Caused by organisms in the Apicomplexa phylum, toxoplasmosis and malaria are parasitic diseases with significant global health impacts. An estimated 1 in every 3 individuals worldwide are infected with toxoplasmosis, and over 600,000 die annually from malaria. Apicomplexa are a group of single-celled parasites distinguished by specialized organelles in their apical region. Among these structures are micronemes and rhoptries, secretory organelles essential for host cell invasion. Though the process of microneme and rhoptry synthesis is not well understood, T. gondii Dynamin Related Protein B (TgDrpB) has been identified as a necessary component in their formation. While TgDrpB is implicated in the biogenesis pathway of micronemes and rhoptries, its structure and molecular mechanism are currently unknown. The goals for this project are therefore to investigate TgDrpB structure and mechanism in microneme and rhoptry synthesis. It is hypothesized that TgDrpB initiates the process of organelle biogenesis by budding vesicles from the Golgi, similar to that of human dynamin. Completion of this study will establish the structural basis by which TgDrpB accomplishes membrane fission necessary for the biogenesis of secretory organelles and that work to facilitate TgDrpB activity.

Kawasaki disease (KD) is a rare autoimmune disorder affecting nearly 20 in every 100,000 U.S. children aged 5 years and under. KD causes a harmful immune response towards blood vessels, which can lead to coronary artery aneurysm (CAA). The causes and mechanism of action of KD remain largely a mystery, despite decades of etiological research posing a range of biological, environmental, and genetic causative factors. Due to this lack of information, there are no specific diagnostic tests for the disease, and it is diagnosed only by the presentation of a combination of symptoms. This represents a major deficiency, considering that timely and specific diagnosis is critical for preventing life-threatening complications. However, original research has suggested the existence of several subtypes with distinct clinical features under the broad categorization of KD. These subtypes associate specific variables of KD patients to 4 disease subtypes through hierarchical clustering on principal components (HCPC). Yet, my findings in the process of reproducing this work demonstrated significant shortcomings in the previous methods. Upon extensive testing, the HCPC method proved too sensitive to produce convincing clusters using the available variables. Therefore, future KD research must not rely on HCPC, given its demonstrated instability and risk of misleading subtyping. I thus employ unsupervised k-means clustering and produce 3 putative distinctive, characteristic patient subgroup clusters, which appear markedly less sensitive, providing a more stable framework for following KD subgrouping research. These results suggest that KD may be a syndrome with several distinct variants, requiring targeted care approaches.

The mammalian gut microbiome is composed of trillions of bacteria that play essential roles in host metabolic functions and immune responses. Changes in the composition of the microbiome have been implicated in diseases including colitis, metabolic disorders, and cancer. The intestinal tract in which the microbes reside is a regionalized structure with distinct cell types lining the intestinal epithelium that produce and secrete antimicrobial proteins (AMPs). The bactericidal activity of these secreted AMPs, together with a mucus layer, form a barrier that protects the host epithelium from bacterial invasion, thus maintaining host-microbiome homeostasis. Prior studies have shown that distinct bacterial species inhabit different regions of the intestine, but whether AMPs have a role in driving bacterial localization patterns is not clearly established. In this work, I build on results acquired from spatial transcriptomics analysis that show that AMPs exhibit differential expression along the length of the large intestine. One such AMP is RELMβ, which is expressed in the large intestine and targets Gram-negative bacteria. Here, I find that localized RELMβ expression is necessary for the spatial separation of bacteria from the host epithelium in the proximal large intestine and that bacterial localization patterns are disrupted in the absence of RELMβ using wild-type and knockout mouse models. These findings suggest that microbial localization is driven by differential host AMP expression and provide novel insight into the spatial regulation of host-microbiome interactions, which has implications for our understanding of intestinal inflammation and disease.

