Molecular Biology, 1954-2026

Post-traumatic stress disorder (PTSD) is associated with alterations in inflammatory and endocrine signaling. However, across studies, findings are highly variable, making it unclear whether biomarker changes are due to a coherent biological state that changes according to context or unrelated heterogeneity. Thus, this study aimed to evaluate whether PTSD is characterized by an integrated, between-system pattern of biological dysregulation that contributes to the experiential outcomes noted in PTSD. A meta-analysis was conducted on immune (IL-6, TNF-α) and endocrine markers (cortisol), along with a qualitative structured synthesis of other markers, such as ACTH. Results indicated that PTSD is characterized by a system-level pattern of dysregulation across interacting biological systems, including consistent inflammatory activation alongside variable endocrine regulatory control. Mechanistically, this pattern is consistent with increased excitatory signaling paired with unstable regulation, which may disrupt neural integration processes that enable experiences to be encoded as self-authored, which becomes particularly notable when applied to sexual experiences. Accordingly, the occurrence of a physical act alone may be insufficient to determine whether an experience was “had” in the sense of being encoded as self-authored and owned. This study thus highlights the importance of internal biological state in shaping conscious experiential outcomes, and in informing the terms used to define those outcomes, such as virginity and its loss.

Germ cell formation is essential for sexual reproduction. In many animals, this process depends on the germ plasm, which is a specialized maternally inherited cytoplasm that directs germ cell fate. In Drosophila embryos, the germ plasm is localized to the posterior pole and is organized into germ granules that concentrate maternal mRNAs needed for germ cell development. Although a few of these transcripts, such as nanos and pgc, have known functions in germ cell specification, the roles of the vast majority remain untested because mutations in germ plasm components disrupt germ plasm assembly altogether, and RNA interference lacks the spatial precision to deplete transcripts specifically within the germ plasm. To overcome these limitations, I aimed to develop a subcellular RNA knockdown system based on Cas7-11 a CRISPR endonuclease that causes degradation of target RNA without the collateral cleavage activity associated with existing Cas13-based systems. To restrict Cas7-11 activity to the germ plasm, I fused it to the pgc 3'UTR and the nanos translational control element, regulatory sequences that together localize the mRNA to the posterior pole and silence translation among unlocalized copies. Immunofluorescence confirms that Cas7-11 protein accumulates in the embryonic germ plasm and is inherited by pole cells. Single-molecule fluorescence in situ hybridization reveals that the crRNA-directed Cas7-11 effect can reduce nanos transcript levels at the posterior pole. nanos knockdown does not diminish levels of non-target germ granule mRNAs, which is consistent with the expected absence of collateral cleavage. Together, these findings demonstrate that Cas7-11 can achieve targeted RNA depletion within a defined subcellular compartment, providing possibilities for systematic functional analysis of individual germ granule mRNAs in the process of germ cell development.

Improper branching due to abnormal cellular signaling is a driving factor for many congenital diseases involving angiogenesis in humans. To better understand these defects, developmental biologists have long used the Drosophila melanogaster tracheal system as a model for branched tube formation. While previous research has demonstrated a requirement for components of the Bnl/Btl (FGF/FGFR) signaling pathway during tracheal network formation, there is limited understanding of its role and essential time frames in promoting unicellular branching of specialized tracheal terminal cells during larval growth. This study investigates the temporal requirements of this pathway and whether it promotes branching through transcriptional or cytoskeletal mechanisms. Using genetic tools to perturb signaling during larval stages, we characterize pathway activity and its role in terminal cell morphogenesis. This work provides insight into how a single cell forms a complex branched structure with subcellular lumens.

