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.

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.

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.

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.

Coronary artery disease (CAD) is the most common type of heart disease, with about 20.5 million adults having CAD in the U.S, as of 2023. This disease is caused by plaque buildup beneath the endothelium of the arteries that supply blood to the heart, called coronary arteries. Currently, durable polymer drug eluting stents (DP-DES) are most typically used in treatment of this disease, due to lower rates of complications compared to older generation stents. However, fully biodegradable stents have recently been developed and studied within preclinical studies and promising clinical trials. In order to evaluate the association between biodegradable polymer stent use and clinical outcomes, as compared to DP-DES use, a meta-analysis of clinical trials and data available in respect to the in-stent restenosis and target lesion failure (TLF) rates of biodegradable stents, and of DP-DESs, was conducted. Critically, this study has found that the efficacy of fully biodegradable stents is not significantly different from the more commonly used DP-DESs, when analyzed in the general patient population, and in the diabetic patient population. In addition, several novel biodegradable polymers were identified, which demonstrate promise in being used to create new and more effective biodegradable stents. Overall, the findings of this study have highlighted the potential of fully biodegradable stents, encouraging future research in the development and clinical testing of new biodegradable stents.

Symmetry breaking events in development are key to establishing new cell fates. In mammalian embryos, the first cell fate decision, taking place during preimplantation development, gives rise to trophectoderm (TE) and inner cell mass (ICM) cells. Without TE formation, an embryo cannot implant into the uterus, leading to its arrest. The formation and inheritance of an apical domain plays a key role in the first decision and is necessary for TE formation. The inheritance of the apical domain has been previously explained using the polarity model, in which the apical domain can be inherited asymmetrically by daughter cells during division. Interestingly, recent work observed disassembly of the apical domain prior to cytokinesis, followed by de novo formation on exposed cell surfaces of daughter cells. Disassembly and reformation, however, is at odds with asymmetric apical domain formation taking place even when cells are allowed to divide in isolation (i.e. without any cell-cell contact). In this project, I used live imaging of isolated polar blastomeres with Ezrin-neonGreen-labeled-apical domains to understand apical domain behavior independent of cell-cell adhesions in both non-dividing and dividing cells. I found that isolated, non-dividing polar cells at the 8-cell, 16-cell, and 32-cell stages were all able to autonomously maintain an apical domain, meaning that polarity is a cell-intrinsic property at these stages of development. The apical domain exhibited spreading behaviors at the 8, but not 16 or 32 cell interphases. Furthermore, during the 8-16 and 16-32 cell divisions, the apical domain exhibited spreading behaviors towards the cytokinetic furrow, which coincided with re-localization of actin (labeled with Lifeact-mCherry) form the apical domain to the cytokinetic furrow. While this actin re-localization-induced spreading occurred at both the 8-16 and 16-32 divisions, it took place only briefly before the division and resulted in limited spreading of the apical domain. Consequently, most 16-32 divisions resulted in asymmetric divisions with only one daughter inheriting the apical domain. In contrast, 8-16 divisions were either asymmetric or symmetric, with symmetric divisions resulting from the apical domain spreading extensively during the 8-cell stage interphase.

In summary, these findings indicate a relationship between the actin-cytoskeleton and apical domain behavior during cell divisions and highlight stage specific behaviors of the apical domain that influence its inheritance in the early preimplantation embryo.

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.

The final event in vesicular trafficking is membrane fusion, facilitated by specific SNARE proteins and SM proteins, which function as SNARE chaperones. A vesicle SNARE and three unique target membrane SNAREs (Q-SNAREs) form membrane-bridging complexes via interactions between their SNARE motifs thereby driving membrane fusion. Some Q-SNAREs form self-interactions via oligomerization, which can inhibit their ability to interact with other SNARE proteins and prevent fusion events. However, the mechanism driving Q-SNARE oligomerization is relatively unknown. Interestingly, SM protein Sly1 prevents Ufe1 from oligomerizing, holding it as a monomer. Here, we show that the removal of the SNARE motif from Q-SNARE Ufe1 prevents its oligomerization. The SNARE motif of Ufe1 forms oligomers in size exclusion chromatography in a concentration-dependent manner but remains monomeric in analytical ultracentrifugation. Our findings suggest that the SNARE motif contributes to Ufe1 self-oligomerization, but additional regions outside the motif are likely necessary to stabilize these interactions. Crystals of a Sly1-Ufe1 complex lacking the SNARE motif diffracted to a resolution of 10 Å by X-ray crystallography. Future optimization of crystallization conditions or structural studies by cryo-EM may clarify how Sly1 inhibits Ufe1 oligomerization and whether this regulation depends on the SNARE motif as well as other structural features of Ufe1. Such investigations will further our understanding of how SM proteins coordinate with Q-SNAREs to promote SNARE complex assembly.

