Current Search: Moore, Sean (x)
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- Title
- Biochemical Studies of ABCE1.
- Creator
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Sims, Lynn, Igarashi, Robert, Ebert, Steven, Self, William, Moore, Sean, University of Central Florida
- Abstract / Description
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The growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and...
Show moreThe growth and survival of all cells require functional ribosomes that are capable of protein synthesis. The disruption of the steps required for the function of ribosomes represents a potential future target for pharmacological anti-cancer therapy. ABCE1 is an essential Fe-S protein involved in ribosomal function and is vital for protein synthesis and cell survival. Thus, ABCE1 is potentially a great therapeutic target for cancer treatment. Previously, cell biological, genetic, and structural studies uncovered the general importance of ABCE1, although the exact function of the Fe-S clusters was previously unclear, only a simple structural role was suggested. Additionally, due to the essential nature of ABCE1, its function in ribosome biogenesis, ribosome recycling, and the presence of Fe-S within ABCE1, the protein has been hypothesized to be a target for oxidative degradation by ROS and critically impact cellular function. In an effort to better understand the function of ABCE1 and its associated Fe-S cofactors, the goal of this research was to achieve a better biochemical understanding of the Fe-S clusters of ABCE1. The kinetics of the ATPase activity for the Pyrococcus abyssi ABCE1 (PabABCE1) was studied using both apo- (without reconstituted Fe-S clusters) and holo- (with full complement of Fe-S clusters reconstituted post-purification) forms, and is shown to be jointly regulated by the status of Fe-S clusters and Mg2+. Typically, ATPases require Mg2+, as is true for PabABCE1, but Mg2+ also acts as a unusual negative allosteric effector that modulates ATP affinity of PabABCE1. Comparative kinetic analysis of Mg2+ inhibition shows differences in the degree of allosteric regulation between the apo- and holo-PabABCE1 where the apparent Km for ATP of apo-PabABCE1 increases (>)30 fold from ~30 (&)#181;M to over 1 mM when in the presence of physiologically relevant concentrations of Mg2+. This effect would significantly convert the ATPase activity of PabABCE1 from being independent of cellular energy charge to being dependent on energy charge with cellular [Mg2+]. The effect of ROS on the Fe-S clusters within ABCE1 from Saccharomyces cerevisiae was studied by in vivo 55Fe labeling. A dose and time dependent depletion of ABCE1 bound 55Fe after exposure to H2O2 was discovered, suggesting the progressive degradation of Fe-S clusters under oxidative stress conditions. Furthermore, our experiments show growth recovery, upon removal of the H2O2, reaching a growth rate close to that of untreated cells after ~8 hrs. Additionally, a corresponding increase (~88% recovery) in the ABCE1 bound 55Fe (Fe-S) was demonstrated. Observations presented in this work demonstrate that the majority of growth inhibition, induced by oxidative stress, can be explained by a comparable decrease in ABCE1 bound 55Fe and likely loss of ABCE1 activity that is necessary for normal ribosomal activity. The regulatory roles of the Fe-S clusters with ABCE1 provide the cell a way to modulate the activity of ABCE1 and effectively regulate translation based on both cellular energy charge and the redox state of the cell. Intricate overlapping effects by both [Mg2+] and the status of Fe-S clusters regulate ABCE1's ATPase activity and suggest a regulatory mechanism, where under oxidative stress conditions, the translational activity of ABCE1 can be inhibited by oxidative degradation of the Fe-S clusters. These findings uncover the regulatory function of the Fe-S clusters with ABCE1, providing important clues needed for the development of pharmacological agents toward ABCE1 targeted anti-cancer therapy.
Show less - Date Issued
- 2012
- Identifier
- CFE0004600, ucf:49204
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004600
- Title
- Evolutionary Relationships Among Staphylococci and the Prevention of Staphylococcus aureus Nasal Colonization.
- Creator
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Lamers, Ryan, Cole, Alexander, Parkinson, Christopher, Chai, Xinqing, Moore, Sean, University of Central Florida
- Abstract / Description
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Staphylococcus is a significant cause of human infection and mortality, worldwide. Currently, there are greater than 60 taxa within Staphylococcus, and nearly all are pathogenic. The collective potential for virulence among species of Staphylococcus heightens the overall clinical significance of this genus and argues for a thorough understanding of the evolutionary relationships among species. Within Staphylococcus, aureus is the most common cause of human infection, where nasal carriage of...