This thesis investigates how the Piwi protein regulates germline stem cells in the ovary of Drosophila melanogaster, with a focus on how its activity is shaped by direct protein–protein interactions. Genetic evidence has shown that Piwi's functionality mainly relies on RNA-guided chromatin repression and interactions with other proteins. To better understand this mechanism, Piwi’s interaction with Arx was analyzed and compared to the Piwi–Papi interaction, which served as a control. Structurally several protein-protein interactions were examined using tools such as AlphaFold, ChimeraX, and Mol*. The Piwi–Arx interaction was modeled and assessed for stability and binding confidence, while Piwi mutants (S606W and R679A) were designed to disrupt predicted amino acids and noncovalent interactions to test their importance in binding. The results suggest that Piwi's function depends strongly on its binding partners. The Piwi–Arx interaction showed high predicted interaction confidence, supporting the idea that Arx may contribute to activation or stabilization of Piwi-mediated silencing. Overall, this work highlights that Piwi’s role in genome regulation is not determined by Piwi alone, but by a network of protein interactions that help regulate silencing roles in the germline.

The germline develops as a functionally distinct population of cells, uniquely tasked with transmitting genetic information across generations. In Drosophila melanogaster, the identity of germ cell precursors – termed pole cells – is established by maternally deposited determinants. A maternal germ plasm containing germ granules – ribonucleoprotein assemblies that guide germline development – accumulates at the posterior, where it associates with syncytial nuclei to induce their cellularization into individual pole cells. In early stages of embryogenesis, all nuclei are transcriptionally quiescent, with maternal factors independently driving development. Pole cells retain this transcriptional quiescence for longer relative to somatic cells, a feature believed to insulate them from somatic induction, thereby safeguarding their fate. While several factors with roles in the regulation of transcription in the germline have been characterized, the full repertoire of responsible molecules remains unknown. According to the demonstrated role of nucleoporins (Nups) – proteins of the nuclear pore complex (NPC) – in gene regulation, this project explored their role in the regulation of transcriptional quiescence in the Drosophila germline. An exploration of phenotypic outcomes resulting from the RNAi-based depletion of individual Nups allowed for the characterization of four Nups as potential regulators of transcriptional quiescence in pole cells. Additionally, the investigation of unexpected phenotypes – namely infertility and defective embryonic development – allowed for the implication of several Nups in a variety of developmental processes, ranging from oogenesis to germ granule localization. These results expand our understanding of the role of the NPC beyond nucleocytoplasmic transport, potentially implicating Nups and nuclear architecture in the regulation of various stages of development across the animal kingdom.

Metabolism is a fundamental process which provides energy to living organisms, and its dysfunction is a shared feature of many noncommunicable diseases and of the degenerative process of aging. Identifying the metabolic hallmarks of aging is an ongoing pursuit, particularly in mice, which are commonly used for modeling human aging. Multiple dietary and pharmacological interventions have been shown to modulate age-related declines in metabolic function and increase lifespan in mice, although the precise mechanism by which this occurs is unknown. Here, we perform a longitudinal metabolomics analysis of aging mice on distinct dietary paradigms: control, methionine restriction (MetR), 15% caloric restriction (PF), and two drug supplemented diets: ZGN1062, and ZGN201, to better understand how age, sex, and dietary interventions modulate the serum metabolome. We report 607 known metabolites and 1004 unknown metabolites with significant associations with age, sex, diet, or the combination of these variables. Moreover, we use decision trees, gradient boosting, and Long Short-Term Memory (LSTM) machine learning models to predict lifespan from metabolomic profiles, finding that gradient boosting is particularly effective at predicting days of life remaining in mice. Overall, this thesis employs a range of computational tools to identify metabolites that are age-associated, sexually dimorphic, lifespan-predictive, or modulated by dietary interventions, allowing us to generate hypotheses about age-induced metabolic dysregulation and how geroprotective interventions may slow this process.

We have come to know that the gut microbiome influences the education of the innate immune system; however, how this education occurs at the molecular level is poorly understood. Previous work by the Brooks Lab has elucidated the role of cytokines in the regulation of key AMPs in the mammalian gut. Here I seek to delineate a spatial profile for several cytokines, IL-4, IL-13, IL-22, and IL-23 and their patterning with respect to the expression profiles of the AMPs they have been shown to regulate. My findings reveal that IL-13 and Retnlb expression are paired across the murine gut and demonstrate elevated levels in colon. Interestingly, IL-4 does not exhibit any patterning with IL-13 or Retnlb. I also showed that IL-23 and IL-22 levels were paired across the length of the gut. Despite previous works demonstrating that the IL-23/IL-22 axis responsible for inducing REG3G, I demonstrate that in healthy mice the IL-22 spatial patterning is decoupled from that of REG3G because baseline levels of IL-22 are not sufficient. However, further work is required to fully determine if these findings are real.