The robustness of a developmental process is defined as the ability of a system to produce a consistent phenotype, despite high variability in input signals. This variability is often absorbed by signal pathways that temper the received signal into an “average” range, which keeps the system’s developmental trajectory leveled at wildtype phenotypes. When signal intensity exceeds the thresholds of this buffering effect, the system’s robustness breaks down, and development becomes disordered. Studying developmental robustness in the Drosophila melanogaster tracheal system has many applications to understanding how human developmental disorders occur. Drosophila tracheal development is dependent upon the FGF/FGFR signaling pathway. By perturbing tracheal development through the use of a heterozygous FGF background, terminal cell specification was discovered to be differentially sensitive to decreases in FGF ligand across metameres of the animal. Additionally, a novel FGFR activity sensor was confirmed to accurately identify active FGFRs. This sensor showed that FGFR activity levels across metameres correlate with these newly found differential metameric rates of terminal cell specification, the differentiation event controlled by FGFR activity. A model was constructed to show how developing tracheal metameres closer to the spiracles of the animal receive less FGF-dependent FGFR activity than metameres closer to the center of the animal. This makes them more susceptible to failure at specifying a terminal cell than middle metameres. This study demonstrates how robustness can fluctuate within the same developing system, leading to areas of the organism that are more prone to fail in achieving proper development.

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.

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.

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.

EGF presents on the cell surface as a pro-ligand, which can be cleaved by proteases such as ADAM10/17. Free EGF ligands can then bind to the EGFR, which allows for EGFR dimerization that induces signal transduction and cellular responses in receiving cells. EGFR activation can be detected via an ERK-KTR biosensor that changes its localization in response to the activation of ERK a downstream EGFR signaling transduction. On a tissue scale, ERK activation has been observed in waves or pulses that can travel tens of micrometers per hour across a sheet of cells. A synthetic extracellular signaling circuit was designed to model a potential mechanism for how these dynamic ERK waves form. To do this, three key signaling components were designed – surface expressed TEVp, synthetic pro-ligand with two receptor binding sites, and an engineered EGFR-like receptor with a synthetic extracellular binding domain. Each component was cloned and expressed in HEK293T cells, where proper localization to the cell surface was verified via confocal microscopy. Surface expressed TEVp was shown to maintain its ability to cleave substrates using the eNRGies biosensor, which serves as a generalized indicator for extracellular shedding via translocation of a fluorescent protein. Additionally, synthetic receptors properly dimerize in response to the addition of purified ligand. Signal transduction in response to receptor dimerization was detected using the ERK-KTR. Ultimately, with the key components successfully constructed, their integration into a complete synthetic signaling circuit would establish a model for the analysis of ERK wave propagation.

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.

Transmembrane receptor proteins are essential for translating extracellular signals into programmed cellular behaviors. Synthetic Receptor Tyrosine Kinases (RTKs) bearing non-native extra- and intracellular domains have emerged as powerful tools for activating novel cell behaviors in response to specified inputs. Endogenous RTK signaling achieves a level of precision in both space and time that synthetic RTKs have yet to display, owing largely to their tight regulation by Protein Tyrosine Phosphatases (PTPs). To achieve precise, tunable signaling with synthetic RTKs, we engineered synthetic PTPs that function as programmable regulators of Epidermal Growth Factor Receptor (EGFR) phosphorylation. We first generated a panel of synPTPs that bind to an auxiliary phosphotyrosine motif on the tail of EGFR in response to its activation with a ligand. We found some of these synPTPs dephosphorylate the auxiliary motif near-completely, but the EGFR-driven binding of these synPTPs perpetually attenuated EGFR phosphorylation. We then generated a light-inducible optoPTP platform for recruiting phosphatase catalytic domains to EGFR independently of EGFR activation. optoPTP1B displays light-inducible dephosphorylation of EGFR on the scale of minutes. optoPTP1B did not, however, completely dephosphorylate cellular EGFR, and we observed no significant change to EGFR-driven ERK activation in response to light-induced PTP recruitment. Additionally, we did not observe any off-target disruption of PDGFR signaling by optoPTP1B. Our synPTP and optoPTP systems demonstrate that engineering the recruitment of phosphatase catalytic domains to the tail of an RTK is a promising strategy for precisely controlling the dynamics of RTK signaling in both space and time.

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.

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.

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.