Browsing by Author "Palmer, Kelli"
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Item Analysis of the Transcriptomics, Physiology, and Invasiveness of Uropathogenic and Non-pathogenic Escherichia Coli(August 2023) Hogins, Jacob Buel 1997-; Reitzer, Lawrence J.; D'Arcy, Sheena; Zimmern, Philippe; Spiro, Stephen; Palmer, KelliEscherichia coli is a disease-causing species that can be divided into several phylogenetic groups (PGs). The B2 group of the Clermont classification scheme causes up to 65% of E. coli caused urinary tract infections (UTIs), with the remaining 35% caused by the summation of groups A, B1, and D. A common set of virulence genes has not been identified. To determine the features of B2 strains that contribute to virulence, I analyzed 35 strains by comparative RNA sequencing. The strains were non-pathogenic and pathogenic, i.e., isolated from individuals with a UTI, from groups A, B2 and D. I established transcriptomic differences of core gene expression between groups B2 and A/D that involve genes for all aspects of macromolecular synthesis, pathways of energy generation, and environment-sensing transcription factors. I propose that these differences are responsible for the B2 group’s enhanced virulence potential. Virulence requires attachment to and the formation of intracellular bacterial colonies within epithelial cells. I found that five of five uropathogenic and one of three nonpathogenic B2 strains had 25-50 times more intracellular colonies than strains from either groups A or D. My analyses of bacterial appendages and the receptors for bacteria on epithelial cells show multiple entry mechanisms through which E. coli can utilize to invade human urothelium. A UTI involves multiple environments where bacteria must grow, including urine. Analyzing growth in urine can be difficult as urine is a highly unreproducible mixture, so pools of human urine are commonly used to minimize individual variation. I analyzed how the bacteria respond to growth in an individual’s urine and in pools of urine from multiple classes of patients. I identified pooled urine as a highly variable environment that does not necessarily mimic the average growth of urine from individual patients. These results suggest that better urine mimics are needed for future studies and that variations in urinary nutrient content could affect the host- pathogen interaction and the outcome of the infection. In the same study, I also observed that group B2 uropathogens are better at obtaining low levels of nutrients. Genetic analyses of uropathogens are difficult because many are resistant to a common form of genetic exchange mediated by a virus, known as transduction. We developed a method by which uropathogens will accept the transducing phage P1 and subsequently allow for alterations of the pathogenic chromosome. With the use of this method, metabolic mutants were constructed, and the use of the genes were studied in motility, another prominent virulence function in uropathogens. This led to the identification of a mechanism of motility, using the pili appendage, previously thought not to be used by E. coli. The regulation of this motility mechanism and alanine synthesis were also studied in part to help identify novel treatment avenues for UTIs. In summary, I provided evidence that suggests the basis for group B2’s virulence: Group B2 strains have an altered transcriptome, and increased invasiveness, metabolism, and nutrient acquisition capabilities. A new approach to the transduction protocol allows genetic alterations that allows easier genetic analysis of these prominent pathogens.Item Beyond Nitrogen Limitation – Novel Mechanisms Regulating Glutamine Synthetase Expression in Escherichia coli and a Possible Alternative Pathway of Glutamine Synthesis(May 2023) Urs, Karthik; Reitzer, Lawrence J.; Conlon, Ronan; Palmer, Kelli; Spiro, Stephen; Kim, Tae HoonThe expression of glnA (ammonia-assimilating glutamine synthetase) is high for uropathogenic E. coli grown in urine. Because glnA is part of an operon that codes for regulators of the nitrogen-regulated (Ntr) response, high glnA expression has been interpreted to suggest nitrogen limitation, which is unexpected because of the high urinary ammonia concentration which should suppress glnA expression. We present evidence that glnA expression does not result from nitrogen limitation. First, in the presence of ammonia, urea induced expression of glnA from the cAMP receptor protein (Crp)- dependent glnAp1 promoter, which circumvents control from the nitrogen-regulated glnAp2 promoter. This urea effect on glnA expression has not been previously described. Second, the most abundant amino acids in urine inhibited GS activity, based on reversal of the inhibition by glutamate and glutamine, and increased glnA expression. The relevance of these inhibitory amino acids in natural environments has not been previously demonstrated. Third, urea and the inhibitory amino acids did not induce other Ntr genes, i.e., high glnA expression can be independent of other Ntr genes. Finally, the urea- dependent induction did not result in GlnA synthesis because of a previously undescribed translational control. We conclude that glnA expression in urea-containing environments does not imply growth rate-limiting nitrogen restriction and is consistent with rapid growth of uropathogenic E. coli. ΔglnA mutants are glutamine auxotrophs, however, UTI89ΔglnA mutants, were unexpectedly able to grow in a synthetic urine medium. This phenotype was conditional