The mammalian intestine integrates dietary, microbial and immune-derived cues to regulate metabolic processes and maintain tissue homeostasis. These signals are sensed in a spatially restricted manner giving rise to region-specific functional specialization along the gastrointestinal tract. This architecture supports essential processes, including nutrient absorption and immune defense. In this work, I build on spatial transcriptomics experiments revealing spatial localization of select genes along the length of the intestine. Strikingly, many of these localization patterns were dampened in animals devoid of a microbiome. Among the genes to exhibit greater localization within the proximal region of the large intestine in the presence of the microbiome is Pparg. The gene Pparg is known to control the expression of several genes involved in lipid storage and mobilization. Here, I confirmed the proximal localization of PPARG at the level of protein expression. To identify potential regulators of this localization, I examined the effect of microbial and immune-derived signals on Pparg expression in intestinal organoids. I find that isolated microbial cues do not significantly alter Pparg expression. Similarly, I demonstrate that immune signal IL-13 is not sufficient to change Pparg expression. These findings suggest that Pparg localization is not directly driven by these candidate microbial or immune signals but rather relies on alternative, more complex mechanisms. This work provides novel insight into the establishment of regionalized metabolic functions, with implications for understanding intestinal inflammation and disease.

The development of multicellular organisms requires highly coordinated tissue movements that depend on the precise alignment of cells within an epithelial plane. Planar cell polarity (PCP) is a conserved signaling pathway that establishes this directional organization through the asymmetric localization of core protein complexes at opposing cell-cell junctions, propagating intercellular asymmetry across entire tissue sheets to guide oriented migration, cell division, and cytoskeletal remodeling. Despite its well-established role in morphogenetic processes ranging from neural tube closure to cochlear patterning, how asymmetric PCP localization is translated into directed cytoskeletal organization remains poorly understood, particularly in mammalian systems. Eyelid closure in the mouse embryo offers an accessible model to address this question. Closure proceeds through localized cell intercalations at the epithelial front that advance the tissue over the cornea, and disruption of PCP produces a fully penetrant eyes-open-at-birth phenotype with disorganized actin arrays and failed fusion. Despite this clear requirement, PCP has never been directly characterized within the eyelid epithelium. Its spatial distribution, layer specificity, and relationship to the actin cytoskeleton remain entirely unexamined. This study provides the first characterization of PCP organization in this system, investigating how core PCP protein localization relates to actomyosin dynamics at the advancing epithelial front. Using Fz6, Celsr1, and Vangl2 reporter mice alongside layer-specific markers, I find that PCP expression is largely restricted to the basal epithelial layer of the outer eyelid, suggesting a spatially regionalized role during closure. Phalloidin staining further reveals that F-actin does not co-localize with any of the three core PCP proteins, instead accumulating apically as a continuous cable at the leading edge, altogether revealing a spatially compartmentalized architecture of PCP-cytoskeletal coupling with implications for wound healing and tissue repair.

Tuberculosis (TB) remains the world’s single deadliest infectious disease in absolute terms, with drug-resistant strains posing a particularly difficult challenge for global health systems. Even bedaquiline (BDQ), the first novel anti-TB agent introduced in four decades and now the cornerstone of the WHO’s second-line treatment guidelines, has encountered alarming rates of emerging resistance. Thus, before subjecting a patient to the steep physical, financial, and time-intensive costs of BDQ-based regimens, it is imperative that drug susceptibility testing (DST) be performed to verify the efficacy of the drug against their specific strain. The only methods available, however, either require long incubation periods or rely on expensive genomic sequencing. Hence, there exists an urgent need for rapid and cost-effective BDQ DST. Here we present a novel molecular DST system that combines the precision of CRISPR-Cas13 with computational sequence design and microfluidic multiplexing to maximize accuracy, efficiency, and modularity. Through extensive screening, we assembled a panel of 60 guide RNAs that successfully distinguished between 81 of the most prevalent BDQ-resistant variants. Recognizing the need to further improve Cas13’s mismatch intolerance, both for DST and other RNA detection tasks, we also evaluated the viability of phosphorothioate (PS) crRNAs as a tool for specificity enhancement. After multiple rounds of rational design and systematic testing, we found that PS modifications offer a substantial discrimination boost across the entire crRNA-target duplex. However, significant disparities exist in the magnitude of the specificity improvement generated by different PS-modified sites and received at different mutation sites, potentially due to variation in Cas13 contact. Despite the need for further optimization, this proof of principle offers a promising path forward for robust single-nucleotide precision with Cas13.

