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The anthrax toxin protective antigen (PA63) is an oligomeric channel that represents an ideal target for multivalent channel-blocking ligand development. PA63 acts as a translocase, delivering enzymatic factors of the tripartite anthrax toxin into the cytosol. We previously described poly(amidoamine) (PAMAM) dendrimers as effective multivalent PA63 blockers. Here, we used single-channel planar lipid bilayer technique to examine the effect of PAMAM surface chemistry on their ability to block PA63. We found that the strength of channel/dendrimer interaction was determined by both binding reaction on- and off-rates. Thus, we observed a three folds decrease in on-rate and a ten times decrease in residence time with generation 2 hydroxyl-terminated PAMAM compared to amino-terminated PAMAM. For both blockers, kon and tres increased dramatically with transmembrane voltage. Considering the electrostatic nature of the interaction, the effect on the off-rate is not surprising. One of the unforeseen findings was the increase in dendrimer capture rate as result of trans-negative voltage and the strong on-rate dependence on dendrimer surface chemistry. Analytical interpretation of current noise data in these studies was complicated by the intrinsic current fluctuation of PA63, described as 1/f noise. We only observed 1/f noise in the binary bacterial toxin channels that are evolved to translocate their enzymatic components and contain functionally critical hydrophobic phenylalanine rings, called Φ clamps. PA63 channel with a mutated Φ clamp was dysfunctional as a translocase and did not exhibit 1/f noise. Using poly(ethylene glycol) partitioning experiments and molecular dynamic simulations, we suggested that the 1/f noise in PA63 channel occurs as a result of ‘hydrophobic gating’ at the Φ clamp region. When studying the small ion transport properties of PA63, we discovered an unusually strong PA63 conductance dependence on the nature of the bathing electrolyte cations. We suggest that this effect is determined by the interplay between cation radii and their binding affinity to the negatively charged residues, proximal to the Φ clamp. Understanding the intrinsic behavior of PA63, including its fluctuation dynamics and ion transport properties, can contribute to the antitoxin development effort and advance this channel as a novel tool for biosensor development and applications.

Biology

Biophysics

Antrhax, channel, conductance, dendrimer, noise, toxin

Biology

Degree Awarded: Ph.D. Biology. The Catholic University of America

Advisors/Committee Members: The Catholic University of America (Degree granting institution), Nestorovich, Ekaterina (Thesis advisor), Tuma, Pamela (Committee member), Choy, John (Committee member).

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Multidrug resistance to antifungals and chemotherapeutic drugs is a huge clinical problem for treatment of fungal infections and cancers. Overexpression or alteration of multidrug efflux ATP Binding Cassette (ABC) transporters is often associated with this broad-spectrum of resistance. Pleiotropic Drug Response 5 gene (PDR5) in Saccharomyces cerevisiae defines a subclass of important fungal efflux pumps. The characterization of mutations in this transporter uncovered two novel mechanism of increased resistance. The first mutation, A666G, resulted in enhanced resistance without increasing either the amount of protein in the plasma membrane or the ATPase activity. In fluorescence-quenching transport assays with rhodamine 6G in purified plasma membrane vesicles, the initial rates of rhodamine 6G fluorescence quenching of both the wild-type and mutant showed a strong dependence on ATP concentration, but were about twice as high in the latter. Plots of initial rate of fluorescence quenching versus ATP concentration exhibited strong cooperativity that was significantly increased in the A666G mutant and accounted for the observed enhancement in resistance. Alanine substitution mutations in six serine residues of linker-2 all exhibited increased resistance to xenobiotic agents. Biochemical studies of these nonsynonymous mutants demonstrated that they have increased steady-state levels of Pdr5 protein expression and thus enhanced resistance. Quantitative-RT PCR and metabolic labelling experiments demonstrated that the mutants had levels of PDR5 mRNA that were two to three times as high as in the isogenic wild-type strain because the transcript half-life was increased. These data demonstrate that the nucleotides encoding unconserved amino acids may be used to regulate expression and suggest that Pdr5 has a newly discovered RNA stability element within its coding region.

Biology

Genetics

Biochemistry

ABC Transporter, Cooperativity, Linker-2, mRNA, Nonsynonymous mutation, Pdr5

Biology

Degree Awarded: Ph.D. Biology. The Catholic University of America

Advisors/Committee Members: The Catholic University of America (Degree granting institution), Golin, John (Thesis advisor), Tuma, Pamela (Committee member), Choy, John (Committee member).