and required the presence of both glutamate and ammonia, the substrates for glutamine synthetase. Additionally, overexpression of proA, which is part of the proline biosynthesis pathway, whose product catalyzes the formation of glutamate-5-semialdehyde, improved growth. In contrast, an increase in proC expression, which directs pyrroline-5-carboxylate, the cyclized form of glutamate-5-semialdehyde to proline, impaired growth. We describe a possible alternate route of glutamine production in these mutants involving the enzymes of proline synthesis and the substrates, glutamate and ammonia, via the intermediate glutamate-5-semialdehyde. The pathway may be facilitated by a putative secondary activity of the ProA enzyme – the reduction of a proposed imine intermediate to ultimately form glutamine. Members of the B2 clade of E. coli exhibit high glnA transcript levels in nitrogen-rich glucose-tryptone medium. This increased expression did not translate to increased protein production and enzyme activity as evidenced by low translation levels and glutamine synthetase activity in these strains. Transcriptomic analysis revealed an inverse correlation between phoB, a phosphate-dependent transcriptional regulator, and glnA expression. Consistent with this, overexpression of phoB, reduced glnA transcription levels. The effect was not a complete repression of glnA transcription. Additionally, translational expression appeared to be stabilized upon phoB overexpression. Our findings suggest a novel mechanism of glnA regulation at both the transcriptional and translational levels that involves PhoB operating either directly or indirectly, and possibly in combination with other unidentified factors.Item Chronic IL-1 Exposure Drives Prostate Cancer Progression(May 2023) Dahl, Haley C 03/03/1993-; Delk, Nikki; Kolodrubetz, Michael; Palmer, Kelli; Kim, Tae Hoon; Winkler, Duane D.Prostate cancer (PCa) is the second most common cause of cancer-related deaths among American men. Androgen Receptor (AR) transcriptional activity is required for PCa tumor growth. Androgens regulate normal prostate tissue growth and differentiation via androgen receptor (AR) activation. Due to the role of androgens in prostate cancer, androgen-deprivation therapy (ADT), either through chemical or surgical castration or the use of anti-androgens, has become the standard therapy. However, ~10-20% of PCa patients will develop treatment resistance, referred to as castration-resistant PCa (CRPC). One mechanism of CRPC is the loss of dependence on AR for cell growth and survival. As such, over 84% of CRPC patients will develop incurable, lethal bone metastasis. Thus, it is important to uncover the mechanisms that drive CPRC. IL-1 is elevated in PCa patient tissue and serum and is associated with disease progression and metastasis. IL-1 is clinically relevant, but the role of IL-1 in CRPC development is not fully elucidated. Chronic inflammation is a known hallmark of cancer initiation and progression. Therefore, we exposed the cancer cells to IL-1 for several months to make the chronic IL-1 sublines, LNas1 and LNbs1. The chronic IL-1 sublines restore AR and AR activity but evolve AR independence and acquire a constitutive p62-KEAP1 interaction. p62 is a multi-domain, multifunctional pro-survival protein that mediates autophagic turnover of damaged proteins and organelles, promotes NF-κB inflammatory signaling and induces NRF2 antioxidant signaling through its binding to and sequestering of KEAP1 from NRF2. Despite constitutive p62- KEAP1 binding, the chronic IL-1 sublines only show elevated NRF2 signaling in the NRF2 target genes, HMOX1 and GCLC. Furthermore, the chronic IL-1 sublines evolve insensitivity to IL-1 extracellular signaling and, thus, do not activate NF-κB nor NRF2 signaling. Thus, the regulation and function of the constitutive p62-KEAP1 interaction in the chronic IL-1 sublines is novel. To investigate the regulation and function of the constitutive p62-KEAP1 interaction in PCa cells chronically exposed to IL-1, I dissected p62-KEAP1 regulation and function under the oxidative stress-inducing stimulus, androgen deprivation. Under androgen deplete conditions, there is attenuation of HMOX1 and overexpression of GCLC in the chronic IL-1 sublines. This suggests that there is active but aberrant NRF2 signaling that may allow these cells to be primed to withstand oxidative stress. Furthermore, knockdown of KEAP1 results in upregulation of HMOX1 suggesting that KEAP1 negatively regulates HMOX1. Both HMOX1 and GCLC function as an antioxidant to attenuate iron-induced lipid ROS and thus regulate iron- dependent cell death known as ferroptosis. Based on what we have found, we hypothesize that the sublines activate a novel pathway that primes them to withstand ferroptosis-induced cell death.Item Computational Simulations of DNA Associated Enzymes Using Both Quantum and Molecular Mechanics Methods(May 2023) Berger, Madison B 04/24/1996-; Cisneros, G. Andres; Palmer, Kelli; Meloni, Gabriele; Torabifard, Hedieh; Slinker, Jason D.This dissertation concerns the development and usage of computational methods to explore a variety of biological systems involved in DNA replication or repair. First, we improve the polarization component of our group’s quantum mechanics/molecular mechanics (QM/MM) software, Layered Interacting Chemical Models (LICHEM), by incorporating the effects of induced dipoles from the MM into the Hamiltonian. We found that one round of induced dipoles does not improve the interaction