Cardiovascular disease (CVD) remains a leading cause of morbidity and mortality worldwide. Novel, non-invasive biomarkers that capture early vascular changes and biological aging may improve risk stratification, particularly in socioeconomically diverse populations. To evaluate associations between grayscale imaging features, cardiovascular health as measured by Life’s Essential 8 (LE8), and DNA methylation (DNAm)- based epigenetic aging. Data were analyzed from 1,421 participants in the Fragile Families and Child Wellbeing Study. Grayscale imaging features, including grayscale median (GSM), entropy, gray level dependence matrix (GLDM), and gray level co-occurrence matrix metrics (SGLD-ASM, SGLD-HOM), were examined in relation to LE8 scores (including sleep) and multiple DNAm clocks. Associations were assessed using linear regression and generalized additive models (GAMs) to capture nonlinear relationships. GSM was the only grayscale feature significantly associated with LE8 (p<0.01), demonstrating a positive relationship in which higher GSM values corresponded to better cardiovascular health. GAM analyses revealed a nonlinear association with a plateau at GSM ~80-90. Other grayscale features were not significantly associated with LE8. Healthmap and regression analyses showed largely non-significant relationships between grayscale features and DNAm clocks; however, select measures of pace of aging and PhenoAge demonstrated modest inverse associations with GSM. Entropy showed weak negative trends with LE8 and certain DNAm measures. Grayscale median is a promising imaging derived marker of cardiovascular health and shows limited but suggestive links to epigenetic aging. These findings support the integration of scalable biomarkers with comprehensive health metrics like LE8 to enhance early detection and prevention of CVD, with applications in global health.

Piwi is a PIWI-clade Argonaute protein that functions in transposon silencing and germline stem cell regulation in Drosophila melanogaster, but the structural basis of many Piwi protein–protein interactions remains unclear. This study used a computational screening strategy to identify candidate Piwi interaction interfaces by combining FlyBase Gene Ontology-based candidate selection with AlphaFold- and ColabFold-based complex prediction. Piwi-centered multimer predictions were ranked using pLDDT, predicted aligned error (PAE), predicted template modeling score (pTM), and interface predicted template modeling score (ipTM), and top-ranked models were further analyzed in ChimeraX through structural superposition, RMSD comparison, and sequence alignment against known experimental Piwi structures. Most candidate complexes showed low-to-moderate interface confidence, with only a small subset emerging as stronger structural candidates. Comparisons with experimental structures demonstrated that the workflow reliably recovered major features of the Piwi core, supporting its use as a proof-of-concept method for structural screening. Candidate complexes nevertheless varied substantially in interface confidence: Piwi–Panx showed weak support for a stable interface, whereas Piwi–Arx emerged as a stronger candidate complex with higher overall confidence. These results show that AlphaFold/ColabFold-based screening can prioritize candidate Piwi partners and structural interfaces for future validation, providing a foundation for downstream mutagenesis and biochemical analysis of Piwi function.