Early in development, cells receive information about their location in an embryo through external cues, which they must interpret to output specific transcriptional responses required for the proper development of the body structures at each position. The same set of signaling cues can give rise to a range of cell fates depending on the specific timing and concentration of the signal, thus the delivery dynamics of an input are essential to inform a cell’s response. Despite their importance in development, these dynamics are difficult to study as small changes can have multi-faceted effects, and signaling pathways often involve complex feedback mechanisms and redundancy in regulation. However, the use of optogenetics to manipulate the amount of signal delivered through precisely timed light inputs offers a novel approach to investigate complicated regulatory networks and directly examine how signaling dynamics impact transcriptional outputs. One system in which these signaling dynamics are incredibly important, yet incompletely understood, is the terminal extracellular signal-regulated kinase (Erk) network in the Drosophila embryo. Early in development, a very low level of Erk activation patterns a narrow stripe of Abdominal-B (Abd-B) expression near the embryo’s posterior, which is required to form posterior spiracle “tail structures” in the adult fly. Abd-B expression requires tailless (tll), a direct Erk target gene and known transcriptional repressor, but it is unclear how this repressor mediates activation of Abd-B expression. In this thesis, I took advantage of the power of optogenetics to dissect the regulatory network that produces a stripe at the correct time in development and position along the embryo’s anterior-posterior axis. I applied specific optogenetic Erk inputs to embryos and then measured the effects on tll and Abd-B expression. I also built new MS2 biosensors for measuring endogenous Abd-B transcription in live embryos to characterize its expression dynamics in relation to tll. I found that Abd-B transcription is promoted by low levels of tll but repressed by higher levels of tll. To make sense of this tll concentration-dependent activation to repression switch, I used optogenetic stimulation to further examine the regulatory relationships between tll, Abd-B, and the gap genes, hypothesizing that a gap gene might be the missing link between a low level of tll expression and activation of Abd-B. I characterized the gap gene giant (gt) as a direct Abd-B repressor and found that expression of tll at even low levels silences gt expression. These findings informed a model for Abd-B regulation in which low levels of tll repress gt to promote Abd-B expression in a narrow stripe. The borders of the stripe are set through repression by gt at the anterior and high levels of tll at the posterior. Overall, this thesis offers a mechanism through which a precisely low level of a signaling input can produce a particular developmental fate.

The development of the immune system begins in utero and maternal exposures to pathogens during pregnancy and lactation influence long-term immunity in offspring, impacting their life-long susceptibility to infection and inflammation. The hygiene hypothesis suggests that modern hygiene practices may hinder proper immune training, raising the risk of immune disorders. Building on this, my thesis investigates how maternal exposure to helminths enhances antiviral immunity in offspring. Specifically, I examined the effects of maternal helminth infection on offspring responses to influenza by monitoring clinical symptoms, immune responses, and inflammatory markers following infection. My findings show that a maternal helminth infection confers protection against influenza in offspring by reducing viral load and lung inflammation. I also found that treatment with the helminth-derived metabolite indole-3-propionate (IPA) recapitulates these protective effects, although at a smaller magnitude. This confirms that the maternal gut microbiota acts as an immune educator during early development. These results build on prior work in our lab showing similar protective effects against respiratory syncytial virus (RSV), indicating that maternal immune education of offspring is broadly important across respiratory viruses. My findings highlight the importance of how maternal microbial exposures influence offspring immune development and underscore the potential of microbiota-derived metabolites to promote tissue immunity. This research provides new insights into maternal-offspring immune education and its implications for maternal and child health in an increasingly hygienic world.

During mitosis, microtubules make up the structural backbone of the mitotic spindle and provide the machinery to segregate chromosomes. The formation of new microtubules (i.e. microtubule nucleation) can happen at centrosomes, near kinetochores of chromosomes, and from pre-existing microtubules (i.e. branching microtubule nucleation) downstream of RanGTP near chromosomes. Branching microtubule nucleation contributes to a majority of microtubules in the mitotic spindle of many cell types and depends on Ran-regulated spindle assembly factors (SAFs) to recruit the universal microtubule template, gTuRC. XRHAMM (Xenopus laevis analog of human RHAMM) is a microtubule associated protein required for early mitotic spindle assembly implied to interact with TPX2 – an essential Ran-regulated SAF – and gTuRC; however, how XHRAMM contributes to mitotic spindle assembly remains to be uncovered. To start addressing this question, my research is focused on if and how XHRAMM is involved in RanGTP-dependent branching microtubule nucleation. Through pull-down assays, I found full-length XRHAMM directly binds to TPX2. To assess how these proteins could be interacting on the microtubule, I performed sequential microtubule binding assays, but my results are inconclusive as to whether TPX2 is recruiting XRHAMM to the microtubule. Also, through pull-down assays, I found the C-terminal domain of XRHAMM directly binds to gTuRC, whereas the N-terminal domain and full-length XRHAMM do not bind to gTuRC, potentially indicating XRHAMM autoinhibition. These results suggest XRHAMM is part of a TPX2-XRHAMM-NEDD1-gTuRC complex and may have a role in branching microtubule nucleation. Understanding XRHAMM’s binding partners and function is important to explain mitotic spindle assembly, and how overexpression of human RHAMM is connected to various forms of cancer.