Show moreStaphylococcus is a significant cause of human infection and mortality, worldwide. Currently, there are greater than 60 taxa within Staphylococcus, and nearly all are pathogenic. The collective potential for virulence among species of Staphylococcus heightens the overall clinical significance of this genus and argues for a thorough understanding of the evolutionary relationships among species. Within Staphylococcus, aureus is the most common cause of human infection, where nasal carriage of this bacterium is a known risk factor for autoinfection. The predisposition to infection by nasal carriers of S. aureus, and the ease with which strains are transferred between individuals, suggests that nasal carriage is a major vector for the transmission of virulent strains throughout the community. This hypothesis, however, has not been assessed in any great detail to identify the genetic relationships between clinical isolates of S. aureus and those strains being carried asymptomatically throughout the community. Also lacking within this field is a unified and robust estimate of phylogeny among species of Staphylococcus.Here, we report on a highly unified species phylogeny for Staphylococcus that has been derived using multilocus nucleotide data under multiple Bayesian and maximum likelihood approaches. Our findings are in general agreement with previous reports of the staphylococcal phylogeny, although we identify multiple previously unreported relationships. Regardless of methodology, strong nodal support and high topological agreement was observed with only minor variations in results between methods. Based on our phylogenetic estimates, we propose that Staphylococcus species can be evolutionarily clustered into 15 groups, and six species groups. In addition, our more defined phylogenetic analyses of S. aureus revealed strong genetic associations between both nasal carriage strains and clinical isolates. Genetic analyses of hypervariable regions from virulence genes revealed that not only do clinically relevant strains belong to identical genetic lineages as the nasal carriage isolates, but they also exhibited 100% sequence similarity within these regions. Our findings indicate that strains of S. aureus being carried asymptomatically throughout the community via nasal colonization are genetically related to those responsible for high levels of infection and mortality.Due to nasal carriage of S. aureus being a risk factor for autoinfection, standardized preoperative decolonization has become a major consideration for the prevention of nosocomial infection. Toward this end, we have identified the macrocyclic ?-defensin analogue RC-101 as a promising anti-S. aureus agent for nasal decolonization. RC-101 exhibited bactericidal effects against S. aureus in both epithelium-free systems, and ex vivo models containing human airway epithelia. Importantly, RC-101 exhibited potent anti-S. aureus activities against all strains tested, including USA300. Moreover, RC-101 significantly reduced the adherence, survival, and proliferation of S. aureus on human airway epithelia without any noted cellular toxicity or the induction of a proinflammatory response. Collectively, our findings identify RC-101 as a potential preventative of S. aureus nasal colonization.
Show less - Date Issued
- 2011
- Identifier
- CFE0004124, ucf:49092
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0004124
- Title
- Behavioral and disease ecology of Gopher Tortoises (Gopherus polyphemus) post exclusion and relocation with a novel approach to homing determination.
- Creator
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Napier, Johnathan, Savage, Anna, Moore, Sean, Vonkalm, Laurence, Fedorka, Kenneth, University of Central Florida
- Abstract / Description
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In the wake of human expansion, relocations and the loss of habitat can be stressful to an organism, plausibly leading to population declines. The gopher tortoise (Gopherus polyphemus) is a keystone species that constructs burrows it shares with 362 commensal species. Frequent exclusions and relocations and long generation times have contributed to G. polyphemus being State-designated as Threatened in Florida. Prior studies have indicated that G. polyphemus may possess homing behavior and...
Show moreIn the wake of human expansion, relocations and the loss of habitat can be stressful to an organism, plausibly leading to population declines. The gopher tortoise (Gopherus polyphemus) is a keystone species that constructs burrows it shares with 362 commensal species. Frequent exclusions and relocations and long generation times have contributed to G. polyphemus being State-designated as Threatened in Florida. Prior studies have indicated that G. polyphemus may possess homing behavior and thus be able to counteract stressors due to relocation and exclusion. I radiotracked a cohort of G. polyphemus for 11 months following excavation, relocation, and exclusion due to a pipeline construction project. In conjunction with analyzing G. polyphemus movement patterns post-release, I developed novel statistical methodologies with broad application for movement analysis and compared them to traditional analyses. I evaluated habitat usage, burrowing behavior, movements, growth, and disease signs among control versus relocated and excluded individuals and among sexes and size classes, forming predictors for behavior and disease risk. I found statistical support that my new methodology is superior to previous statistical tests for movement analyses. I also found that G. polyphemus engages in homing behavior, but only in males. Behavioral differences were also found between the sexes with respect to burrowing behavior. Overall health, disease prevalence, and immune response were unaffected by relocation and exclusion, nor were they statistically correlated. Signs were unreliable as etiological agents, outperformed by serological detection. I determined that the Sabal Trail pipeline as a potential stressor did not affect movement behavior, homing, nor the disease/immune profile of G. polyphemus in this study.