Electrotaxis is the directed movement of cells in response to an electric field and is a crucial biological process underlying processes such as tissue morphogenesis, wound healing, and cancer metastasis. Despite its significance, the mechanisms that enable cells to sense the electric field and facilitate directional migration are still not fully understood. Recently, transmembrane protein 154 (TMEM154) was identified as a driver of electrotaxis directionality in the human immune cell line, HL60s, where TMEM154 knockout resulted in the cells no longer exhibiting directed migration under an electric field. However, Madin-Darby Canine Kidney (MDCK) epithelial cells which are also capable of electrotaxis on the collective level, but not as single cells, have very low native expression of the TMEM154 ortholog. Additionally, TMEM154 was not upregulated upon electrical stimulation, signifying that MDCKs do not require TMEM154 for electrotaxis. To understand how TMEM154 abundance shapes electrotaxis behavior, this research will generate a stable MDCK-CRISPR inference (CRISPRi) cell line that can be used to knockdown TMEM154 in high-expressing MDCK-TMEM154 cells to evaluate how knockdowns can alter electrotaxis dynamics. Additionally, TMEM154 will be used as a design scaffold to engineer synthetic biosensors to visualize cell dynamics immediately following electrotaxis to provide another method of understanding cell response dynamics. Through this thesis we were able to create a synthetic TMEM154 inspired biosensor that localizes to the cell membrane and a stable MDCK-CRISPRi cell line. Understanding the mechanisms by which cells sense and react to an electric field has significant implications for fundamental biology, particularly in biological processes where electric fields guide cell migration such as wound healing and tissue patterning. As such, this research will also discuss the relevance of electrotaxis in chronic wound healing and emerging bioelectric therapeutic approaches in the global context.

Bacteria frequently form multicellular, surface-associated communities called biofilms. Biofilms provide advantages to residing cells, protecting them from environmental stresses such as antibiotics and mechanical perturbation. It has been proposed that these advantages stem from limited diffusion of small molecules into and out of the biofilm. However, due to the challenge of probing events occurring inside the biofilm structure, direct measurements of diffusion through the biofilm are limited. Here, I use single-molecule fluorescent in situ hybridization (smFISH) to measure spatiotemporal transcriptional responses of quorum-sensing (QS)-regulated genes in Vibrio cholerae biofilms following perturbations in levels of the QS signaling molecule called autoinducer-2 (AI-2), allowing gene expression to be used as a proxy to infer the spatial distribution of AI-2 over time within biofilms. Spatial position within the biofilm influenced QS response kinetics, with peripheral cells responding more rapidly than interior cells, particularly during the early response to AI-2 addition. Transitions between QS states were also asymmetric, occurring more rapidly following AI-2 addition than removal. Increased extracellular matrix production also slowed these responses following AI-2 addition. Collectively, these results suggest that diffusion limitations within the biofilm matrix restrict autoinducer movement and modulate QS responses. This study establishes a framework for linking gene expression dynamics to spatial transport processes and demonstrates how physical constraints within biofilms shape collective bacterial behavior.

In its natural environment, C. elegans is exposed to both pathogenic and non-pathogenic bacteria and must survive by learning how to distinguish between the types as a nutrient source. Our lab previously found that C. elegans can learn to avoid a pathogen, Pseudomonas aeruginosa, after relatively short exposure and then pass on this learned avoidance pattern to its offspring for four generations before the behavior must be relearned. The mechanism through which this transgenerational epigenetic inheritance functions and where the components necessary for this process act, are not well understood. NRDE-3 is a siRNA-binding Argonaute protein in the Nuclear RNAi Defective (NRDE) pathway and NRDE-4 is a nuclear RNAi factor that is responsible for chromatin association. This project will determine where and how NRDE-3 and NRDE-4 function in C. elegans to produce a pattern of learned avoidance behavior.

For productive viral replication, both an infected cell and its surrounding environment are metabolically and defensively rewired. Human cytomegalovirus (HCMV) is a ubiquitous beta herpesvirus across the global population that is the leading infectious cause of birth defects and extensively remodels its surrounding microenvironment throughout infection. Cells proximal to an HCMV infection exhibit decreased immune signaling and increased viral protein abundance, priming them for a subsequent infection. In my thesis work, I sought to understand the mechanisms behind this remodeling of the viral microenvironment (VME) using a multidisciplinary approach including microscopy, proteomics and method development. Preliminary findings revealed that cell-cell contact dependent mechanisms are an important axis of intercellular communication deserving of more rigorous investigation. My project focuses on the role of mitochondria, tunneling nanotubes, and direct cell-cell contacts more broadly in altering the immune signaling pathways of cells proximal to a site of infection. I developed a cell line that labels mitochondria using HaloTag technology that allowed for the first known visualization of mitochondrial transfer during HCMV infection. Further exploring cell-cell communication within a VME, I leveraged fluorescence-activated cell sorting and proteomics to investigate the relative contributions of contact-mediated and contact-independent communication on bystander cells. Finally, I developed innovative strategies to visualize tunneling nanotubes, study the effects of cell density on immune signaling, and spatially pattern the VME. This work ultimately provides evidence that contact-dependent mechanisms of cell-cell communication during HCMV infection are crucial for modulating the immune response in neighboring cells.