Hepatitis B Virus (HBV), the major etiologic agent of acute and chronic hepatitis and end-stage liver disease, constitutes a major global health crisis, with over 296 million chronic carriers worldwide. HBV infection can be prevented through prophylactic vaccination, but currently, there is no cure for the disease due to the persistence of the highly stable covalently closed circular DNA (cccDNA). cccDNA serves as the template for the majority of HBV transcripts and its persistence is the root cause for HBV chronicity. By hijacking host DNA repair machinery, cccDNA is formed from lesion-bearing viral relaxed circular DNA (rcDNA), which enters the cell as part of the HBV virion. Our lab has previously identified 5 host factors sufficient for cccDNA biogenesis in vitro. However, in cellulo, it is conceivable that other host factors and redundant pathways may function in this process. In this thesis, we aimed to define all critical host factors essential for rcDNA to cccDNA conversion in cellulo. We first established a CRISPRi platform to downregulate host factors of interest and study its impact on HBV infection. We then validated this platform by silencing DNA Damage Binding Protein 1 (DDB1), a host factor necessary for robust HBV infection, and observed a robust decrease in HBV infection. We utilized this platform to study the impact of silencing 4 out of the 5 factors previously demonstrated to be sufficient for cccDNA biogenesis in vitro as well as several host cellular polymerases. Finally, we developed two novel reporters: one that monitors HBV infected cells by taking advantage of a key viral mechanism, and the other that monitors rcDNA repair by expression of a fluorescent reporter when repaired. Ultimately, the results from this thesis suggest the presence of redundant pathways and factors that function in rcDNA repair in cellulo, and the methodologies established will be utilized in future studies to define a comprehensive set of host factors key for rcDNA repair. These results thus will aid towards a better understanding of key steps in HBV infection and a curative treatment for HBV.

Animals sense signals from their environment to inform their behavior and decision-making. Many species have evolved to detect alarm cues released from injured or deceased conspecifics, triggering defensive responses that help them to avoid the same threats that led to the demise of other individuals in their population. Understanding the molecular nature of these signaling molecules and the methods of signal transduction provides insight into the mechanisms behind such behaviors. In Caenorhabditis elegans, the Murphy Lab identified a novel signaling molecule released from lysed worms that living conspecifics avoid. We termed this necrotaxis cue “Todstoff” (death substance). This thesis seeks to characterize the molecular identity of Todstoff, as well as its sensory signal transduction. Analysis of the Todstoff signal suggests that it is proteinaceous and smaller than 20 kDa in size, distinct from known ascaroside signals and alarm pheromones. Building upon previous work by the Murphy lab, which established that Todstoff is sensed by the ASH sensory neuron, I further elucidate the intracellular signal processing pathways involved in Todstoff detection. I demonstrate that necrotaxis requires G-protein-coupled receptor signaling, mediated by the ODR-3 G alpha subunit, as well as synaptic transmission via glutamatergic signaling. Finally, I found that C. elegans avoid lysate from other species of Caenorhabditis, but are attracted to protein precipitated from these worms, indicating that necrotaxis in response to related nematode species is likely directed by a signal separate from Todstoff. Together, the results of this thesis illustrate the existence of a novel, post-mortem inter- animal necrotaxis cue that promotes organismal longevity and survival.

