Molecular Biology, 1954-2024

The antibiotic resistance crisis demands novel therapeutic methods that can provide mechanisms of action to which bacteria have not yet become resistant. Here, we begin to characterize two drugs, fenobam and cisplatin, in their ability to effectively target log-phase and/or biofilm MRSA both independently and within a co-treatment using various classical antibiotics. These drugs were identified in a previous small molecule drug screen against non-growing MRSA in which compounds were applied to a 1:10^6 dilution of cells in PBS to assess for non-growing antibacterial activity. To assess the extent to which each drug is inhibited by the inoculum effect against non-growing MRSA, we applied ranging drug concentrations to various dilutions of cells in PBS. We found that while cisplatin exhibits activity against cell densities up to 3.189E+08 CFU/mL, fenobam experiences a larger inoculum effect, only being effective for up to 3.116E+07 CFU/mL. Furthermore, against MRSA in PBS fenobam was found to stimulate cell proliferation at concentrations 32 and 64 µg/mL. To begin the characterization of drug interactions between cisplatin and three different classical antibiotics (vancomycin, clindamycin, and trimethoprim), we applied ranging concentrations of cisplatin against ranging concentrations of classical antibiotics to log-phase MRSA in TSB. We found that instead of increasing the efficacy of any antibiotic tested, cisplatin stimulated cell proliferation in a dose-dependent way for antibiotic concentrations below MIC at earlier timepoints. Later in the incubation, cisplatin exhibited a dose-dependent OD600 reduction in cell density plateau. To examine the activity of fenobam both independently and within a co-treatment with vancomycin, mature MRSA biofilms were treated for 2 hours with subsequent cell viability assessment with alamarBlue. Fenobam exhibited activity against MRSA biofilms not only within a co-treatment with vancomycin, but also independently. Unfortunately, vancomycin and fenobam were found to hinder the ability of each other to target the biofilm, where the improved reduction of biofilm viability post-treatment was less than expected based on the individual effects artificially combined. To limit the variations in the result due to variations in biofilm maturation or inconsistent cell loss at wash steps, we began optimizing a live vs. dead staining protocol for fluorescence microscopy resulting in preliminary imaging. Lastly, we proposed a cyclical resistance protocol for the development of resistance mutations for non-growing specific antibacterial agents. These data help to inform the field on new possible directions to take antibiotic therapy development.

Diseases affecting the central visual field, including macular telangiectasia type 2 (MacTel), Stargardt macular dystrophy, and age-related macular degeneration, are a leading cause of blindness globally and significantly diminish quality of life. While advanced imaging tools like OCT provide anatomical insight into the physical basis of these diseases, they often overlook a critical component: the patient's subjective visual experience. Visual field distortion, where straight lines appear wavy or portions of images appear missing, is a common symptom that clinicians assess using the Amsler grid—a simple but powerful paper test. The Amsler grid test is a standard visual distortion screening practice and often complements high-resolution retinal imaging tests. Despite its widespread use, the Amsler grid relies on qualitative interpretation and verbal patient reports, which can be inconsistent and imprecise—in fact, subjective patient reporting often leads clinicians to misdiagnose macular disease or miss it altogether. There is a need for a quantitative framework to analyze patient-annotated Amsler grids. In this thesis, we propose a novel computational and statistical framework to characterize visual distortions observed on patient-annotated Amsler grids. We applied this framework to a patient with MacTel and found that our framework effectively captured variation within eyes and asymmetries between eyes. By demonstrating the feasibility of drawing clinically relevant insights from hand-drawn patient data, we show that our framework not only has the potential to enhance diagnostic sensitivity but also bridges the gap between objective imaging and the subjective patient experience. Ultimately, our framework could reshape the current landscape of retinal disease monitoring and diagnosis.