Underlying cellular form and function are complex networks of Protein-Protein Interactions (PPIs). These PPIs are constantly fluctuating, exhibiting dynamic responses to maintain normal cellular processes and respond to environmental stimuli. The introduction of disease-specific proteins often disrupts these already dynamic networks, broadly reprograming the cell and exacerbating disease progression. As obligate intracellular parasites, viruses are a prime example of such disease-driven remodeling of protein interaction networks, with viral proteins introducing new layers of PPIs that broadly alter cellular function to support viral replication. Human Cytomegalovirus (HCMV) presents one of the most profound examples of such virus-induced remodeling of the host cell, causing profound morphological changes throughout its replication cycle. Notably, HCMV infection induces severe mitochondrial fragmentation, simultaneously disrupting immune responses from this compartment while enhancing mitochondrial bioenergetics. This presents an interesting paradox to well established mitochondrial structure-function relationship paradigms across multiple model systems. Critical to our mechanistic understanding of mitochondrial dynamics during HCMV infection is elucidating the temporal PPI network disrupted by viral proteins localized to the mitochondria. Previous studies from our lab found that the viral protein pUL13 plays a key role in the HCMV-driven increase in mitochondrial bioenergetics. pUL13 alone is sufficient to upregulate oxidative phosphorylation and remodel the mitochondrial architecture. However, the mechanisms underlying the impact of pUL13 on mitochondrial bioenergetic and its contribution to HCMV-induced changes in mitochondrial structure have yet to be investigated. To understand how HCMV ‘s pUL13 alters mitochondrial protein interaction networks, we developed a novel platform integrating mitochondrial fractionation and Thermal Proximity Coaggregation Assay (TPCA) methods, herein referred to as mito-TPCA. We demonstrate that our optimized experimental framework improves the detection of mitochondrial-specific PPIs compared with whole-cell TPCA analysis. Following optimization, we leveraged this mito-TPCA workflow to compare the mitochondrial PPIs composing infections with wild-type HCMV strain AD169 or an AD169 virus lacking pUL13 (ΔUL13). Using these virus strains we uncovered the specific contribution of pUL13 on HCMV-driven changes in mitochondrial networks. Using this platform, we uncovered previously unreported pUL13 interactors, including the uncharacterized viral protein pUL15a, and reveal that pUL13 expression modulates cristae architecture and several ETC respiratory complexes during HCMV infection. We further validated these interactions between pUL13 and ETC complexes via orthogonal confocal microscopy and biochemical approaches. Furthermore, results from our mito-TPCA analysis revealed that the uncharacterized pUL15a is not only an important interactor of UL13 but also interacts with several subunits of Complex I, suggesting a role for this factor in the HCMV-driven rewiring of cellular metabolism. To better understand the contribution of pUL15a, we generated a stable pUL15a expressing cell line and mapped its impact on the host cell proteome via liquid-chromatography based mass spectrometry (LC-MS) analysis. Finally, to better understand how the clinical significance of HCMV-driven alterations in mitochondrial PPIs, we conduct a whole-cell TPCA analysis in cells infected with a clinical HCMV strain TB40/E and conducted a comparative analysis between our findings for both HCMV TB40/E and AD169 strains. Our findings have uncovered infection specific differences in mitochondrial complex architecture between the two strains.