▼ HIV-1 continues to be a public health concern, causing 1.8 million new infections per year globally. Despite four decades of research around the globe, an effective HIV-1 vaccine is still elusive. The only HIV-1 vaccine trial that showed promise after dozens of failures is the RV144 study conducted in Thailand which showed a modest efficacy of 31.2%. Subsequent analyses revealed a positive correlation between the V2-directed antibodies (Abs) and protection from HIV-1 acquisition. The V2 region is part of the variable V1V2 domain located at the apex of the HIV-1 envelope glycoprotein trimer (Env), the key target of HIV vaccine design. While several studies explored the positive correlates for the success of this vaccine, studies investigating the limited protection and occurrence of breakthrough infections are largely missing. Here, we aim to address this gap in knowledge through deeper understanding of the structure and function of Env of transmitted/founder (T/F) viruses that cause infection by escaping host immune pressure. This might reveal mechanisms that provide insights into the design of an effective HIV vaccine.We have examined the env genes of evolving T/F viruses longitudinally at an acute stage of infection for V1V2-specific immune escape mutations. For this, we evaluated the. Phylogenetic analyses of env sequences from four participants of an acute HIV infection cohort (RV217) identified a key viral escape mutation, H173Y, in one of the participants (40007). This mutation is located in the conserved V2 epitope (C-β strand) of Env that is also a target of RV144 vaccine pressure. The H173Y mutant not only evaded binding to the V2-directed antibodies (Abs) elicited by this patient but also poorly reactive to V2 Abs isolated from the RV144 vaccinees. To explore the mechanism of escape, mice were immunized with the recombinant V1V2 of the T/F virus of 40007 (T/F07) and its escape V1V2-variants, scaffolded to the dodecameric phage T4 terminase, gp16. Ab responses generated by these immunogens were distinct and mimicked the escape phenomenon observed in this patient. Furthermore, surprisingly, the H173Y escape mutant induced broadly reactive Abs. Characterization of these Abs, structural modeling, and molecular dynamics simulations indicate a significant conformational change in the V2 region of the H173Y mutant, which might be responsible for the observed Ab evasion. Based on these data, we hypothesized that the conformational dynamism in the V2 region might be one of the mechanisms of immune escape and breakthrough infections in vaccinees. To test the above hypothesis, we constructed a combinatorial mutant library of V2 conformational variants mimicking the naturally existing HIV population and evaluated their immunogenicity and breadth in the context of H173 residue in a mouse model. Remarkably, the gp16-scaffolded V2 domains generated Abs that reacted strongly with the Env trimer, and consistent with our hypothesis, certain groups with the H173Y mutation further increased the breadth of the V2 Abs. This… Advisors/Committee Members: The Catholic University of America (Degree granting institution), Rao, Venigalla (Thesis advisor), Greene, James (Committee member), Choy, John (Committee member).

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Nearly 38 million people across the globe are currently living with HIV-1 infection, but there is yet no cure. During HIV-1 infection, HIV enters the body and targets CD4+ T cells, and once inside the cell, the virus makes its way to the nucleus and inserts its genome into the host cell's genome. Most of the infected CD4+ T-cells actively produce new viruses, but in a fraction of cells, the virus enters into a latent phase where the integrated viral genome remains transcriptionally silent and does not produce progeny viruses. A collection of such cells carrying the HIV-1 “provirus” constitutes the latent “reservoir.” Although combinational antiretroviral therapy (cART) is effective in controlling the infection, it is not curative as it does not target the integrated proviruses that remain in the latent reservoir. Hence, viral rebound occurs by activation of a small pool of these proviruses when treatment is discontinued. Reducing or eliminating the latent reservoir, therefore, is essential to achieve “remission” or “cure.” An effective curative approach would be to activate the latent reservoir and selectively disrupt the proviral genome as well as elicit immune responses to neutralize any remaining viruses.The main goal of the present thesis is to elucidate the ability of 120 x 86 nm bacteriophage T4 capsid (head) nanoparticle to deliver a combination of HIV cure molecules; proteins, DNAs, and RNAs to target to CD4 + T cells and achieve curative outcomes. About 170 kb DNA can be packaged inside the T4 capsid and ~1,025 proteins fused to the outer capsid proteins Soc (small outer capsid protein, 870 copies) and Hoc (highly antigenic outer capsid protein, 155 copies) can be displayed on the capsid of this large capacity platform. In vitro assembled CD4 receptor targeting T4 nanoparticles with CD4 DARPin-Hoc and/or HIV-1Env-Soc allowed targeted gene delivery into CD4+ T cells. Customization of CD4 receptor-targeting T4 nanoparticles by incorporating genome editing molecules resulted in disruption of the HIV-1 provirus genome in CD4+ HEK293T cells. Unexpectedly, T4 nanoparticles also activated the provirus of latent CD4+ T cells in an in vitro Jurkat cell model for latent HIV-1 infection. Furthermore, the latency reversal was also indicated in CD4+ resting T cells of HIV-infected ART suppressed patients. A series of experiments suggest that T4 nanoparticles appear to activate a novel pathway that is distinct from the classical signaling pathways reported thus far. Our studies demonstrate that T4 bacteriophage nanoparticles can be programmed to target CD4+ T cells, mediate provirus disruption, and cause latency reversal.