Influenza A Virus (IAV) infection poses an ongoing threat to human health. The IAV genome is contained in its negative-sense RNA, which replicates using its RNA-dependent RNA polymerase. However, during IAV genome replication, shorter aberrant RNA such as mini viral RNA (mvRNA) can be produced. Previous study has found that mvRNAs PA-60 and PA-66 were detected in mitochondrial fractions of infected cells. However, the method of transportation of mvRNA to mitochondria is not yet understood. In order to detect mvRNA, an LbuCas13abased detection system was optimized for mvRNA detection, testing for conditions including Mg2+ source, Mg2+ concentration, pH-stabilizing and crowding agent buffers, and LbuCas13a:crRNA ratios. It was found that only LbuCas13a:crRNA ratio yielded a significant difference in mvRNA detection. This research also sought to determine the method of localization of mvRNA to mitochondria using components of the innate immune signaling pathway. Retinoic acid-inducible gene 1 or mitochondrial antiviral signaling protein were knocked out in cells expressing the synthetic mvRNA NP61. mvRNA detection found no significant difference in mitochondrial detection in the absence of retinoic acid-inducible gene 1 but potentially suggests an increase in mitochondrial detection in the absence of mitochondrial antiviral signaling protein. These results provide insights into optimal conditions of mvRNA detection using LbuCas13a, and a potential relationship between the innate immune signaling pathway’s involvement in mvRNA mitochondrial localization.

Atopic dermatitis (AD) is a chronic skin condition characterized by dry, itchy, and inflamed skin. It affects about 15% of children and 10% of adults around the world, decreasing quality of life. It is more common in young children, and presents differently with age: younger individuals experience more edema, while older individuals exhibit more desiccated lesions. The three etiologies of skin barrier dysfunction, immune dysregulation, and skin microbiome dysbiosis amplify each other through various molecular pathways, worsening disease symptoms and preventing recovery. In particular, colonization of the skin by Staphylococcus aureus is associated with disease severity and persistence. Previous studies have confirmed specific mechanisms of S. aureus modulation of the disease, but have not investigated how S. aureus gene content varies with age on AD patients. This study fills this gap by analyzing whole-genome sequencing data of 1584 S. aureus isolates previously cultured from young participants with AD. Results from microbial GWAS methods reveal that several S. aureus genes are differentially present in young children vs. adolescents with AD. One particularly strong association was found for saeS, which regulates gene expression of over 20 virulence factors related to AD. Future studies can build on these results to elucidate the clinical basis for AD age endotypes, and perhaps develop personalized therapies for AD patients, stratified by age.

The basement membrane (BM) is a specialized extracellular matrix that underlies diverse tissues and shapes distinct biological environments. Though the BM acts as a selective barrier to cells and molecules, certain cells— such as immature melanocytes during embryogenesis and epithelial tumor cells during metastasis— transit across the BM to migrate through tissue compartments. Currently, the time- and context-dependent transcriptional regulation driving melanoblast (Mb) BM crossing is poorly understood. Moreover, potential differences in the crossing patterns of embryonic Mb populations due to oncogenic transformation have not yet been defined. Thus, an enriched understanding of the gene expression patterns underlying compartmentally distinct Mb identities, and how these characteristic patterns change upon introduction of an oncogenic mutation, is important from both a developmental and disease perspective. First, I aim to spatially validate transcriptional signatures linked to BM transit. Preliminary scRNA-seq findings support the existence of putative dermal and epidermal Mb subpopulations, each displaying distinct transcriptomes that match the cells’ activity in these respective compartments; scRNA-seq provides no spatial information, so it is necessary to spatially validate these BM crossing signatures, which are hypothesized to be exclusively expressed in dermal populations due to the unidirectional nature of Mb BM crossing. Utilizing HCR RNA-FISH technology on E13-E15 mouse embryonic backskins to perform spatial validation of putative dermal Mb gene candidates, this study documents the validation of selected mRNA probes targeting Dct and Cdh1— respective dermal and epidermal Mb signatures— that successfully function as positive control gene markers. Furthermore, the first experimental mRNA probe that was tested— targeting putative dermal Mb signature Sema3C— produced fluorescent signal in E13.5 backskins, however no Sema3C expression was detected in dermal Mbs, indicating the need for further optimization of the HCR technique before successfully validating putative dermal Mb expression profiles. Secondly, I characterize the patterns by which oncogenically transformed V600EBRaf-expressing Mbs cross the BM, by comparing the relative Mb numbers, compartmental distributions, and cell-division frequencies of wild-type (WT) and mutant models across E14.5 and E15.5 timepoints. I show that at both E14.5 and E15.5, V600EBRaf Mb counts were significantly elevated in the dermis compared to those of WT Mbs, with no significant differences in both the counts of epidermal Mb, and in the amount of Mb proliferation across mutant and WT groups. Results suggest that V600EBRaf selectively alters the dermal Mb population size. Future work, including additional biological replicates and earlier timepoints (E13.5), is needed to confirm these results and conclusively establish the mutation’s impact on Mb spatial distribution and BM crossing. Together with the spatial validation of dermal Mb gene signatures using HCR RNA-FISH, these findings pave the way for continued exploration of transcriptional regulation of Mb behavior during BM crossing and how oncogenic mutations may disrupt this process.