energy between the QM and MM regions. The polarization catastrophe results in much higher interaction energies. Second, we explore the effects of mutations on the β-clamp of DNA Polymerase III (E. coli). We used molecular dynamics (MD) to simulate 6 different variants and found that there is a large reduction in the dynamic motion of all variants as well as a change in the networks formed between the domains of each monomer. Third, we used MD and QM/MM methods to study the effects of 2 different mutations on the β-clamp. This work simulated both the β-clamp and the Pol III core subunits including α, ϵ and θ. These distal mutations change the DNA conformation found within the system altering the exonuclease reaction within the ϵ subunit. Fourth, we used MD to determine how the drug, remdesivir, and two analogues affect the structure of RNA-dependent RNA Polymerase (RdRp) when saturated throughout the dsRNA. Incorporation of this drug along the RNA chain was found to have lead to an overall destabilization of the polymerase. Additionally, remdesivir and the two analogues studied had higher binding affinities than the natural substrate indicating their effectiveness. Fifth, we explore the mechanism of primer synthesis in the CRISPR-Associated Primase Polymerase from Marinitoga piezophila. Here, we used SAPT0 calculations to determine the important components of the primer initiation complex. Last, we provide a review of all computational studies carried out on 2 families of Iron and α–ketoglutarate–Dependent enzymes. We also discuss new results for 2 enzymes not yet explored in the literature.Item Dentification of Novel Regulators of Nitrate Respiration in Paracoccus Denitrificans: Roles of DksA, (p)ppGpp, and RegAB(May 2023) Ray, Ashvini 1993-; Spiro, Stephen; Minkoff, Susan E.; Reitzer, Lawrence J.; De Nisco, Nicole; Palmer, KelliParacoccus denitrificans is a metabolically versatile Gram-negative alpha-proteobacterium that is used as a model organism for studying respiratory metabolism. One of the most notable features of P. denitrificans is its capacity to use nitrate or nitrogen oxides as terminal electron acceptors in the process of denitrification, which is the sequential conversion of nitrate (NO3-) to gaseous dinitrogen (N2). The P. denitrificans genome encodes three members of the DksA/TraR family of transcription factors, two of which appear to be bona fide DksA proteins (hence designated DksA1Pd and DksA2Pd). There is a single relA/spoT gene in the genome encoding a predicted bifunctional (p)ppGpp synthetase and hydrolase (designated RSH, for RelA/SpoT Homolog). Both DksA1Pd and DksA2Pd can rescue the amino acid auxotrophy of an Escherichia coli dksA mutant. Deletion of either dksA1 or relA/spoT eliminates the upregulation of expression of the periplasmic nitrate reductase (NAP) that is seen when cultures are grown on a reduced substrate such as butyrate. Thus, we conclude that DksA1Pd and (p)ppGpp activate nap transcription in response to growth on a reduced substrate and that these proteins therefore help to maintain redox homeostasis by activating a mechanism for the disposal of excess reductant. Moreover, we show that the redox- sensing RegAB two-component pair acts as a negative regulator of nap expression under anaerobic growth conditions. Under the same conditions, RegAB acts as a positive regulator of the expression of the membrane-associated nitrate reductase Nar, mediating reciprocal regulation of the two nitrate reductases that have distinct physiological roles. The dksA1 and relA/spoT genes are conditionally synthetically lethal; the double mutant has a null phenotype for growth on butyrate and other reduced substrates while growing normally on succinate and citrate. The flavohemoglobin gene hmp is modestly upregulated in dksA2 and relA/spoT mutants, which are also defective for biofilm formation (possibly because of enhanced scavenging of nitric oxide). In conclusion, we have successfully identified two functional DksA homologs in P. denitrificans - DksA1Pd and DksA2Pd, along with a (p)ppGpp synthetase homolog (RSH) and a two-component RegAB system. These regulators were previously unknown and found to be responsible for regulating the expression of enzymes involved in nitrate reduction and nitric oxide metabolism.Item Discovery and Targeting of Strict Glucose Reliance in Lung Squamous Cell Carcinoma and Taxonomic and Genomic Ecology of Recurrent Urinary Tract Infection(2020-04-21) Neugent, Michael Lee; Palmer, Kelli; De Nisco, NicoleTumors are dynamic metabolic systems which require highly augmented metabolic fluxes and nutrient needs to support cellular proliferation and physiological function. For many years, a central hallmark of tumor metabolism has emphasized a uniformly elevated aerobic glycolysis as a critical feature of tumorigenicity. This led to extensive efforts of targeting glycolysis in human cancers. However, clinical attempts to target glycolysis and glucose metabolism have proven to be challenging. Recent advancements revealing a high degree of metabolic heterogeneity and plasticity embedded among various human cancers may paint a new picture of metabolic targeting for cancer therapies with a renewed interest in glucose metabolism. Adenocarcinoma (ADC) and squamous cell carcinoma (SqCC) are the two predominant subtypes of non-small cell lung cancer (NSCLC) and are distinct in their histological, molecular and clinical presentation. However, metabolic signatures specific to individual NSCLC subtypes remain unknown. We show