Pole cell development in Drosophila is key to establishing the germ line. Within pole cells are ribonucleoprotein granules, known as germ granules, that contain key maternal determinants for pole cell formation, including nanos and pgc. It is not fully understood as to the role of every maternally provided mRNA that is enriched within the germ granules. RNA knockdown is a method by which researchers can understand the function of an mRNA. However current RNA knockdown tools are limited and cannot adequately be subcellularly localized. The CRISPR/Cas13 system is a method by which RNA can be subcellularly depleted, but its collateral activity once activated makes phenotype attribution difficult. This paper worked to optimize Cas13 for usage in subcellular knockdown of mRNA within the Drosophila germ granules. In particular, this paper looked at Gwl, a protein kinase previously reported to be found in the germ granules. I showed that gwl mRNA is expressed broadly across the early Drosophila embryo with weak but detectable enrichment in the posterior pole. I then laid the foundation for knockdown of gwl by selecting hfCas13X, a modified version of Cas13X that has been shown to have reduced collateral activity in-vitro, and testing translational control and localization by appending nanos and pgc regulatory sequences to the hfCas13X sequence. Despite generating flies with this transgene, hfCas13X was not expressed. I was able to successfully design crRNA arrays target gwl and nanos and generate transgenic fly stocks for both of these constructs, which can be used in future experiments with hfCas13X.

Dengue virus (DENV) is the most common arthropod-borne virus globally, accounting for 400 million infections each year in over 100 countries. Caused by four distinct serotypes (DENV1-4) with increasing co-circulation in recent decades, dengue poses a unique immunological challenge given the possibility for mixed infections to occur. Despite decades of research, fundamental questions about how these serotypes interact at the cellular level during mixed infections remain unresolved. Existing methods cannot simultaneously provide serotype specificity, single-cell resolution, and live infection dynamics. Studies have also been largely confined to mosquito cell lines or patient samples with unknown infection history, making controlled investigation in human-relevant cell types difficult. In this thesis, we developed and validated two molecular tools to enable serotype-specific tracking of DENV infection in human hepatocytes. Using Circular Polymerase Extension Reaction, we generated mScarlet- and mGreenLantern-expressing reporter viruses for all four DENV serotypes by inserting spectrally orthogonal fluorescent proteins between the E and NS1 genes of infectious cDNA clones. We performed coinfection experiments using fluorescent DENV2 reporter pairs at defined multiplicities of infection, and population dynamics were analyzed by flow cytometry. We additionally developed a genome-modification-free approach, validating an ER-tethered GFP reporter that undergoes nuclear translocation when a linker sequence containing the capsid cleavage site is recognized by the NS2B/3 viral protease. Reporter viruses reliably tracked infection dynamics, and within-serotype coinfection experiments demonstrated that relative population abundance is tunable by inoculum dose. The ER-tethered reporter produced robust nuclear translocation in response to infection, while substitution with a heterologous cleavage sequence led to a six-hour delay, reflecting sequence specificity for NS2B/3 substrate recognition. These data have established a modular approach for studying DENV coinfections as well as inter-serotype protease activity, with possible extensions to the broader Flaviviridae family.

Mitotic cell division relies on complicated interactions between motor proteins and spindle assembly factors (SAFs) that result in the rearrangement of the microtubule cytoskeleton. A majority of microtubules (MTs) for spindle assembly are generated by branching MT nucleation at branch sites marked by Targeting Protein for Xklp2 (TPX2) condensates. The resulting dense MTs are slid apart by molecular motors to form a bipolar spindle. A previous study observing binding between the motor protein Kif15 and TPX2 suggests that Kif15 may bind during branching MT nucleation, prompting investigation into the Kif15-TPX2 interaction. This project aimed to identify the site on Kif15 that mediates its binding with the SAF TPX2 and determine how this interaction influences recruitment to the MT. Using TIRF microscopy, I found that the presence of TPX2 enhances Kif15 tail localization to MTs. Since Kif15 has been previously shown to cooperate with the motor Eg5 to build and maintain the bipolar mitotic spindle, I performed additional pulldown assays with both Kif15 and Eg5 to provide insight into how multiple motor proteins interact with SAFs to build and maintain the bipolar mitotic spindle. I found that Eg5 motor binding to the C terminal portion of TPX2 decreases when in the presence of Kif15 motor and tail domains. This research furthers the model of Kif15 as a tethered MT crosslinker, providing a better understanding of how nucleation and motor molecules work together to orchestrate spindle assembly.