Passive flight has evolved multiple times across different species through the use of gliding membranes called patagia, but there is little known about the development of these structures. Although there is existing research on mammal patagium development that provides insight into its tissue structure and cellular activity patterns, there is no similar data for other gliding organisms. This research aims to uncover the developmental mechanisms underlying patagium formation in reptiles. Specifically, I compare the development of the patagium of a gliding gecko, Hemidactylus platyurus, to the lateral trunk skin of its non-gliding relative, Hemidactylus turcicus. Using histological and fluorescent assays, I characterize the tissue composition, growth patterns, and cell density of developing gecko trunks across embryonic stages. These results reveal that H. platyurus patagium development is marked by the early formation of a dermal condensate of cells early in development, which persists and expands as the patagium grows and is absent in non-gliding trunk tissue. This pattern of increased cell density mirrors the development of the patagium primordium seen in marsupial sugar gliders, suggesting that there exist shared developmental features underlying patagium formation across distant lineages. Although attempts to detect patterns of cell proliferation and apoptosis in H. platyurus tissue were inconclusive, this work highlights potential early developmental constraints in the evolution of gliding membranes and offers new insights into how convergent structures repeatedly arise across vertebrates.

The human gut microbiome is made up of a diverse set of bacterial species that have the potential to impact biological processes such as drug metabolism. Gut bacterial species and their enzymes have been shown to metabolize some drugs through enzymatic conversion into their metabolites, known as microbiome-derived metabolism (MDM). One such drug is the immunosuppressant mycophenolate mofetil (MMF), commonly used in organ transplantation and known to be metabolized into its active metabolite, MPA, by bacterial esterases. However, the specific bacterial strains and their corresponding enzymes responsible for this metabolism remains unclear. This thesis identifies a bacterial strain isolate and an enzyme responsible for MMF metabolism by the gut microbiome. Using MDM assays, I screened six gut bacterial strain isolates for their ability to convert MMF to MPA, identifying the gut bacterial strain, Prevotella copri, as a robust metabolizer of MMF. To investigate which P. copri enzyme drove this metabolism, I heterologously expressed six P. copri-encoded esterase enzymes and similarly screened their ability to convert MMF to MPA. This revealed a P. copri carboxylesterase, PC5, as the primary enzyme responsible for MMF metabolism by the strain. To evaluate whether these findings of MMF metabolism in bacterial isolation predict MMF metabolism in gut microbial communities, I correlated P. copri relative abundance and PC5 abundance with MMF metabolism across 20 individual gut microbial communities. Neither P. copri nor PC5 significantly predicted MMF metabolism across gut microbial communities, suggesting that additional bacterial strains and enzymes also contribute to MMF metabolism when in complex microbial communities. These findings provide insight into the role of P. copri in MMF metabolism and highlight the need for further characterization of MMF metabolism within microbial communities to improve predictions of microbiome-driven drug metabolism.

The nucleolus is a multiphase nuclear condensate and the site of ribosome production in the cell. After ribosomal RNA (rRNA) is transcribed in the innermost phase of the nucleolus, it moves to the outer phases where it undergoes cleavage, modification, and folding events. This directional rRNA movement is critical to the export of correctly processed ribosomes to the cytoplasm. However, it is unclear how this directionality is established. Here, I provide evidence for my hypothesis that specific rRNA processing events (cleavage, modification, and folding) are required for directional rRNA movement through the nucleolar phases. Testing this hypothesis has previously been challenging due to a lack of tools capable of resolving where and when rRNA processing events occur in each nucleolar phase. Therefore, I co-develop* a genomic-imaging platform to map the cleavage, modification, and folding state of nascent rRNAs in time and space with base-pair resolution. Using this novel method, I* create a map of the rRNA processing steps occurring in each nucleolar phase. I* then systematically perturb specific rRNA processing steps and show their necessity to directional rRNA movement. Based on these findings, I propose a biological model for how rRNA processing enables directional rRNA movement and export of only correctly processed ribosomes to the cytoplasm. This work 1) provides novel mechanistic insight into how directional rRNA movement is maintained in the nucleolus and 2) establishes a novel method for studying RNA kinetics in the nucleolus and other cellular condensates in normal, perturbed, and diseased systems. *In collaboration with Sofia Quinodoz, Lifei Jiang, and Troy Comi.