that a critical reliance on glycolysis renders lung SqCC vulnerable to glycolytic inhibition, while lung ADC exhibits significant glucose independence. Clinically, elevated GLUT1-mediated glycolysis in lung SqCC strongly correlates with high 18F-FDG uptake and poor prognosis. This previously undescribed metabolic heterogeneity of NSCLC subtypes implicates significant potential for the development of diagnostic, prognostic and targeted therapeutic strategies for lung SqCC, a cancer for which existing therapeutic options are clinically insufficient. Recent advances in the analysis of microbial communities colonizing the human body have identified a resident microbial community in the human urinary tract (UT). Compared to many other microbial niches, the human UT harbors a relatively low biomass. Studies have identified many genera and species that may constitute a core UT microbiome. However, the contribution of the UT microbiome to urinary tract infection (UTI) and recurrent UTI (rUTI) pathobiology is not yet clearly understood. Evidence suggests that commensal species within the UT and urogenital tract (UGT) microbiomes, such as Lactobacillus crispatus, may act to protect against uropathogen colonization. However, the mechanisms and fundamental biology of the UT and UGT microbiome-host relationship are not understood. Urinary tract infections (UTIs) are the most commonly reported infections in adult women and have high rates of recurrence, especially in postmenopausal women. Recurrent UTI (rUTI) greatly reduces quality of life, places a significant burden on the healthcare system, and contributes to antimicrobial resistance. Because treatment of rUTI by long-term antibiotic therapy is often ineffective or poorly tolerated in elderly women, new therapies must be developed. The molecular basis of rUTI, especially in postmenopausal women, has remained unclear because modeling rUTI in mice is difficult, and human data are limited. Invasion of the urothelium and induction of host inflammation are hypothesized to be key mechanisms by which bacterial pathogens cause rUTI. We provide direct evidence that bacteria reside in the bladder wall of postmenopausal rUTI patients and that diverse bacterial species can be isolated from the bladder tissue. These data provide conclusive evidence that bacteria invade the human urothelium and suggest that diverse bacterial species and the adaptive immune response play important roles in rUTI in humans. The microbial ecology associated with rUTI remains uncharacterized. The ability to measure and quantify the composition of the UT microbiome has been enabled through the development of next generation sequencing and bioinformatic platforms that allow for the unbiased detection of resident microbial DNA. While numerous studies have used 16S rRNA amplicon sequencing to taxonomically profile the microbial taxonomic ecology of the human urogenital microbiome in health and disease, few whole genome metagenomic (WGM) datasets have been generated to profile the genomic ecology of this understudied niche. We set out to study the urogenital microbiome of PM women through WGM to not only determine the taxonomic ecology (microbial species present) of rUTI but also the genomic ecology (microbial genes present) that may contribute to rUTI pathobiology and susceptibility. When completed, this metagenomic dataset along with detailed clinical metadata and a curated biobank of more than 2000 patient-derived isolates will serve a larger role as a publicly available resource that will further the field’s understanding of rUTI and the PM urogenital microbiome. Translating technological advances into clinical insight will require further study of the microbial and genomic ecology of the UT and UGT microbiomes in both health and disease. Future diagnostic, prognostic, and therapeutic options for the management of UTI may soon incorporate efforts to measure, restore and/or preserve the native, healthy ecology of the UT and UGT microbiomes.Item Examining the Effect of Microbial Community on CRISPR-Cas Activity in Enterococcus Faecalis(May 2023) Byregowda, Shrinidhi 1998-; Palmer, Kelli; Dillon, Nicholas; De Nisco, NicoleEnterococcus faecalis is an opportunistic pathogen involved in causing various hospital-acquired infections. Their propensity to develop antibiotic resistance by horizontal gene transfer via conjugative mobile genetic elements makes the treatment of E. faecalis infection increasingly difficult. Pheromone-responsive plasmids are involved in the transfer of mobile genetic elements in E. faecalis. CRISPR-Cas acts as a barrier against foreign genome transfer in bacteria. E. faecalis CRISPR-Cas activity varies in vivo (murine intestinal model) and in vitro. The functional differences may be due to various biotic and abiotic factors such as nutrient availability, community diversity, donor-to-recipient ratios, and cas9 regulatory differences. A previous study has shown that the efficacy of CRISPR-Cas varies in the presence of a microbial community. Here we study the effect of Escherichia coli, also a resident of the mammalian gut, on CRISPR-Cas defense in vitro using E. faecalis containing pAM714 plasmid. Through our work, we are able to demonstrate that CRISPR-Cas efficacy is not altered by the presence of E. coli during conjugation between donor OG1SSp (pAM714) and recipients T11RF and T11RFΔcas9. Our goal is to provide insights into CRISPR-Cas function of Gram-positive bacteria in the microbial community.Item Genomic analyses of Enterococcus