Biology

Biology

Degree Awarded: Ph.D. Biology. The Catholic University of America

Advisors/Committee Members: The Catholic University of America (Degree granting institution), Rao, Venigalla (Thesis advisor), Tuma, Pamela (Committee member), Choy, John (Committee member).

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Twist family members are highly conserved basic helix-loop-helix (bHLH) transcription factors that are important for mesoderm development in all animals. Twist either forms homodimers or heterodimers with other bHLH proteins through their HLH domains. Twist-containing dimers regulate the expression of downstream target genes by binding an enhancer element called the E-box (CANNTG). Humans have two paralogous genes, TWIST1 and TWIST2, and mutations in each gene have been identified in specific craniofacial disorders. Sweeney-Cox (SwCoS) and Barber-Say syndrome (BSS) are craniofacial disorders that are caused by heterozygous mutations in TWIST1 and TWIST2, respectively. C. elegans has a single Twist homolog, known as HLH-8. C. elegans strains with the analogous disease alleles in HLH-8 that are found in SwCoS and BSS are referred to as Glu29 mutants. When expression of an HLH-8 target gene, arg-1, was examined in these mutants, it was found in the head mesodermal cell (HMC) and enteric muscles (EMs) but not in vulval muscles (VMs). These observations indicate that these Glu29 alleles retain some tissue-specific transcriptional activity. The studies in this dissertation have been aimed to better understanding the mechanism of how Glu29 mutants are activating transcription of arg-1. First, we wanted to determine if Glu29 mutants maintain their dimerization properties. Thus, to test protein-protein interactions, we used biomolecular fluorescence complementation (BiFC) method. We established this method and tested proteins dimerization pattern for wild-type and Glu29 HLH-8 in vivo. During these studies, we discovered that as human TWIST1/2, HLH-8 partner choice can be determined by its phosphorylation state. Although wild-type and Glu29 HLH-8 proteins are not able to form homodimers in vivo, Phospho-null HLH-8 proteins do form homodimers. The 385 bp arg-1 promoter region contains three different E box DNA sequences (E1-E2-E3) bound by either HLH-8/HLH-8 homodimers or HLH-8/HLH-2 heterodimers. Upon examining the region near the E-boxes, a GT box was identified previously that promotes arg-1 expression in the VMs. The GT box isn’t bound by any of HLH-8-containing dimers, suggesting that there is an additional factor (X) that contributes to arg-1 expression in the VMs. To further understand the molecular basis of how Glu29 mutants activate arg-1 expression, we used the yeast one-hybrid assay (Y1H) to identify (X). Results from this experiment revealed HMG-11 as an accessory protein that binds to the GT box sequence, cooperate with HLH-8, and regulates arg-1 expression in VMs. Therefore, in Glu29 mutants, this cooperation may be defective which disrupts arg-1 expression in VMs.

Developmental biology

C. elegans, HLH-8, TWIST

Biology

Degree Awarded: Ph.D. Biology. The Catholic University of America

Advisors/Committee Members: The Catholic University of America (Degree granting institution), Corsi, Ann (Thesis advisor), Golin, John (Committee member), Choy, John (Committee member).