Endocytosis is a vital cellular process during which vesicles form at the plasma membrane to internalize nutrients, signaling molecules, and transmembrane proteins. Bridging integrator 1 (BIN1) is a key protein that forms a helical polymer around the necks of budding vesicles and interacts with dynamin 2, a large GTPase responsible for vesicle scission. While BIN1’s recruitment and regulation of dynamin 2 is critical for endocytosis to occur, little is known about BIN1’s structure and domain organization to evaluate the mechanism by which it carries out its functions. Therefore, this study aims to use cryogenic electron microscopy (cryo-EM) to (1) determine the 3D structure of BIN1 on lipid tubules and (2) BIN1 in complex with dynamin 2 on lipid tubules to evaluate the structural basis of BIN1's mechanism of action during endocytosis. Our hypothesis is that BIN1’s src-homology 3 (SH3) domain is exposed and extended outwards from the membrane to enable efficient recruitment of dynamin 2’s proline rich domain (PRD). Additionally, we anticipate that BIN1 will promote dynamin 2’s GTPase activity by co-assembling in an intercalated pattern with its SH3 domain, stabilizing dynamin 2’s PRD to promote dimerization of its GTPase dimers. This study lays the groundwork for understanding the SH3-PRD interaction and explores optimization approaches for achieving high-resolution structural data. Ultimately, our findings will provide valuable structural insights into the molecular coordination between BIN1 and dynamin 2 in membrane remodeling during endocytosis.

The symbiotic relationship between bacteria and humans can be readily observed in the human gastrointestinal (GI) tract, where gut bacteria play a role in immune system functions and metabolism. Healthy individuals have diverse and stable microbiomes, and disruption of this microbiome composition correlates with disease. To understand bacterial involvement in human processes and elucidate microbe-host interactions, it is important to study how bacteria regulate their own behavior. Small regulatory RNAs (sRNAs) are one of the mechanisms by which bacteria control gene expression and respond to environmental stimuli. Cis-encoded antisense sRNAs bind mRNA targets with full complementarity and post-transcriptionally regulate mRNA translation. Since few asRNAs have been experimentally confirmed, this study computationally selected two potential mRNA-asRNA pairs from Bacteroides thetaiotaomicron using sequencing data of bacterial RNA transcripts from human fecal samples. A previously designed computational pipeline annotated noncoding RNAs in this metatranscriptomic dataset by potential gene, identifying the selected sod and cshA genes as encoding superoxide dismutase [Mn/Fe] and ATP-dependent RNA helicase CshA, respectively. This work establishes a model for experimentally identifying biologically relevant asRNAs by co-expressing asRNAs and their mRNA targets in Escherichia coli.

Obesity has reached epidemic proportions in the United States and is associated with an increased risk of several cancers, including postmenopausal breast cancer. Regardless of menopausal status, obesity leads to worsened breast cancer progression due to its associations with systemic metabolism and inflammation, which affect the tumor microenvironment. Dietary interventions such as fasting, calorie restriction, and bariatric surgery have shown promise in improving outcomes and response to treatment for obese and lean patients, though their invasiveness and poor patient compliance limit clinical utility. Incretin agonist drugs, once confined to the treatment of Type 2 diabetes, have come to the forefront of public conversation for their remarkable ability to affect weight loss in overweight or obese patients; Tirzepatide, a dual-agonist of GLP-1 and GIP receptors, has been shown to be even more effective than earlier approved Semaglutide (GLP-1 agonist alone). To evaluate Tirzepatide as a metabolic therapy for breast cancer, a clinically relevant mouse model resembling the comorbidities associated with diet-induced obesity (DIO) is required. However, female DIO models are understudied due to delayed onset, high variability, and protective profiles of female mice compared to males. To combat this, fructose was administered in combination with a high-fat diet (HFD) for both its relevance to the Western diet and its indirect effect on weight gain by encouraging overconsumption. Fructose supplementation accelerated the onset and severity of obesity phenotypes in C57BL/6 and BALB/c models. FVB/N GEM models also responded well to HFD-feeding alone. C57BL/6 and BALB/c models were chosen for further tumor induction studies with EO771 or 4T07 triple-negative cell lines. HFD or HFD plus fructose (HFD + F) mice were given Tirzepatide injections either pre- or post-tumor inoculation. As well, mice on a purified control diet (PC) were similarly treated to observe the effects of the drug in a lean model. Though the experiment is ongoing, obese mice (HFD or HFD + F) treated with Tirzepatide show significant reductions in tumor burden, with outcomes comparable to lean controls. These findings support the utility of fructose in female DIO modeling and future investigations into Tirzepatide as a metabolic adjuvant therapy.