faecium: vancomycin-resistant isolates from Dallas, Texas and occurance of CRISPR-Cas(2021-05-04) Islam, Moutusee Jubaida; Palmer, KelliThe Gram-positive bacterium Enterococcus faecium is among the most prominent causes of hospital-acquired infections (HAI). Multi-drug resistant (MDR) strains have emerged, including those resistant to last-resort antibiotics like vancomycin. Therefore, infections caused by MDR strains leave very few treatment options and impose a major health problem. The vancomycin resistance genes and many other antibiotic resistance genes in E. faecium are encoded on mobile genetic elements (MGEs) such as plasmids or transposons that can be rapidly disseminated. The MGEs themselves continue to evolve through mutation and recombination, changing their mobility, maintenance, and host range. Therefore, surveillance of MDR strains, and specifically their MGEs, is critical. The genome defense mechanism, CRISPR-Cas, is an established system of defense against MGE acquisition in many bacteria but has not been explored in E. faecium extensively. The primary focus of the research presented here was the genomics analysis of vancomycin-resistant E. faecium strains (VREfm) obtained during routine fecal surveillance of high-risk patients from a Dallas, Texas area hospital system, with an emphasis on their plasmids and the transposon Tn1546, which are responsible for dissemination of vancomycin resistance genes. High-quality genome sequences were achieved for 47 E. faecium isolates through the hybrid assemblies of long (Oxford Nanopore Technology) and short (Illumina) sequence reads. A total of 251 closed plasmid sequences were generated and were assigned to 12 previously described and 9 newly defined rep family types. New variants of the Tn1546 were described that harbored a combination of 7 insertion sequences (IS) including 3 novel IS elements reported in this study. Overall, it was concluded that the VREfm isolates analyzed in our collection possess novel arrangements of the Tn1546-like elements and the vanA operon, which have evolved independently of the pRUM-like backbone carrying them. A second part of the research analyzed the occurrence of a previously reported Type II CRISPR-Cas system among E. faecium genomes and experimentally assessed its activity against plasmid acquisition. Publicly available genome sequences of 817 E. faecium strains were screened. Only 2.9% of the strains were identified to harbor the Type II EfmCRISPR-Cas system, which is characterized by the cas9 gene. Of the 61 non-redundant CRISPR spacers identified, 32.8% (20 out of 61) had sequence identity with bacteriophage genomes, indicating the hypothetical anti-phage role of CRISPR-Cas in E. faecium. The activity of the EfmCRISPR-Cas system was experimentally determined in the E. faecium strain 1,141,733. A cas9-dependent significant decrease in the acquisition of a small mobilizable plasmid was observed. Overall, this study identified and classified diverse plasmids harbored by Dallas, Texas, VREfm and demonstrated that the EfmCRISPR-Cas is able to defend E. faecium against plasmid acquisition, thus this system could be engineered to prevent antibiotic resistance dissemination among E. faecium strains.Item Lipidomic Analysis of the Streptococcal Cellular Membrane(2020-11-11) Joyce, Luke R; Palmer, KelliStreptococci are Gram-positive bacteria that natively colonize niches throughout the human body. While carriage of streptococci is mostly asymptomatic, they can be major human pathogens causing a variety of diseases across all age groups. Research on streptococcal mechanisms of pathogenicity, colonization, and transmission have identified virulence factors such as proteins, extracellular polysaccharides, intercellular communication pathways, and gene regulation pathways. These virulence factors have been demonstrated to aid in adherence to and invasion of human tissues and evasion of the immune system. A major component of the streptococcal cell is poorly understood: the cellular membrane. The cellular membrane is a critical site in host-pathogen interactions, providing an anchor for extracellular polysaccharides, protecting the internal workings of the bacterial cell, aiding transport of nutrients, and promoting survival in harsh conditions. The overarching goal of my dissertation work was to elucidate the biosynthetic pathway of phosphatidylcholine (PC) in Streptococcus mitis and S. oralis, members of the Mitis group streptococci, and to characterize the streptococcal cellular membrane using a culture medium that mimics the human host. In my research, I utilized lipidomics coupled with isotope tracing to discover that the glycerophosphocholine (GPC) pathway is required for PC biosynthesis in the Mitis group streptococcal species S. mitis, S. oralis, and S. pneumoniae. Briefly, the GPC pathway scavenges the major human metabolites GPC and lysophosphatidylcholine (lysoPC) and acylates them to form PC. Additionally, I determined that S. pneumoniae, S. pyogenes, and S. agalactiae synthesize PC when cultured in the presence of human serum. I demonstrate that lysoPC is the major substrate scavenged for an abbreviated GPC pathway in S. pyogenes and S. agalactiae. Furthermore, I characterized the structure of the novel aminoacylated glycolipid lysylglucosyl-diacylglcyerol (Lys-Glc-DAG) in S. agalactiae. I experimentally confirmed that the enzyme multiple peptide resistance factor (MprF) is the biosynthetic enzyme responsible for the lysine modification of Glc-DAG, establishing