More 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.

The 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.

Human 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.

Formins 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.

Structural 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.

Varicella 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.

Long 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.

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.

Managing chronic wounds is a complex challenge, often aggravated by the presence of inflammation and infection. Hydrogel materials stand out for their remarkable properties such as biocompatibility and drug-loading capabilities, making them ideal for applications in wound dressing and tissue regeneration. A compelling treatment strategy involves not only resolving persistent hyperinflammation but also effectively eliminating infection. The demand for an "all-in-one" hydrogel platform with dual functions, targeting both inflammation and infection in chronic wounds, is huge in clinical settings. This thesis presents two promising dual-functional hydrogel platforms specifically crafted for potent topical application in the treatment of wounds. First platform was named "pull and push" hydrogel, which is essentially micro-sized hydrogel functionalized with innovative and versatile telodendrimers (TDs). By rational design and synthesis, these conjugated TDs can strongly grab or trap inflammatory factors such as cytokines and endotoxins, as well as hold drug payload such as antibiotics and release out in a desired manner. We demonstrated the TD hydrogel's capability to scavenge proinflammatory cytokines using in vitro assays, and shown the TD hydrogel's effect in scavenging cytokines in a mouse model with skin inflammation. We also demonstrated TD hydrogel's structure dependent capabilities in encapsulating different antibiotics, and sustainable release profile. The drug maintained its efficacy with prolonged therapeutic window and reduced toxicity, as evidenced by in vitro assays and in vivo animal skin and soft tissue infection model. Second hydrogel platform was termed "kill and block", which is essentially a bulky photo-crosslinked hydrogel incorporated with telodendrimer nano-formulated antibiotics. Nanodrug released from topically applied bulky photogel can further dissociate into drug and nanocarrier telodendrimer. The released drug functions as antimicrobial to "kill" bacteria. Dead bacteria still potentially stimulate inflammation. The payload-free nanocarrier can simultaneously modulate local immune response by "blocking" inflammatory pathway. In addition, this platform address issues of wound morphology adaptiveness. We demonstrated the tunable drug release profiles, reserved drug efficacy after TD encapsulation and phtogel incorporation, and superior anti-inflammation efficacy in vitro and in vivo. Collectively, we organically combined the advantages of nanotechnology and hydrogel platform, providing promising solutions for the chronic wound treatment from immune modulation and drug delivery.

RELN is strongly associated with Autism Spectrum Disorder (ASD). Homozygous loss of the encoded protein REELIN is associated with severe neurodevelopmental phenotypes characterized by lissencephaly and cerebellar hypoplasia, yet the ASD linked variants are typically heterozygous and appear to require additional genetic risk to cause ASD. To functionally characterize a RELN variant in a patient with ASD, we used induced pluripotent stem cells (iPSCs) from a family of non-autistic parents and their son who had ASD (the proband). The proband has a maternally-inherited missense variant (R2457C) in the RXR motif of the REELIN protein. We differentiated the iPSCs into two types of neurons, inhibitory neurons which model the inhibitory forebrain neurons that secrete REELIN, and excitatory neurons which model the cortical pyramidal neurons that respond to REELIN. Immunoblotting revealed that the proband inhibitory neurons had a lower ratio of extracellular/intracellular REELIN compared to that of the parental neurons, suggesting a decreased REELIN secretion. Sholl analysis on the proband excitatory neurons showed reduced dendritic complexity and reduced total length compared to the parental neurons. REELIN treatment increased the dendritic length and complexity in proband neurons up to the level of parental neurons. CRISPR/Cas9-mediated RELN KO did not change the dendritic phenotype in the excitatory neurons, ruling out a cell autonomous role for REELIN in these neurons. The proband excitatory neurons also had lower mRNA expression of WNT target genes in response to WNT3a, suggesting an underactive WNT signaling, as well as higher total GSK3β protein and lower phosphorylation at the inhibitory S9 site, indicating an overactive GSK3β signaling. Inhibition of GSK3β improved the proband neurons dendritic complexity in the proximal parts of the dendritic arbor. However, inhibition of mTOR signaling, which has shown to regulate REELIN signaling, did not change the dendritic morphology. In conclusion, the pathophysiology of ASD in the proband likely consists of a reduced REELIN secretion from the inhibitory neurons and an additional vulnerability in the REELIN-responding excitatory neurons, the latter likely being an overactive GSK3β and an underactive WNT signaling, all of which result in reduced dendritic complexity.