Single-cell RNA sequencing (scRNA-seq), a technology used to study the transcriptome at single-cell resolution, has allowed for many advances in biomedicine. However, the dissociation of cells in the scRNA-seq protocol takes away spatial information. Spatial transcriptomics technologies emerged as a way to fill this gap. Because spatial transcriptomics technologies do not capture the transcriptome at a single-cell resolution, integration with scRNA-seq data is needed for a deeper understanding of the transcriptome of a region of interest with a spatial context. In order to explore the spatial dynamics that contribute to cell differentiation during branching morphogenesis of the mouse lung, we applied a spatial transcriptomics technology, deterministic barcoding in tissue for spatial omics sequencing (DBiT-seq), to sections of the mouse lung at timepoints E11.5-E.13.5. The DBiT-seq replicates were integrated with scRNA-seq data from published papers to form an object for transcriptomic analysis. The presence of DBiT-seq data and scRNA-seq data from the same region and timepoints allowed for comparison of the two technologies. DBiT-seq data has a higher and more variable proportion of unspliced mRNA. There is also a set of genes that is expressed only in the DBiT-seq data, which does not align with the fact that DBiT-seq has less sampling-depth. Investigation of this gene set revealed that these genes were smaller and enriched for septins. Spatial mapping of the gene set to the replicates reveals no clear localization pattern. An optimization step added to our DBiT-seq protocol was hypothesized to be the reason why these genes were captured. However, comparison of this gene set to that of an older DBiT-seq paper which lacked the optimization step ruled this theory out.

The bacterial CRISPR-cas9 system has revolutionized our ability to manipulate the genome through precise RNA-guided editing. Despite its success in cutting precisely at target loci, RNA-guides also bind to off-target genomic regions, inducing undesired, and potentially genotoxic, double stranded breaks (DSBs). Currently, there exists no method for predicting guide RNA (gRNA) on-target and off-target efficiency with sufficient accuracy for all types of gRNA (i.e. gRNA targeting coding and non-coding regions) across cell-types. In this thesis, I present the development of a high-throughput, pooled GUIDE-seq assay to experimentally profile Cas9 on and off-target cleavage across a diverse set of gRNAs. This assay builds on the established GUIDE-seq protocol, which labels DSBs in vivo with a double-stranded oligodeoxynucleotide (dsODN) tag, enabling genome-wide identification of cleavage sites via amplicon sequencing. As a proof-of-principle, I demonstrated successful GFP knockout (KO) in a reporter cell line using both plasmid transfection and ribonucleoprotein (RNP) nucleofection, validated by flow cytometry and next-generation sequencing (NGS). I then cloned a pilot guide pool and generated a stable cell line expressing the pool via lentiviral infection. A preliminary GUIDE-seq experiment confirmed successful dsODN integration at a target site, though integration rates remain below the threshold necessary for robust off-target detection. This work lays the foundation for a pooled GUIDE-seq pipeline, with future efforts focused on optimizing the assay to generate comprehensive off-target profiles. These data will support the development of a predictive model – GuideScan3 – for the rational design of highly specific gRNAs, facilitating the broader use of CRISPR-cas9 editing in therapeutics and basic science without inducing harmful off-target effects.