Show less - Date Issued
- 2018
- Identifier
- CFE0007581, ucf:52581
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0007581
- Title
- Analysis of the Effects of Formative Assessment in Promoting Transfer of Learning in an Undergraduate General Microbiology Laboratory Course.
- Creator
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Rediske, Andrea, Butler, Malcolm, Boote, David, Sivo, Stephen, Chini, Jacquelyn, Moore, Sean, University of Central Florida
- Abstract / Description
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The undergraduate microbiology lab serves an important role in establishing a foundation of best practices in aseptic technique and infection control for pre-medical, pre-nursing, pre-pharmacy, or pre-allied health students. The high incidence of hospital-acquired infections (HAIs) in the US and evidence in the literature of less effective implementation of proper aseptic technique among apprentice doctors and nursing students suggests that more effective transfer of learning could improve...
Show moreThe undergraduate microbiology lab serves an important role in establishing a foundation of best practices in aseptic technique and infection control for pre-medical, pre-nursing, pre-pharmacy, or pre-allied health students. The high incidence of hospital-acquired infections (HAIs) in the US and evidence in the literature of less effective implementation of proper aseptic technique among apprentice doctors and nursing students suggests that more effective transfer of learning could improve implementation of these procedures in the clinical setting. The research described in this study aimed to assess learning transfer as it applied to aseptic techniques and infection control skills learned in the undergraduate microbiology lab from pre- and post-lab formative assessments to midterm and lab practical summative assessments. Assisting students in building connections between the aseptic techniques learned in general microbiology and their application in the clinical setting through pre-lab formative assessments and reflective practices may lead to improvements in use of aseptic techniques and infection control measures as they progress into clinical careers and may ultimately reduce infection rates and mortality rates due to HAIs.The first major aim of this study was to explore the experiences of students with respect to learning transfer through qualitative analysis of student responses to post-lab free-response questions regarding difficulties faced in the lab and the relevance of microbiology to students' future careers. The second major aim of this study was to determine if the implementation of an in-class pre-lab formative assessment facilitateslearning transfer as evidenced by significant improvements on summative lab midterm and final lab practical exam scores.Qualitative analysis of student responses to open-ended reflection questions indicated evidence of predominantly low-road transfer with respect to transfer of automaticity. Additionally, qualitative analysis of student responses indicated evidence of lateral transfer regarding transfer of complexity. Finally, there was evidence of an evolution from near to far transfer of context indicating that students were able to perceive the application of the knowledge gained in the microbiology lab in contexts similar to the lab as well as contexts outside of the lab. Evidence from student responses suggested that primarily students intending to pursue careers in healthcare fields were able to perceive specific applications of the microbiology lab to their future careers. Further, evidence from student responses suggested that students predominantly had difficulties with procedures, interpretation of results, manual dexterity with microbiological equipment and materials, and expressed the need to practice these procedures and techniques.Statistical analyses provided quantifiable evidence that the implementation of pre-lab quizzes had both a statistically significantly positive impact and a practically positive impact on lab practical final scores in both of the semesters studied as compared to historical control groups with a large effect size. The statistically and practically significant impact of the pre-lab quizzes on lab practical final exams is an important finding and will add to the current literature on the importance of formative assessment in undergraduate microbiology education.
Show less - Date Issued
- 2017
- Identifier
- CFE0006784, ucf:51824
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006784
- Title
- The role of a highly conserved eubacterial ribosomal protein in translation quality control.
- Creator
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Naganathan, Anusha, Moore, Sean, Cole, Alexander, Teter, Kenneth, Roy, Herve, Koculi, Eda, University of Central Florida
- Abstract / Description
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The process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of...