a novel glycolipid substrate for the S. agalactiae MprF. The MprF of S. agalactiae also catalyzes the addition of lysine to phosphatidylglycerol, forming lysyl-phosphatidylglycerol (Lys-PG), as expected based on prior knowledge of MprF in other bacteria. Using in vitro assays, I show that the lysine lipids, Lys-PG and Lys-Glc-DAG, impact the cellular membrane physiology such that S. agalactiae lacking the two lysine lipids are unable to survive in acidic conditions, have a more net negative outer surface charge, and exhibit significantly reduced human cell adherence and invasion. Taken together, my research provides critical insight into the cellular membrane of streptococci, evidence of cellular membrane remodeling through scavenging of major human metabolites, and the characterization of a novel aminoacylated glycolipid which impacts S. agalactiae colonization and invasion potential.Item Microfluidic Platform for Analyzing Chemotaxis of Bone Marrow Derived Murine Neutrophils Towards S. Aureus(December 2022) Beach, Taylor C. 1998-; Jones, Caroline N.; Palmer, Kelli; Rodrigues, DanieliWith the rise of MRSA (Methicillin-Resistant Staphylococcus aureus), there is a need for investigating alternatives to traditional antibiotics. A growing alternative exists in the form of chemoattraction based therapeutics which enhance the immune system’s response to infection. Novel methodologies will need to be developed to measure the increased activity. Our microfluidic system was used to show that the introduction of chemoattractants increased neutrophil migration towards Staphylococcus aureus, pointing to potential utility in therapy. Traditional migration assays, such as Boyden chambers are not ideal for measuring chemotaxis from mouse cells as they require a large number of cells (>100,000 per condition), make it difficult to pay discern the movements of individual cells, and cannot be used to measure directionality or retrotaxis. Mature neutrophils, band cells, and other cells from the marrow microenvironment were harvested from murine bone marrow and placed in a microfluidic chip in order to measure and quantify their migration patterns. We establish a baseline reaction of migration towards media populated by S. aureus and chemoattractants separately. Then we combined both stimuli to demonstrate how chemoattractants improved immune response and to confirm that this migration rate was four times higher than what neutrophils would respond without the aid of additional stimuli. Experiments found that the average quantity of neutrophils migrating increases when chemoattractants are added to media where S. aureus was present in comparison to media where S. aureus was injected without any chemoattractants. These results confirmed that chemoattractants improved cell migration, offering a potential mechanism for treatment of disease, and showed how our platform was capable of quantifying migrations towards pathogens.Item Multifaceted Roles of Environmental Factors toward Dental Implant Performance: Observations from Clinical Retrievals and In Vitro Testing(Elsevier Inc.) Sridhar, Sathyanarayanan; Wang, Frederick; Wilson, T. G., Jr.; Valderrama, P.; Palmer, Kelli; Rodrigues, Danieli C.; Sridhar, Sathyanarayanan; Wang, Frederick; Palmer, Kelli; Rodrigues, Danieli C.Objective: Oral bacteria and periodontal pathogen have been predominantly linked with early- and late- stage failures of titanium (Ti) dental implants (DI) respectively. This study is based on the hypothesis that bacterial colonization can damage the surface oxide (TiO₂) layer. Early-failed DI were compared with DI post-in vitro immersion in early colonizing oral bacteria; late failed DI were weighed against DI immersed in late colonizing anaerobic pathogens. Methods: Retrieval analysis: Seven early- stage failed implants with five of them connected to healing abutments (HAs), and ten late- stage failed retrievals were subjected to surface analysis. Bacteria immersion test: Three dental implants each were immersed in polycultures containing (i) early colonizers (Streptococcus mutans, S. salivarius, S. sanguinis) (ii) late colonizers (Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans). The implants were immersed for 30 days to simulate the healing period and bacterial biofilm adhesion. Optical microscope, x-ray photoelectron spectroscopy (XPS), and electrochemical test were performed to analyze the surface- morphology, chemistry, and potential respectively. Results: Early colonizers inflicted surface morphological damage (discoloration and pitting). Even though, XPS detected thinner oxide layer in 2/3 early retrievals, XPS and electrochemical tests illustrated that the TiO₂ layer was intact in HAs, and in DI post- immersion. Late colonizers also caused similar morphological damage (discoloration and pitting), while mechanical wear was evident with scratches, cracks, and mechanical fracture observed in late-stage retrievals. XPS indicated thinner oxide layer in late-stage retrievals (3/4), and in DI post-immersion in late colonizers. This was reflected in electrochemical test results post-immersion but not in the late-stage retrievals, which suggested an intact surface with corrosion resistance. Significance: This study concluded that bacteria could negatively affect implant surface with late colonizers demonstrating more pronounced damage on the surface morphology and chemistry. ©2018 The Academy of Dental MaterialsItem Pharmacological Regulation of Protein Translation in Fragile X