Tubular networks are implicated in the function of multiple organs, arising from diverse tube topologies. Branching morphogenesis and tubulogenesis of these seamless tubes are processes that remain open to further investigation. The tracheal system of Drosophila melanogaster is an exemplary model of tubular networks that arise from seamless or unicellular tubes; its terminal cells have even been evidenced in cytoskeletal mechanisms of tube formation. Septins are GTP-binding proteins that function by associating with themselves and other cytoskeletal components, such as microtubules and actin. By facilitating crosstalk between actin and microtubules, which are heavily involved in cell elongation and lumen growth, septins like Sep2 become compelling effectors of seamless tube formation. Preliminary findings indicate that Sep2 is required for proper branching morphology and for the coupling of branch and lumen growth in Drosophila terminal cells. In the following research, we characterize the phenotype of Sep2 knockdown using two distinct RNAi lines and confirm RNAi silencing of Sep2. A Sep109 mutant allele is identified in CRISPR-edited fly lines and its effects are characterized in tracheal terminal cells. Further experimentation with septin depletion helps elaborate on the role of these proteins in the mechanisms that underlie the formation of seamless tubes.

Human cytomegalovirus (HCMV) is a master manipulator of host cell biology, not only altering directly infected cells but also reshaping the surrounding tissue environment through secreted factors. In this study, we uncover how conditioned media (CM) from HCMV-infected fibroblasts exerts rapid and transient effects on neighboring uninfected cells, reprogramming their proteome and phosphoproteome in a manner that bypasses classical immune signaling. Central to this paracrine reprogramming is the activation of mitotic kinases such as CDK1, CDK4, AURKB, and particularly Aurora kinase A (AURKA); a master regulator of mitosis and interphase remodeling. We demonstrate that AURKA is both induced and phosphorylated in recipient cells exposed to CM, even in the absence of extracellular vesicle (EV) trafficking and type I interferon pathways. These findings suggest that HCMV-infected cells secrete non-vesicular, non-canonical factors capable of mimicking proliferative cues. Beyond its well-characterized mitotic roles, AURKA also governs mitochondrial dynamics, ciliary disassembly, and DNA replication licensing; functions that viruses increasingly exploit to favor replication and immune evasion. The engagement of AURKA by paracrine signaling thus represents a novel strategy by which HCMV may prime the surrounding cellular landscape for enhanced viral fitness or persistence. Our data support a model in which the virus-modified environment (VME) serves not only as a reservoir of viral proteins and EVs but also as a conduit for soluble signals that transiently rewire the cell cycle and stress response pathways of uninfected bystanders. These insights position AURKA as a potential therapeutic node at the intersection of viral pathogenesis, host cell cycle control, and long-term tissue homeostasis.

Across the animal kingdom, germ cells contain ribonucleoprotein (RNP) condensates necessary for proper germline development called germ granules. In some organisms like the Drosophila melanogaster, germ granules form as early as oogenesis, and in other organisms, like the mouse, they form much later, after primordial germ cell (PGC) specification. Despite this difference in developmental timeline, germ granules across species share many components, like a Tudor domain scaffolding protein and piRNA binding proteins like Aubergine. Although hypothesized to be hubs of post-transcriptional regulation, the role of germ granules in PGCs, especially after their specification, remains largely unknown. Investigating the role of conserved proteins, like Tudor and Aubergine, after PGC specification in Drosophila may provide further insight into the more conserved functions germ granules have across species. Here, I show that Tudor and Aubergine persist in Drosophila germ granules until PGCs have reached the gonads, indicating they may be needed after PGC specification. I then lay the groundwork for investigating the roles of Tudor and Aubergine in pole cells and germ granules after PGC specification. Using CRISPR Cas9 Scarless editing methods and classical Drosophila genetics, I generate the necessary CRISPR lines needed to deplete Tudor and Aubergine in the PGCs after their specification using an auxin inducible degradation system. These Drosophila lines can be used for future experimentation to help determine the roles of Aubergine and Tudor past PGC specification, potentially pointing to conserved roles they play in the germ granules of other organisms.