Show moreThe process of decoding is the most crucial determinant of the quality of protein synthesis. Ribosomal protein L9 was first implicated in decoding fidelity when a mutant version of L9 was found to increase the translation of a T4 phage gene. Later studies confirmed that the absence of L9 leads to increased translational bypassing, frameshifting, and stop codon readthrough. L9 is part of the large subunit of the prokaryotic ribosome and is located more than 90 (&)#197; from the site of decoding, making it difficult to envision how it might affect decoding and reading frame maintenance. Twenty years after the identification of L9's putative function, there is no mechanism for how a remotely located L9 improves translation fidelity. This mystery makes our picture of translation incomplete. Despite the high conservation of L9 in eubacteria, E.coli lacking L9 does not exhibit any obvious growth defects. Thus, the evolutionary advantage conferred by L9 in bacteria is masked under laboratory conditions. In order to uncover unique L9-dependent conditions, a library of E. coli mutants was screened to isolate those that rely on L9 for fitness. Interestingly, factors found to be synergistic with L9 had no known role in fidelity. Six independent mutants were isolated, each exhibiting a severe growth defect that is partially suppressed in the presence of L9. One class of L9-dependent mutations was present in an essential ribosome biogenesis factor, Der. Der's established function is in the maturation of the large ribosomal subunit. The identified mutations severely impaired the GTPase activity of Der. Interestingly, L9 did not directly compensate for the defective GTPase activity of mutant Der. The second class of L9-dependent mutations was present in EpmA and EpmB, factors required to post-translationally modify elongation factor, EF-P. EF-P's established function is in the translation of poly-proline containing proteins. EF-P deficient cells were nearly inviable in the absence of L9; however, L9 did not directly influence poly-proline translation. Therefore, in each case, L9 improved cell health without altering the activity of either Der or EF-P. Remarkably, the der mutants required only the N domain of L9, whereas the absence of active EF-P required full-length, wild-type L9 for growth complementation. Thus, each mutant class needed a different aspect of L9's unique architecture. In cells lacking either active EF-P or Der, there was a severe deficiency of 70S ribosomes and the indication of small subunit maturation defects, both of which worsened upon L9 depletion. These results strongly suggest that L9 plays a role in improving ribosome quality and abundance under certain conditions.Overall, the genetic screen lead to the discovery that bacteria need L9 when either of two important translation factors (Der or EF-P) is inactivated. This work has characterized the physiological requirement for L9 in each case and offers a new insight into L9's assigned role in translation fidelity.
Show less - Date Issued
- 2015
- Identifier
- CFE0005674, ucf:50169
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0005674
- Title
- Monitoring Crystal Structure Refinements Using Solid-State NMR Chemical Shift Tensors.
- Creator
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Kalakewich, Keyton, Harper, James, Campiglia, Andres, Elsheimer, Seth, Chumbimuni Torres, Karin, Masunov, Artem, Moore, Sean, University of Central Florida
- Abstract / Description
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Inclusion of lattice-fields in density functional theory (DFT) methods has enabled the accurate calculation of solid-state nuclear magnetic resonance (SSNMR) chemical shift tensors. Calculated 13C and 15N tensors (i.e. 3 principle values per nucleus) can be used to monitor crystal structure refinements and to select the correct structure from a large population of computationally generated candidates. In this dissertation, chapter 2 describes a methodology to improve established crystal...
Show moreInclusion of lattice-fields in density functional theory (DFT) methods has enabled the accurate calculation of solid-state nuclear magnetic resonance (SSNMR) chemical shift tensors. Calculated 13C and 15N tensors (i.e. 3 principle values per nucleus) can be used to monitor crystal structure refinements and to select the correct structure from a large population of computationally generated candidates. In this dissertation, chapter 2 describes a methodology to improve established crystal structures from three different diffraction techniques involving geometric refinement monitored using SSNMR tensor values. The calculated 13C tensors for three relatively simple organic compounds (i.e. acetaminophen, naphthalene, and adenosine) are shown to markedly improve upon DFT refinement. The so-called GGA-PBE functional provided the best agreement with experimental data. The use of the three principle values of the tensor is required for such results as the average (i.e. the isotropic) is less accurate. Chapter 3 applies this method to differentiate between hundreds of computationally predicted crystal structures. Typically, lattice energy of each candidate is used to select the correct structure, a process which is seldom successful. Herein, it is demonstrated that when 13C tensors from DFT refined structures are used for structural ranking by comparison to experimental data, only the correct structure agrees with experimental data in all cases. Chapter 4 illustrates the use of 15N tensors to monitor DFT refinement as an alternative to the 13C approach of Chapter 2. 15N tensors have been very difficult to obtain previously, thus a novel experimental method is developed here which improves signal-to-noise by as much as 300% and allows routine measurement. This improvement also improves the accuracy of the tensor values. Overall, the 15N tensors are found to be at least 5 times more sensitive to DFT refinements than 13C values.
Show less - Date Issued
- 2017
- Identifier
- CFE0006888, ucf:51726
- Format
- Document (PDF)
- PURL
- http://purl.flvc.org/ucf/fd/CFE0006888