▼ ATP-binding cassette (ABC) transporters are present in all known organisms and comprise one of the largest families of integral membrane proteins. Clinically, ABC transporters are associated with numerous diseases, cause multidrug resistance in various types of cancer, and cause antifungal resistance in pathogenic yeasts. Pdr5 is a plasma membrane bound multidrug transporter and the founding member of the Pdr subfamily of ABC transporters.In ABC transporters, the nucleotide binding domains dimerize to form two nucleotide binding sites, which are composed of highly conserved motifs. The deviant ATP-binding site, a unique feature of the Pdr subfamily, has been found to transmit signals between the various domains of Pdr5, specifically through the Q- loop (Ananthaswamy et al. 2010) and D-loop (Furman et al. 2013) motifs, and to facilitate ATP hydrolysis at its canonical ATP-binding site (Gupta et al. 2014). A Pdr5 deviant signature mutant, E1013A has reduced drug resistance, drug transport capability, and ATPase activity. In order to elucidate the role of the deviant signature motif, we isolated suppressors of E1013A. Two suppressors, SUP5 and SUP6, exhibited dominant gain-of-function phenotypes that significantlyrestored E1013A drug resistance independent of overexpressing Pdr5. Sup5 suppression fully restored the ATPase activity of Pdr5, while Sup6 suppression occurred via a different mechanism. Significantly, Sup5 and Sup6 were unable to rescue the loss-of-function phenotype present in the canonical signature mutant, G312A, suggesting that both are extragenic suppressors that interact specifically with the deviant ATP-binding site of Pdr5.We verified and characterized the drug resistance phenotype, ATPase activity, and plasma membrane abundance of Pdr5 for SUP5 and SUP6. Several genetic mapping and screening methods were utilized to identify SUP5 and SUP6, but a successful Yep24 overexpression screen identified SKS1, a putative serine/threonine kinase. The overexpression of SKS1 in the E1013A mutant strain mediated multidrug resistance to clotrimazole and cerulenin, possibly by phosphorylating the linker 2 region of Pdr5 (Johnson et al. 2014). Sks1-mediated phosphorylation also conferred hyper-resistance to WT Pdr5 in a manner that did not increase the ATPase activity or increase the abundance of Pdr5 in the plasma membrane. A Sks1 K39I mutation was unable to suppress the E1013A mutant. Despite common expectations, knocking out SKS1 in WT had no decrease in drug resistance, which suggests that Sks1 is not essential for Pdr5-mediated drug resistance.We proposed a mechanism for in vivo regulation of Pdr5 in low glucose and potential drug conditions based on evidence that positions Sks1 in the cAMP-PKA pathway, a pathway that controls cell division and stress responses. Sks1 has a homolog, SHA3, in C. albicans which functions in the cAMP-PKA pathway and is required for hyphal development and virulence. An SKS1 homolog, SNFK, was also found in humans, thus providing greater relevance for elucidating the Sks1 signaling… Advisors/Committee Members: The Catholic University of America (Degree granting institution), Golin, John (Thesis advisor), Corsi, Ann (Committee member), Choy, John (Committee member).