Biomolecular condensates, membraneless compartments found ubiquitously within eukaryotic cells, regulate cellular organization and biochemistry. Forming via liquid-liquid phase separation (LLPS), these complexes typically behave like droplets, fusing and flowing the way a liquid might. However, issues within the cell, including neurodegenerative diseases like ALS, induce a liquid-to-solid transition within condensates known as aging. Aging is detrimental to condensates as they rely on their liquid properties to adequately function, yet the molecular mechanism that drives these droplets to enter a solid-like, arrested state remains poorly understood. We seek to propose a valid mechanism for condensate aging that is both generalizable and agrees with observed biological behavior. Using coarse-grained molecular dynamics simulations through the program LAMMPS, we show that condensates become dynamically arrested through the formation of laddered constructs within the system that effectively freeze polymer movement. Building on a theoretical framework of conformational entropy, we find that aging is the result of molecular-level choices and dynamics. Crosslinkers between polymers converge to adjacent binding sites in order to minimize the entropic cost of forming bonds. Following the formation of these bonds, we see that more rigid polymers experience fewer fluctuations and can extend the lifetime of these linker ladders. The accumulation of linkers and maintenance of ladders through the simulation ultimately leads to the slowing of dynamics and structural hardening associated with the macroscopic event of condensate aging. By illuminating how simple physical mechanisms motivate complex cell-level outcomes, we hope to lay the groundwork for future research in biomolecular condensates and neurodegenerative diseases.

Having to adapt to a foreign land, immigrants in the United States often experience elevated stress levels. Migration is a complex psychosocial process with long-lasting health consequences. Paradoxically, immigrants arrive healthier than U.S.-born individuals, but this advantage declines over time. As chronic stress can induce epigenetic changes such as hypermethylation, I hypothesize that children of immigrant mothers exhibit altered DNA methylation patterns and worse cardiovascular outcomes compared to children of U.S.-born mothers. This thesis investigates whether maternal nativity predicts cardiovascular health and biological aging in children, using data from the Future of Families and Child Wellbeing Study and its cardiovascular subsample (FFCVH). Cardiovascular health was assessed at Year 22 using the American Heart Association’s Life’s Essential 8 (LE8) score; biological aging was measured at Years 9 and 15 using six DNA methylation clocks: Horvath Pan-Tissue, Horvath Skin and Blood, DNAmPhenoAge, DNAmGrimAge, DunedinPoAm, and DunedinPACE. A 2:1 propensity score matching approach compared LE8 and DNA methylation age acceleration (EAA). Multivariable regressions tested whether maternal nativity predicted LE8, whether LE8 predicted EAA, and whether LE8 mediated the nativity–EAA relationship. Children of immigrant mothers had significantly higher LE8 scores, indicating better cardiovascular health. Higher LE8 scores were associated with lower EAA across multiple clocks, with stronger effects at Year 15. While maternal nativity alone did not consistently predict EAA, it became significant in adjusted models for select clocks. These findings suggest maternal nativity shapes cardiovascular health and contributes to differences in biological aging in the second generation.

With the increasing prevalence of metabolic disease globally, fasting has been identified as a potential tool to effectively improve metabolic health. Fasting drives pleiotropic effects across all organ systems, including altered metabolome profiles and gut microbiome composition. Both of these changes on their own can significantly impact metabolic health. Changes in metabolomics and microbiome composition are complex, as the host metabolome shapes microbiome composition via nutrient inputs, and the microbiome can affect the host metabolome via bacterial outputs. However, the interaction between these two areas in fasting has not been studied. Thus, this study sought to investigate potential causal roles of the gut microbiome in the systemic metabolic response to fasting. To carry out this task, we subjected mice with an intact or antibiotic-depleted microbiome to 48 hours of water-only fasting, followed by metabolomic profiling of multiple tissues and serum. We identified fasting-induced increases of fatty acids and acylcarnitines in intestinal content, with microbiome depletion-mediated blunting or exacerbation of the fasting effect. We also found significant changes in microbiome composition and gene expression related to fatty acid and acylcarnitine metabolism, suggesting possible mechanisms for the microbiome-mediated fasting effect. Finally, we found evidence suggesting that our observed murine patterns of fatty acid and acylcarnitine metabolism are relevant for human metabolism. Overall, these results suggest that the gut microbiome plays an important causal role in regulating fatty acid and acylcarnitine metabolism during fasting. This work serves to improve our understanding of how the diet and the microbiome of an individual interact to regulate the metabolic activity of the host.