Syndrome(2022-12-01T06:00:00.000Z) Shukla, Tarjani; Dussor, Gregory; Price, Theodore; Delk, Nikki; Palmer, Kelli; Sapkota, DarshanThe behavioral hallmarks of Autism Spectrum Disorder (ASD) are driven by molecular mechanisms that remain largely unknown. One model of ASD involves the deletion of the Fmr1 gene. Mutations in Fmr1 cause Fragile X Syndrome (FXS) and are the most common monogenetic source of intellectual disability in humans. The Fmr1 gene encodes for Fragile X Mental Retardation Protein (FMRP). FMRP is a conserved RNA-binding protein that binds the ribosome and attenuates translation. Loss of FMRP results in aberrant protein translation. This genetic lesion results in widespread cognitive deficiencies, specifically with learning, memory, and social interaction. A deeper understanding of the mechanism driving FMRP-dependent synaptic plasticity enables identification of highly specific therapeutics that ameliorate core neurological deficits associated with the disorder. In FXS, eIF4E is hyperphosphorylated and has emerged as a therapeutic target. Yet, specific inhibitors of Mitogen-Activated Protein Kinase Interacting Protein Kinase (MNK) have not been examined in the context of Fmr1 -/y mice. The goal of this research is to understand if compensation of FMRP loss can be achieved through manipulation of translation by the MNK-eIF4E regulatory axis. The results of this dissertation outline the viability of a novel therapeutic for the reversal of behaviors associated with Fragile X Syndrome, for which there are no FDA-approved treatments.Item Post-transcriptional Controls in Nociceptive Signaling(2022-12-01T06:00:00.000Z) Kunder, Nikesh Prakash 1991-; Delk, Nikki; Campbell, Zachary; Dodani, Sheel; Delk, Nikki; Dodani, Sheel; Dodani, Sheel; Palmer, Kelli; Morcos, Faruck; Misra, Jyoti; Palmer, KelliChronic pain is a condition wherein pain continues beyond the completion of the healing process. It is a debilitating condition that diminishes quality of life and is highly prevalent. Persistent pain is characterized by nociceptor plasticity. Dorsal root ganglion (DRG) neurons are responsible for generating nociceptive signals and undergo plasticity changes following injury. These changes are intimately linked to persistent pain. Translational regulation of mRNA permeates pain plasticity. Yet, the identity of translationally regulated mRNA that mediates plasticity is unknown. In this study, we used ribosome profiling to determine the protein landscape of sensory neurons after a brief exposure to the inflammatory mediators, NGF and IL-6. We observed preferential translation of a variety of transcripts. We focused on two immediate early genes, Arc and c-Fos that play a role in neuronal plasticity. These proteins have two very distinct functions: Arc regulates neuroinflammation while c-Fos regulates neuronal excitability. We also observed ribosomal occupancy on long non- coding RNAs as well as uORF utilization in certain mRNA transcripts. Among the various uORF containing transcripts, we identified a novel peptide generated from a uORF present in the 5ˊUTR of Calca mRNA. This short peptide generated from Calca is responsible for nociceptor sensitization via Gq signaling. Finally, we also identified that the 3’UTRs of preferentially translated mRNA contained a U-rich element for a RNA- binding protein called HuR. This protein contributes to nociceptor firing and mechanical hypersensitivity in mice. Our work provides insights into new key players that govern neuronal functions.Item Probing Dynamic Cellular Properties Using Genome Editing and Systems Biology(2021-12-01T06:00:00.000Z) Nowak, Chance Michael; Bleris, Leonidas; Lee, Mark; Palmer, Kelli; Kim, Tae Hoon; Campbell, ZacharyGenome editing has revolutionized not only the future of biological research, but also holds the promise of being a powerful therapeutic for genetic diseases. When considering the multitude of genetic regulations that contribute to various biological processes and their individual contributions that permit diseased cellular states, especially in instances where more than a single genetic aberration is attributed to the diseased phenotype, it is crucial to consider the interconnectivities of gene regulators and their individual contributions to cell health. Biological network maps that reveal the relation of gene products to one another can provide insight into the biological properties they govern. A biological network map consists of nodes (gene products) connected by edges that are dictated by the nature of the interaction between the two nodes. Nodal ablation (i.e., knocking out a gene to render it non-functional) has been crucial in understanding diseased states. However, this type of mutational analysis essentially disregards the impact that individual edges have on the network as a whole. The goal of my dissertation work was to utilize the genome editing tool Cas9 to disrupt the p53-miR-34a network in an edge-specific manner in order to demonstrate not only the complexity of these networks, but to also underscore the importance that individual edges have on the tumor suppressor phenotype. To this end, I, along with a team of researchers, developed a genetic screen using Cas9-bearing lentiviral vectors to disrupt 93 miR-34a binding sites within the 3’ untranslated region (UTR) of 71 genes impactful to cell survival under apoptotic conditions. I quantified the degree of apoptosis in two colorectal cancer cell lines that differ in functional p53 status, and that each harbored miR-34a binding site mutations