▼ Antimicrobial resistance (AMR) occurs when a microorganism gains resistance to antimicrobial agents that were originally designed to cure infections caused by these microorganisms. Overexpression of ATP-binding cassette (ABC) multidrug transporters is a mechanism of AMR, which acts by pumping the drugs out of the cell. ABC transporters are a superfamily of polytopic proteins that uses the energy of ATP hydrolysis to transport the substrates across the membrane against a concentration gradient. The typical ABC transporters consist of two transmembrane domains (TMDs), composed of six transmembrane helices (TMHs) that are connected by extracellular loops (ECLs) and intracellular loops (ICLs), and two nucleotide-binding domains (NBDs). ABC transporters export substrates through a series of conformational changes. Many ABC transporters shift between an inward-facing, drug-binding (high-affinity) conformation to an outward-facing, drug-releasing (loose-binding) conformation using ATP binding and hydrolysis. When the drug is released from the drug-binding pocket, it must be prevented from reentering through the transporter. The outward-facing or drug-releasing conformation results in only a 10-to-30-fold reduction in binding affinity, which is not sufficient to prevent some rebinding of the drugs if the extracellular concentration is high (Sauna and Ambudkar, 2000). For this reason, it was proposed that ABC transporters have a molecular gate or diode that prevents reflux (Gupta, et al., 2011). The first evidence for a molecular diode mechanism was the observation that a Ser 1368 Ala (S1368A) mutation in the yeast multidrug transporter Pdr5 exhibited significant drug reflux during transport (Mehla, et al., 2014). More recently, multiple cryo-electron microscopy structures of the ABCG2 mammalian multidrug transporter clearly showed the presence of a molecular gate between an inner and outer drug-binding pocket. Two prominent residues—Leu-554 and Leu-555—were identified as constituents of this gate (Manolaridis, et al., 2018). When the sequences of Pdr5 and ABCG2 are aligned, Ser-1368 is quite close to Leu-554 and Leu-555. The corresponding residues in Pdr5 are Val-1372 and Met-1373. Bioinformatics analysis of the Pdr subfamily (Lamping, et al., 2010) revealed highly conserved residues adjacent to Ser-1368, including Phe-1369, Gly-1371, Val-1372, and Met-1373. The corresponding residues to this S1368 adjacent motif in the amino-terminus of Pdr5, extending from Tyr-680 to Ilv-685, also exhibit conservation. The first goal of this study is to determine whether the conserved set of amino acids spanning residues (1369–1373) in the carboxyl-terminus motif of Pdr5 make up a molecular diode motif. The second goal is to investigate whether the molecular diode is composed of residues from both halves of this large, polytopic protein. Our results suggest that this conserved motif plays a variety of roles including signal transmission, protein folding, and molecular gating. Our results also suggest that Ile-685, Val-1372, and Met-1373… Advisors/Committee Members: The Catholic University of America (Degree granting institution), Golin, John (Thesis advisor), Nestorovich, Ekaterina (Committee member), Choy, John (Committee member).

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Human immunodeficiency virus (HIV) is the causative agent of Acquired Immune Deficiency Syndrome. Combination antiretroviral therapy (cART) blocks virus replication and halts disease progression but is not curative. Low-level HIV viremia persists in most individuals after years of cART. Upon treatment cessation, viremia rebounds and disease progression continues. The persistence of cells carrying replication-competent virus in a long-lived reservoir poses a major obstacle towards a cure. HIV mostly infects CD4+ T cells which divide in response to homeostatic signals and upon recognition of antigen. We previously determined that HIV-infected cells frequently undergo proliferation, a mechanism promoting the persistence of infected cells despite therapy. To investigate the forces that shape HIV-infected cell populations after treatment initiation, we measured HIV DNA-containing cells in vivo and determined the fraction that express HIV RNA. Few HIV DNA+ cells in the blood contribute to plasma viremia and are lost after treatment begins. Before and after treatment initiation HIV RNA production is heterogenous. HIV RNA-expressing cells are rapidly eliminated when therapy is initiated. The HIV reservoir resides within the population of infected cells that remain. To determine the composition of these infected cells, we quantified populations of largely-intact versus deleted proviruses. Largely-intact proviruses are eliminated faster than highly-deleted proviruses, suggesting that immune selection pressures and/or viral protein toxicities shape infected cell populations during therapy. Over two-fold accumulations of deleted proviruses occurred after the majority of HIV-infected cells were eliminated. To investigate the role of clonal expansion on shaping the populations of infected cells, we quantified an HIV-infected cell clone from one individual. This cell clone harbored a deleted provirus and underwent a massive expansion during treatment, shifting the HIV-infected cell population. The clone was detected in effector memory cells, suggesting it expanded upon antigen stimulation. In another individual, deleted proviruses dominated the HIV-infected cell population after a Hepatitis C infection. While the total number of HIV-infected cells remains stable during treatment, the composition of the infected cell population is highly dynamic. Widespread clonal expansion of infected cells and accumulations of deleted proviruses occurs in the majority of individuals undergoing long-term cART.

Virology

Microbiology

Clonal Expansion, ddPCR, Decay, HIV-1, Persistence, Reservoir

Biology

Degree Awarded: Ph.D. Biology. The Catholic University of America

Advisors/Committee Members: The Catholic University of America (Degree granting institution), Rao, Venigalla (Thesis advisor), Maldarelli, Frank (Thesis advisor), Corsi, Ann (Committee member), Choy, John (Committee member).