Early embryonic avian lung development is regulated by a host of biochemical mechanisms and signaling pathways, many of which are conserved across species. The role of fibroblast growth factor (FGF), wingless integration site (WNT), and retinoic acid (RA) are well characterized within avian lung development, but little is known about the role of the Hippo pathway in branching morphogenesis in the avian lung. Here, we investigate the role of the Hippo pathway co-transcription factor Yes-associated protein (Yap) in avian lung development using immunostaining and microscopy to determine whether its function is conserved. We find that branch initiation is not accompanied by changes in the Yap signaling, suggesting that Yap does not drive the initial emergence of secondary bronchi. We also find that the spatial pattern of unphosphorylated, active Yap is dynamic across developmental time during branch elongation, from enrichment at distal tips at earlier time points to accumulation in regions of high epithelial cell density at later stages, suggesting a heterogeneous role in elongation. Furthermore, observed changes in Sox2 expression are not paralleled by changes in active Yap patterning, suggesting that Yap-driven transcription and Sox2-mediated cell fate patterning are regulated separately. Our investigation into the patterning of active Yap and Sox2 during chick lung development provides new insight into the role of Hippo signaling in branching morphogenesis in the avian lung.

Chronic hepatitis C virus (HCV) infections often lead to significant long-term complications, including permanent liver damage and hepatocellular carcinoma. Currently, the molecular mechanisms driving cancer development in patients with chronic HCV are unclear. To untangle the contributions of direct effects on infected cells and bystander effects on cells near the infection site, I developed components of what will be an inducible molecular recorder for interferon signaling in human hepatocytes. By designing, cloning, and testing a Cas12a effector and guide RNAs, as well as cloning and evaluating several liver cell lines containing the necessary genome editing machinery, I identified a highly efficient guide RNA to be used in the final recorder system. This larger project led by Dr. Aaron Lin will ultimately allow for greater insights into the molecular mechanisms underlying the development of hepatocellular carcinoma from chronic HCV infections.

TACC3 (Transforming Acidic Coiled-Coil protein 3) is a conserved mitotic protein that plays a key role in microtubule nucleation and spindle organization during cell division. It belongs to a class of proteins known as spindle assembly factors (SAFs), which promote microtubule formation and ensure proper spindle assembly during mitosis. SAFs are tightly regulated by nuclear import receptors to prevent premature activation and maintain mitotic timing and fidelity. In this thesis, I investigated the regulation of Xenopus laevis TACC3 by the nuclear import receptors importin-α and importin-β. To do this, I generated a GFP-tagged TACC3 fusion construct, expressed and purified the protein using affinity and size exclusion chromatography, and performed pulldown assays to test for direct interactions with importin-α, importin-β, and a truncated ∆IBB-importin-α variant under various salt and concentration conditions. My results show that TACC3 specifically interacts with importin-β, but not with importin-α or ∆IBB-importin-α. The interaction is salt-sensitive and concentration-dependent, indicating it is electrostatic in nature and relatively weak. A GFP-only negative control confirmed that importin-β binding is specific to the TACC3 portion of the fusion protein. These findings challenge the traditional model in which SAFs are regulated via direct interaction with importin-α and instead suggest that TACC3 may be controlled directly by importin-β. This mechanism resembles importin-β–mediated regulation of other SAFs such as TPX2, and may reflect a broader regulatory pathway for spindle assembly. Further work is needed to map the binding interface, assess the role of regulatory factors like Ran–GTP, and ascertain any post-translational modifications. Overall, this study reveals a novel regulatory interaction between TACC3 and importin-β that advances our understanding of SAF control during mitosis.

Epithelial cell migration is a striking phenomenon in which cells that are tightly adhered in an epithelium, can undergo dynamic movements while generally maintaining their structural morphology. In development, epithelial cell migration can occur without the activation of a full epithelial to mesenchymal transition (EMT), through a “partial-EMT”. Hair follicle morphogenesis is a useful model for studying epithelial cell migration and partial-EMT, because it has been shown to demonstrate EMT characteristic behaviors . In this study, we investigated the spatial expression and WNT-dependence of pro-migratory genes in early hair follicle progenitors. Using hybridization chain reaction to determine gene expression in mouse embryonic skin explants, we identified that Myosin-X (Myo10), Snai2, and S100A9 are upregulated in inner cells of placodes. Our results revealed the previously unknown spatial expression patterns of pro-migratory genes, linking them to a population of cells proven to exhibit migration. Further, our work supports a role for Wnt signaling and/or positional location inside a developing placode in the modulation of these genes. Inner cells must activate a transcriptional program that grants migration among cells closely bound in an epithelium. Our findings start to characterize the expression and regulation of genes involved in a p-EMT-like event resulting in motility. These findings are not only crucial to developmental biologists, but also for cancer research as partial-EMTs are frequently implicated in cancers with increased metastatic potential.