Pyrimidine antimetabolites such as 5-fluorouracil and Trifluridine have long been used in cancer therapy due to their ability to disrupt nucleotide metabolism, inhibit thymidylate synthase (TS), and incorporate into DNA and RNA. However, the precise impact of structural modifications on their pharmacological behavior and cellular responses remains incompletely understood. Here, we systematically investigated the metabolic fate, DNA/RNA incorporation, and cytotoxic mechanisms of a panel of clinically relevant thymidine analogs and their 4’-sulfur-substituted counterparts across multiple cancer cell lines. Using several liquid chromatography mass spectrometry methods, immunoblotting, and both in vitro and in vivo cancer models, we identify key structure–function relationships that govern analog activity. We find that sulfur substitution enhances metabolic stability by reducing thymidine phosphorylase (TP)–mediated degradation, enabling prolonged TS inhibition, sustained DNA damage, and apoptosis. Specifically, the efficacy of thio-analogs correlates more strongly with the persistence of DNA damage response activation than with peak analog incorporation. Careful kinetic profiling and supplementation studies further reveal that sulfur-modified analogs disrupt both thymidine and uridine biosynthesis pathways, suggesting broader metabolic engagement than their parent compounds. We report that in vitro, 4’-thio-floxuridine did not demonstrate superior efficacy to the parent compound, floxuridine. In contrast, 4’-thio-trifluridine demonstrates superior efficacy to the parent oxygen containing compound, trifluridine, as well as to all other thymidine analogs tested. The compound is well tolerated in vivo and exhibits a longer half-life than conventional pyrimidine analogs. Taken together, our findings establish thio-nucleosides as stabilized derivatives that are mechanistically distinct with unique cellular and metabolic signatures, offering a framework for the design of next-generation antimetabolite therapies.

Quorum sensing (QS) is a process of bacterial cell-to-cell communication that allows bacteria to coordinate group behaviors and serves as a trigger for bacteriophage lysis. Bacteriophages monitor host bacterial QS signals, called autoinducers (AIs); in response to AI accumulation, phages produce proteins known as smORFs which sequester and inactivate lytic repressor proteins (cI), resulting in lysis at high host cell density. The exact mechanisms underlying this process in vivo are unclear, particularly in polylysogens. Previous work has shown that cI repressor proteins are specific to their partner smORFs and vice versa, despite the predicted similarities between cI proteins. This thesis aims to uncover the molecular basis of binding specificity displayed between cI proteins and their smORF partners. I first purified cI protein constructs of the full length and truncated cI proteins of two bacteriophage phage species. I then performed a co-elution assay using these purified protein constructs and determined that the N terminal domain of both species retained binding activity and specificity for its partner smORF protein. Furthermore, I developed a bioluminescence assay to test libraries of mutagenized cI proteins for altered smORF binding activity. The identification of specificity in this binding domain has implications for phage-phage and phage-bacteria interactions in the environment.