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During pregnancy, the fetus acquires maternal antibodies by Fc receptor transfer across the placenta. Many of these Fc receptors are also expressed on immune cells and are responsible for recognition, activation and killing of HIV-1 infected cells as demonstrated in antibody dependent cellular cytotoxicity (ADCC). This study evaluated HIV+ maternal and respective HIV+ or HIV- infant plasma antibodies to understand potential factors that contribute to protection from HIV vertical transmission. Plasma antibodies were assessed for the functional capacity of mediating ADCC against heterologous and autologous HIV-1 infected target cells. While no differences in ADCC mediation were found between transmitting and nontransmitting mothers, HIV- infants had antibodies with greater ADCC potency and breadth. Both HIV+ and HIV- infants had natural killer cells with cytotoxic maturation markers, indicating necessary cellular maturity to implement ADCC. However, no differences were found in cytotoxic frequency. Maternal and infant antibody binding features were distinct in maternal transmission and infant infection status. Nontransmitting mothers and their HIV- infants had greater HIV-1 antigen-specific antibodies that engaged common placental transport and immune cell activation receptors. Nontransmitting mothers transferred higher HIV-specific antibodies that engaged the neonatal transport receptor (FcRn). HIV-1 recognizing antibodies that engage placental FcRn were found to be the greatest predictive feature of reduced MTCT. This study supports that HIV- infants acquire antibodies with greater recognition of HIV-1 antigen and mediation of ADCC.

Immunology

Virology

ADCC, HIV-1, MTCT, Placental Transport

Biology

Degree Awarded: Ph.D. Biology. The Catholic University of America

Advisors/Committee Members: The Catholic University of America (Degree granting institution), Rao, Venigalla (Thesis advisor), Wieczorek, Lindsay (Committee member), Tovanabutra, Sodsai (Committee member), Chung, Byung Min (Committee member), Choy, John (Committee member).

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Staphylococcus aureus is a Gram-positive bacterium that has emerged as a significant problem outside of traditional hospital acquired (HA) infections. Infections by Methicillin Resistant S. aureus (MRSA) strains result in a significant health burden around the world due to its high level of antibiotic resistance, ability to evade the immune system and expression of multiple toxins. The well-characterized USA300 strain of S. aureus is the leading cause of MRSA infections in North America. S. aureus USA300 isolates have acquired genomic elements that have remarkably increased the fitness potential and virulence when compared to other MRSA strains. More recently the USA800 ST5-IV MRSA strain has successfully emerged and has become a great concern in HA and community acquired (CA) MRSA infections in Southeastern Asia, Australia and New Zealand. Both MRSA strains have become a significant health risk but USA800 is less known when compared to USA300. Emergence of S. aureus USA800 ST5-IV has provided a scientifically advantageous opportunity to fully characterize the possible clinical and molecular reasons why this strain has successfully emerged. As S. aureus is a common skin bacterium, colonization through the use of different pathogenicity islands and adhesion factors, is critical for pathogenesis to be successful. Through the use of whole genome sequencing and comparative genome analysis we were able to detect several key adhesion factors and pathogenicity islands that are present in USA800 but absent in USA300, in HA-MRSA USA400, and the common lab strain RN4220. From our sequence data, we have discovered that the USA800 ST5-IV strain has multiple uncharacterized adhesion and LPXTG domain proteins and pathogenicity islands. Using intron sequence insertion gene editing technology, we disabled these gene functions which allowed us to determine if the mutants show a phenotypic response when attempting to colonize our in-vitro skin model system. From our confrontation and crystal violet assays, we determined that adhesion protein 417, a purported SasG adhesion protein, is essential to attachment to human keratinocytes in-vitro. Results also indicate that target genes 146, 147, and 417 are likely part of a larger regulon and would have the potential to affect colonization due to their proposed association with each other. Our findings indicate that these genes provide novel targets for improvement of therapeutics and potentially demonstrating improvement to discovering novel prevention approaches for MRSA infections.

Biology

Microbiology

Biology, Genomics, Microbiology, MRSA, Staphylococcus

Biology

Degree Awarded: Ph.D. Biology. The Catholic University of America

Advisors/Committee Members: The Catholic University of America (Degree granting institution), Tuma, Pamela (Thesis advisor), Jones, Marcus (Thesis advisor), Portugal, Franklin (Committee member), Choy, John (Committee member).