within the pro-survival gene Bcl-2 3’UTR, demonstrating the importance of the miR-34a-Bcl-2 edge on apoptotic progression. Concurrently, I investigated the phenomenon of cell cycle desynchronization by tracking the DNA distribution of a population of cells starting from a synchronized state until asynchrony with flow cytometry analysis. In doing so, I utilized statistical tools to quantify the degree of desynchronization that does not rely on individual cell cycle phase labeling. Additionally, with the help of my peers, tested and validated a mathematical model the capitulates experimental observations. I explored the sensitivity of the model to changes in its parameters to reveal that cell cycle variability within the population is a main contributor to cell cycle desynchronization. Furthermore, I tested this model prediction by treating cells with lipopolysaccharide to enhance cellular noise, resulting in a greater variability of cell cycle duration, which was also shown to increase the rate of cell cycle desynchronization. Taken together, my research provides insight into the importance individual edges have to biological networks and their resulting phenotypes, as well as the underlying sources of cell population heterogeneity and its contribution to cell cycle variability.Item Role of FimK in Mediating Host Urinary Bladder Epithelial Cell Association of Uropathogenic Klebsiella Pneumoniae and Quasipneumoniae(December 2021) Venkitapathi, Sundharamani; De Nisco, Nicole; Lou, Yifei; Palmer, Kelli; Delk, Nikki; Spiro, StephenKlebsiella spp. commonly cause both uncomplicated urinary tract infection (UTI) and recurrent UTI (rUTI). K. quasipneumoniae, a relatively newly defined species of Klebsiella, has been shown to be metabolically distinct from K. pneumoniae, but its urovirulence mechanisms have not been defined. Type 1 and type 3 fimbriae, encoded by fim and mrk operons respectively, mediate attachment of Klebsiella spp. to host epithelial cells. fimK is a regulatory gene unique to the Klebsiella fim operon that encodes an N-terminal DNA binding domain and a C-terminal phosphodiesterase domain that has been hypothesized to cross-regulate type 3 fimbriae via modulation of cellular levels of cyclic di-GMP. Comparative genomic analysis between K. pneumoniae and K. quasipneumoniae revealed a conserved premature stop codon in K. quasipneumoniae fimK that results in loss of the C-terminal phosphodiesterase domain (PDE). We hypothesized that this truncation would ablate cross-regulation of type 3 fimbriae in K. quasipneumoniae. Here, we report that K. quasipneumoniae KqPF9 bladder epithelial cell association and invasion is dependent on type 3 but not type 1 fimbriae. Further, we show that basal expression of both type 1 and type 3 fimbrial operons as well as bladder epithelial cell association are higher in KqPF9 than in K. pneumoniae TOP52. Interestingly, complementation of KqPF9∆fimK with the TOP52 fimK allele markedly reduced type 3 fimbrial expression and bladder epithelial cell attachment, a phenotype that was rescued by mutation of the C-terminal PDE active site. Taken together these data suggest that C-terminal PDE of FimK modulates type 3 fimbrial expression in K. pneumoniae and its absence in K. quasipneumoniae leads to a loss of type 3 fimbrial cross-regulation.Item Tracking Dissemination of Plasmids in the Murine Gut Using Hi-C Sequencing and Bayesian Landmark-based Shape Analysis of Tumor Pathology Images(2020-12-01T06:00:00.000Z) Zhang, Cong; Zhang, Michael Qiwei; Chen, Min; Yang, Duck Joo; Palmer, Kelli; Li, Qiwei; Shin, SunyoungStarting from the experimental design and simple group result comparison using studentst test to the analysis need of explosively growing digital information in the big data era, extensive statistical approaches have been developed and incorporated into biology studies in order to understand the mechanism of life processes and disease. The term ”omics” refers to the various disciplines in biology performing a comprehensive, or global, assessment of a set of biological features in a high-throughput way, such as genomics, transcriptomics, proteomics and metagenomics. When analyzing such a huge amount of data, a proper framework needs to be developed with a thorough knowledge of the associated biology as well as statistical models. In this work, I focus on two critical health-related problems, antibiotic resistance spread in microbial communities by conjugative plasmids, and the association between tumor shape and prognosis in pathology images. In my first work, metagenomics and Hi-C sequencing were employed to analyze plasmid dissemination from Enterococcus faecalis donor strains in the murine intestine. I clustered assembled contigs into metagenomeassembled genomes (MAGs) and showed that the quality of obtained MAGs was improved by combining those two types of sequencing techniques. Then, I demonstrated that Hi-C is able to detect the in situ hosts of native resistance genes in the murine gut microbiota. We also confirmed the association between introduced E. faecalis plasmids and the donor strains and found potential new gram-positive host for the pAM830 resistance plasmid. In my second work, we developed a framework with a novel automatic landmark detection model for tumor shape boundary in pathology image called Bayesian LAndmark-based Shape Analysis (BayesLASA). Two types of landmark-based boundary roughness features were proposed, and we demonstrated the predictive value of them in a large cohort of lung cancer patients.