Kaposi’s sarcoma-associated herpesvirus (KSHV) is an oncogenic gammaherpesvirus and remains one of the leading causes of cancer among immunocompromised individuals world-wide. Like all herpesviruses, KSHV maintains a biphasic life cycle, consisting of a life-long latency sporadically broken by active lytic replication. Like all viruses, KSHV infection induces the remodeling of multiple organellar compartments to either maintain viral latency or promote lytic replication.However, how KSHV drives these remodeling events and how they promote virus-induced tumor progression remain poorly understood. Recently, our group has uncovered that organelle remodeling during herpesvirus infection is directly linked to virus-induced alterations in membrane contact sites (MCS), protein-to-protein or protein-to-membrane connections that form dynamic organelle-organelle communication networks. Here, we use super resolution confocal microscopy to characterize dynamic KSHV-induced remodeling events of mitochondria, peroxisomes, and endosome populations throughout KSHV infection. Then, using siRNA-based knockdown approaches, we demonstrate that MCS proteins that form contacts between the ER and each of these compartments have a significant impact on KSHV lytic gene expression, virion production, and cell survival during lytic replication. Lastly, we find that knockdown of these MCS proteins can not only partially reverse KSHV induced alterations of mitochondria and peroxisomes but also significantly impact the abundance of multiple oncogenic and antiviral cell signaling receptors. Collectively, these findings demonstrate that membrane contacts play a critical role both in driving organelle remodeling events during both phases of KSHV infection that may promote both oncogenic cell signaling and successful lytic replication. Findings from this study have significantly improved our understanding of how KSHV infection alters subcellular compartments to promote diverse replication states and how these changes may drive viral oncogenesis.

Bacterial cells communicate using quorum sensing, a process that involves the exchange of signal molecules known as autoinducers. While autoinducers were once thought to be species specific, the Bassler lab has characterized a “universal” autoinducer called AI-2. Interdomain communication was discovered when mammalian cells were found to produce a mimic of AI-2 capable of binding to AI-2 receptors in Vibrio harveyi, inducing bioluminescence. While the structure of this mimic has not been reported, recent data suggest that the mimic is xylosone. Despite the absence of a known enzyme for xylosone production in mammalian cells, previous literature characterizes pyranose oxidases as potential xylosone synthases, found throughout fungi and bacteria. In this thesis, it is shown that a fungal pyranose oxidase produces an AI-2 mimic, as measured by both high resolution mass spectrometry and a bioactivity assay in V. harveyi. This research lays the foundation for future work that could provide insights into AI-2 mediated chemical communication between bacteria and eukaryotes.

Cardiovascular disease (CVD) remains the leading cause of death worldwide, with atherosclerosis as its primary underlying pathology. Since evidence of disease can manifest at a young age, understanding its early-stage mechanisms of pathogenesis is crucial for preventing and managing CVD. To contribute to this goal, this thesis leverages both epigenetic and genetic data from over 1,200 individuals in the Future of Families and Child Wellbeing Study (FFCWS), whose carotid intima-media thickness (CIMT) was measured as a marker for atherosclerosis. Three complementary epigenomic and genomic methods were employed: a candidate CpG analysis to identify trends in site-specific methylation over time, an epigenome-wide analysis to detect differentially methylated regions that recur throughout adolescence, and Mendelian Randomization to elucidate potential causal effects of methylation on CIMT. Across the three methods, sets of CpGs on 15 genes—SOCS3, CPT1A, HIVEP3, PLEKHB1, SPATC1L, TSBP1, FADS1/FADS2, HCG27, HOXA-AS2, HOXA3, HOXA4, HOXA6, MGST3, TNXB, and ZFP57—were significantly associated with CIMT. These genes emerge as promising candidates for further investigation into the mechanisms of atherosclerosis development, which is especially important given the study’s focus on an ethnically diverse and historically underrepresented cohort. These findings provide a foundation for future research into gene regulation and epigenetic biomarkers for early-stage CVD, which may ultimately improve overall tracking, prevention, and management of the disease.