developed a protocol for long-term subcutaneous EEG/ECG implantation. To better understand the molecular profile of WT vs. mutant rabbits, the relative expression of WT vs mutant Kcnh2 transcripts was evaluated using quantitative PCR (qPCR) with verification via Oxford Nanopore Technology (ONT) sequencing. Additionally, 44 RNA sequencing libraries were prepared and sequenced for further analysis of the molecular profile of WT vs mutant rabbits. Micro-C and high molecular weight DNA libraries were also prepared for the construction of a more thorough rabbit genome. In mutant rabbits, total Kcnh2 expression is roughly half that of WT rabbits. In mutant rabbits, the mutant Kcnh2 RNA represents 11% of the total Kcnh2. These data suggest that most mutant Kcnh2 RNA is degraded shortly after generation. To continuously monitor the rabbits’ cardiac and neuronal electrical function in vivo, a method of constant EEG/ECG recording was designed and implemented. It involves the surgical placement of subdermal electrodes and the design and manufacturing of a wiring system. The surgical placement of electrodes has been optimized to minimize time and number of incisions and improve outcomes. The wiring system enables the rabbits to have free range of motion within the housing cage and keeps all wires protected and out of reach of the rabbits. This system is functional and generates high quality continuous EEG/ECG recordings.
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The murine absolute visual threshold: behavior & retinal pathwaysConnexin 36 (Cx36) gap junctions are important for governing the sensitivity of the dark-adapted retina. Despite its importance for physiological sensitivity, the degree to which retinal Cx36 governs the psychophysical absolute threshold is not known. The purpose of this work is to study to what extent inner- and outer-retinal Cx36 governs the absolute visual threshold. In Chapter 2 we developed a one-alternative forced choice (1AFC) task for measuring murine absolute visual thresholds to full-field flash stimuli. We found that our 1AFC task, in conjunction with the theory of signal detection, gave response bias-independent absolute visual threshold estimated. Using this assay, we found that decision criteria are related to response times. In Chapter 3 we used the 1AFC task and the power of transgenic mice to assess the relative contributions of inner and outer retinal Cx36 to the absolute visual threshold. We concluded that inner, not outer, retinal Cx36 is most responsible for governing the absolute visual threshold. In parallel, by testing mice with disrupted rod vision, we determined that rod OFF pathways, and not cones, set the absolute visual threshold in the absence of Cx36. Finally, we studied the impact of Cx36 on temporal summation at absolute threshold, by obtaining thresholds for a range of flash durations. Threshold-vs-duration data was then fit with a model of temporal summation that allowed us to determine whether Cx36 influences the temporal filtering properties of scotopic vision. Our model fits suggest that photoreceptor Cx36 may play a role in temporal processing at absolute visual threshold. Overall, this work sheds new light on the behavioral dynamics and neural underpinnings of rod mediated vision.
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Battle of arms: human cytomegalovirus manipulates monocyte survival for viral dissemination.Human cytomegalovirus (HCMV) is a highly prevalent pathogen with seropositivity rates reaching upwards of 90% in the United States. Most primary infections are asymptomatic in immunocompetent individuals, but HCMV poses a significant risk in immunocompromised and immunonaїve individuals including transplant patients and developing fetuses in utero. The key to systemic dissemination of HCMV relies upon the infection of monocytes, which function as non-permissive vehicles to deliver virus to end organ tissues. These primary infected monocytes also travel to the bone marrow, infecting CD34+ stem cells, leading to the establishment of a lifelong HCMV infection. HCMV can reactivate at any point throughout the host's lifetime, leading to HCMV-infected stem cells exiting the bone marrow as monocytes, disseminating to end-organ tissue, and perpetuating HCMV disease. In circulation, monocytes have a short life span of 48 hours that can be accelerated by the cellular death pathway, apoptosis, as a cellular defense mechanism against viral infection. Our lab has shown during primary infection, HCMV circumvents intrinsic apoptotic pathways, however, the mechanism by which HCMV blocks extrinsic apoptosis is unclear. The studies in this thesis reveal that HCMV induces cFLIP expression, inhibiting extrinsic apoptosis effector caspase 8. This effective inhibition of intrinsic and extrinsic apoptosis prompts trap-door death pathway necroptosis. However, the mechanism in which this pathway is activated and how HCMV modulates this pathway to promote cell survival is unknown. In these works, we identified TLR3 as the death receptor responsible for inducing necroptosis. To circumvent this activation, HCMV upregulates autophagy, a ubiquitous cellular recycling process. We saw the inhibition of autophagy altered nucleocytoplasmic shuttling and activation of executioner kinase, MLKL, culminating in necrotic cell death. This work highlights the delicate balance between pro-survival and pro-death elements in HCMV infected monocytes. However, investigating how HCMV modulates cellular death pathways in a primary infection monocyte model does not fully encapsulate the role of monocytes in HCMV dissemination. Once HCMV latency is established in CD34+ stem cells, this allows HCMV the ability to persist in the host for their entire life span as monocytes derived from latently infected stem cells that can re-seed HCMV to peripheral organs to establish a chronic lytic infection. To investigate this understudied secondary population of HCMV-infected monocytes, we developed a model in which primary HCMV-infected monocytes and infected monocytes derived from latently infected stem cells are on the same genetic background by differentiating a CD34+ myeloblastic cell line. Though preliminary, we believe that this model, combined with investigations of mechanisms in which HCMV promotes survival in primary infected monocytes, will allow for the development of novel therapies that specifically target HCMV-infected monocytes, thus preventing viral dissemination and the establishment of disease.
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Novel signaling pathways driving experience-dependent maturation in dentate gyrus granule cells: a deep-sequencing approachThe granule cells in the dentate gyrus of the hippocampus are a cell type that is critical for learning and memory ability. Dentate gyrus granule cells exhibit the unique capacity to differentiate and mature throughout an individual's lifetime. Decades of dedicated research has revealed many transcription factors that facilitate the differentiation of granule cells from neural progenitors. The goal of this research project is to identify key molecular and transcriptional pathways that contribute to the maturation of dentate gyrus granule cells. We performed single-cell RNA sequencing and multiomic single-nuclei RNA and ATAC sequencing in an activity-dependent mouse reporter model to examine the influence of neuronal activity on the transcriptome and chromatin accessibility within individual granule cells. These experiments were performed between postnatal day 14 and postnatal day 24 due to the increased abundance of developing granule cells. We implemented an environmental enrichment paradigm where mice were reared in complex, dynamic, and socially-enriched housing. This paradigm allowed us to examine the impact of chronically increased circuit activity on granule cell maturation. This study reveals novel heterogeneity in the maturing granule cell population that is associated with previous neuronal activity and synaptic function. A follow-up investigation identifies new transcription factor candidates that appear to orchestrate the transition between maturity stages. We propose a new model where granule cell identity is established through an activity-independent transcriptional network. The subsequent experience of neuronal activity appears to drive the emergence of a distinct transcriptional network that is poised to facilitate long-lasting granule cell maturity.