Preterm birth is the leading cause of infant mortality worldwide and is a strong risk factor for serious short and long-term health concerns for the child. However, the cause of many preterm births is unknown. Two-thirds of preterm births occur spontaneously, and half of those occur in low-risk pregnancies. Commonly implicated pathways to preterm birth include infection or inflammation, stress, and uterine overdistention. Many studies attempting to target idiopathic spontaneous preterm birth fail to account for confounding maternal medical conditions that can lead to premature labour and delivery, therefore conflating idiopathic cases with those driven by known risk factors. This meta-analysis identified genetic associations with idiopathic spontaneous preterm birth by ruling out genetic loci associated with the high-risk pregnancy conditions of preeclampsia, gestational diabetes mellitus, hypertension, type I diabetes, and placental abruption. The implicated gene loci were subjected to a gene ontology and pathway analysis, revealing three overrepresented functional groupings: neuronal synapses, calcium and ion homeostasis, and metabolism and endocrine signaling. Analysis of these functional groupings and the implicated genes indicates that the placental environment; calcium dysregulation in the myometrium; and possibly maternal mental health contribute to spontaneous preterm birth. Additionally, this thesis highlights the significance of immune biomolecules in the sequence of events that lead to preterm labor. Overall, this thesis represents a novel approach to understanding the pathology of idiopathic spontaneous preterm birth and establishes associations that should be further investigated. The results of this study identify potential diagnostic biomarkers that may help reduce the rates of preterm birth through early detection and intervention.

The life cycle of sexually reproducing organisms relies on the distinct development of germ cells. Specialized ribonucleoprotein assemblies called germline granules are essential for this development across the animal kingdom, as they facilitate the inheritance, storage, and regulation of RNAs to establish a unique gene expression profile. In Drosophila, different types of germline granules have been described at various stages of development, from gametogenesis through specification of the primordial germ cells (PGCs) in early embryogenesis. While granules are known to exist throughout embryogenesis, their role during the late stages remains unclear. I investigate this function by knocking down several transcripts that have localization patterns consistent with late-stage embryonic granules. I show that Nucleoplasmin-like protein (Nlp) is essential for germline development, beginning with PGC migration and gonad formation and persisting through gamete production. Ultimately, disrupting Nlp function results in sex-independent infertility. I also find that Nlp localizes in puncta in the gonadal germ cells that are distinct from previously described germline granules. These results suggest that new, Nlp-containing germline granules are critical for germline development during the late stages of embryogenesis, establishing a continuum of germline granules throughout the Drosophila life cycle.

Influenza A virus (IAV) replicates its genome in the nucleus of a host cell, producing viral RNA (vRNA) transcripts that are packaged and released into neighboring cells. While these vRNA transcripts are typically full-length RNA molecules produced by normal IAV replication, oftentimes the replication machinery may also produce aberrant, shorter RNA fragments, including mini viral RNAs (mvRNAs). Some mvRNAs have a high propensity to act as innate immune agonists, triggering retinoic acid-inducible gene I (RIG-I) activation, downstream innate immune signaling, and cytokine storms, a conserved pathological profile of pandemic-causing IAV strains. While mvRNA-induced RIG-I activation is well-studied, it remains unclear how mvRNAs produced in the nucleus are transported to the cytoplasm where they exert their immunostimulatory effects. Here, we identify a potential trafficking route by treating IAV infected A549 cells with Leptomycin B (LMB), a Chromosomal Maintenance Region 1 (CRM1) inhibitor, and compare their mvRNA production levels and innate immune activation patterns with untreated, infected cells. Infected cells treated with LMB had a steeper distribution skew of mvRNA across the nucleus and cytoplasm, with strong nuclear sequestration of mvRNAs. Although high technical variability limited the statistical power of this observation, the trend seen may point to the role of the CRM1 pathway in mediating mvRNA nuclear export during IAV infection. Luciferase based assays of IFN-β promoter expression revealed a significant fold reduction in IFN-β activity in LMB-treated cells, confirming a potential role of the CRM1 pathway in increasing the accessibility of the cytoplasm, and its innate immune receptors, i.e. RIG-I, to mvRNAs. RPISeq, HDOCK analysis, and ChimeraX predicted CRM1 as a potential direct binding partner for mvRNA, suggesting that mvRNA and its full-length vRNA counterpart are trafficked via mechanistically distinct, yet CRM1-mediated nuclear export routes. Put together, this research provides preliminary insight into IAV mvRNA transport, and paves the way for the development of both, therapeutic agents that optimize the balance between CRM1-pathway inhibition and cytotoxicity, as well as wide-ranging, anti-inflammatory molecules that lower the cytokine storm disease pathology across several viral contexts.