Protein-based conformational switches serve as powerful tools for the construction of biosensors and for the control of cellular processes. These proteins feature a binding domain that recognizes a specific analyte and is coupled to an output domain in such a way that the binding event causes the output domain to provide an observable signal. These signals can either be turn-on of fluorescence, luminescence, or enzymatic activity or consist of the sensor changing its color. A challenge in constructing these protein switches is finding binding domains capable of relaying a ligand binding event to the conformational change of an output domain. Generalized binding domains can address these challenges by providing a scaffold that can easily be modified to detect a different ligand. These generalized binding domains are small proteins with modifiable residues that can be selected to bind a ligand of choice, usually through phage display and similar selection techniques. Here, we present two approaches to make generalized protein switches. In the first approach, antibody mimetics nanobodies and monobodies are inserted in fluorescent proteins such that binding of their ligand causes an increase in fluorescence. This technique, named adaptable turn-on maturation (ATOM), was used to develop biosensors for WD-40 repeat protein 5 (WDR5), c-Abl src homology 2 (SH2) domain, hRas, postsynaptic density protein 95 (PSD95), gephyrin, HOMER1, and mCherry for use in mammalian cells. ATOM is, therefore, compatible with a variety of ligands due to its input domain being a generalized binding domain. Additionally, the ATOM mechanism can be used to convert many fluorescent proteins into biosensors. For demonstration, we made biosensors from Clover, mTurqoise, mTagRFP-t, mStayGold, mBaoJin, and GCaMP6s. In the second approach, we develop a luminescent protein switch from the enzyme nanoluciferase (nLucAFF) that switches color from green to blue upon DNA binding. We show that DNA-based devices can then be used to detect various ligands and relay that event to nLucAFF, which provides an output easily quantifiable by a cell phone. The nLucAFF protein was used to detect DNA sequences amplified from cytomegalovirus (CMV), dengue, and nCoV. Additionally, aptamers binding to serotonin and aptamers were used to detect these molecules by directing the nLucAFF color change. The initial version of nLucAFF was slow, dim, and had low sensitivity. These drawbacks were resolved in the next version, nLucAFF2, to achieve turn-on within 5 minutes and detect ligands down to 40 pM with a cell phone camera. The last chapter combines two ligand-binding domains to activate a small cytotoxic RNase, barnase, and paves the way for the development of multi-input protein switches that can potentially be generalized ligand-binding domains.

Due 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.

Background Millions of U.S. residents experience increasingly more prevalent weather events due to climate change, however, there is limited research exploring the vulnerability to multiple extreme weather events using a national U.S. sample. Aims Identify patterns in exposures to climate events, and examine sociodemographic factors associated with increased climate event vulnerability. Method Data was retrieved from the May 2022 American Trends Panel, a nationally representative sample of 10,282 United States adults. We performed a latent class analysis, a statistical method used to identify unobserved subgroups (latent classes) within a population, to group respondents by patterns in five climate event experiences (heatwave, intense storm, wildfire, drought, and sea level rise), and analyzed variables associated with vulnerability to climate events using weighted multinomial logistic regression, a statistical method that models the probability of membership in one of several outcome categories (climate vulnerability groups) relative to a reference category, while accounting for survey weights to ensure generalizability to the U.S. population. Results Respondents were categorized into four latent classes, which are unobserved subgroups identified through patterns in exposures to five climate events (heatwave, intense storm, wildfire, drought, and sea level rise). These subgroups were based on exposures to heatwave (42.5 %), intense storm (43.2 %), wildfire (21.3 %), drought (30.8 %) and sea level rise (15.8 %): high (9.8 %), heat-storm (22.2 %), heat-drought (13.4 %), and low (54.6 %) climate event vulnerability. Relative risk for high climate event vulnerability refers to the likelihood of belonging to the “high vulnerability” group compared to the “low vulnerability” group. It is assessed using the relative risk ratio (RRR), which is a measure of the association between a particular sociodemographic factor (e.g., age, gender, region) and the likelihood of being in a specific vulnerability group relative to the reference group. For instance, an RRR <1 indicates a reduced risk, while an RRR >1 indicates an increased risk compared to the reference category. Relative risk for high climate event vulnerability was lower for older adults (RRR = 0.39, p < 0.001), potentially reflecting a greater capacity to cope with certain climate events, such as access to stable housing or resources. However, this finding should not be interpreted as older adults being universally less vulnerable. Numerous studies have shown that older adults are at significantly higher risk during heatwaves due to physiological and social factors, which our analysis may not fully capture. Relative risk for high vulnerability was higher for females (RRR = 1.42, p = 0.01) and residents in the South (RRR = 2.05, p = 0.003) and West (RRR = 9.31, p < 0.001) geographic regions. Relative risk for heat-drought was higher for Hispanic adults (RRR = 1.51, p = 0.03), but lower for high school graduates (RRR = 0.40, p = 0.01) compared to those who did not complete high school. Conclusions We identified several underlying climate event exposure subpopulations, ranging from low to high vulnerability. As climate-related events become more frequent, our results provide critical insights for stakeholders to identify high-risk individuals and prioritize resources for disaster management.

Schizophrenia 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.

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.

V-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.

Background: The effectiveness of the clinical outcome of CN (Cytoreductive Nephrectomy) in cases of mccRCC (Metastatic Clear Cell Renal cell Carcinoma) is still uncertain despite two trials, SURTIME and CARMENA. These trials, conducted with Sunitinib as the standard treatment, did not provide evidence supporting the use of CN. Methods: We queried the NCDB for stage IV mccRCC patients between the years of 2004 to 2020, who received (immunotherapy) IO with or without nephrectomy. Overall survival (OS) was calculated among three groups of IO alone, IO followed by CN (IOCN), CN followed by IO (CNIO). Cox models compared OS by treatment group after adjusting for sociodemographic, health, and facility variables. Results: From 1,549,101 renal cancer cases, 7983 clear and nonclear cell renal cell carcinoma cases were identified. After adjusting for sociodemographic and health covariates, patients who received IO followed by CN or CN followed by IO had a respective 64% (adjusted Hazard Ratio [aHR] = 0.36, 95% CI = 0.30-0.43, P = .006] and 47% (aHR = 0.53, 95% CI = 0.49-0.56, P = .001) mortality risk reduction respectively compared to patients who received IO alone. Compared to White adults, individuals who identified as Black exhibited 17% higher risk mortality (aHR = 1.17, 95% CI = 1.06-1.30, P = .002). Patients who received CN prior to IO had a 59% associated mortality risk compared to patients who received IO followed by CN who had a lower risk, 35.7% (P < .001). Conclusions: Patients receiving CN regardless of sequence with IO did better than IO alone in this national registry-based adjusted analysis for mccRCC. Presently available data indicates that the combination of CN and IO holds promise for enhancing clinical results in patients with mRCC.

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.

Evolution 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.