Carolina Digital Repository

    The Yersinia pestis autotransporter YapG contains a fast folding [beta]-helix domain

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    Autotransporter proteins are the most widely secreted protein family in gram-negative bacteria; their passenger domains are predicted to be [beta]-helical in 97% of cases. The [beta]-helical fold has been hitherto understudied with respect to protein folding, which typically is centered on small [alpha]-helical, low contact order proteins. In contrast, the [beta]-helical portions of passenger domains are typically large, and made up of unique structural repeats with high contact order. Here, we have studied the in vitro folding of the passenger domain of YapG, an autotransporter from Yersinia pestis, via thermodynamic and kinetic approaches. We have identified YapG as the fastest refolding passenger domain to date. Steady-state fluorescence and circular dichroism indicate a one-step folding process; however, stopped-flow fluorescence indicates a one-step unfolding and a two-step refolding. Neither proline isomerization nor aggregation is associated with YapG refolding, suggesting this fast folding [beta]-helix may experience a general collapse followed by a slower fine tuning folding step. In addition, gel filtration studies of the refolded state indicate that YapG may refold into two different folded species. Taken together, these results provide the first biophysical analysis of an autotransporter passenger domain from Y. pestis and provide new insight into the folding process in [beta]-helical folds

    Defining the Role of the Cytokinin Response Factors (CRFs) in Arabidopsis thaliana

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    Cytokinin is a phytohormone that plays an integral role in regulating the growth, development and physiological responses of a plant. Among the processes linked to cytokinin signaling are meristem maintenance, root growth, rosette size, seed count, pathogen defense and leaf senescence. The signaling pathway consists of a hybrid two-component system consisting of the histidine kinase receptors, phosphotransfer proteins and response regulators. Numerous genes are activated or repressed downstream of the primary signaling pathway. A large number of these genes are characterized as transcription factors and therefore drive appropriate downstream gene expression in response to cytokinin. This transcriptional cascade is complex and consists of many components and feedback loops, many that have yet to be determined The Cytokinin Response Factors (CRFs) are a family of genes activated downstream of the cytokinin signaling pathway and were first identified by their induction in response to cytokinin. The CRFs are members of the AP2/ERF transcription factor family, one of the largest found in plants. Through mutant analysis, we show that the CRFs negatively regulate several cytokinin related processes, dependent on their interaction with the Arabidopsis Histidine Phosphotransfer proteins (AHPs). The CRFs positively regulate meristem size, hypocotyl elongation in the dark and the rate of leaf senescence. Additionally, through expression analyses and protein binding microarray, we were able to uncover some of the downstream processes and targets with which the CRFs are involved. We show that CRFs bind the GCC box with high affinity, as is common of other AP2/ERF members. Microarray results show the CRFs regulate genes involved in many downstream processes and also regulate some of the cytokinin signaling components. Combining both phenotypic and expression analyses, we are able to elucidate many developmental processes in which the CRFs participate, as well as to define them as negative regulators of many cytokinin regulated processes.Doctor of Philosoph

    Improving the validity of nonexperimental comparative effectiveness research: The impact of calendar time on prescribing of novel chemotherapeutic therapies for stage III colon cancer

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    Oxaliplatin was rapidly adopted for stage III colon cancer treatment after FDA approval in 2004. Uncertainty remains regarding oxaliplatin's superiority to the former chemotherapeutic standard in older patients, the most affected population. The relationship between calendar time and treatment receipt during oxaliplatin's dissemination presents a challenging yet rich methodological research opportunity for comparative effectiveness research (CER). Stage III colon cancer patients aged 65+ initiating chemotherapy from 2003-2008 were studied using U.S. population-based cancer registry data linked with Medicare claims. We examine changes in treatment receipt using a novel calendar time-specific (CTS) propensity score (PS), which allows covariate predictive values to change over time. We compare this method and a calendar time instrumental variable (IV) with traditional adjustment to enhance understanding of oxaliplatin effectiveness for reducing cancer mortality, a strong driver of all-cause mortality among stage III patients. PSs for treatment receipt were constructed using logistic models with key components of demographics, tumor substage, grade, and comorbidities. The CTS PS was used to match oxaliplatin-treated and untreated patients within 1-year intervals. The two-level calendar time instrument was anchored at oxaliplatin's approval and based on IV strength and plausibility of assumptions. PS-matched hazard ratios (HR) were estimated using Cox models. Risk differences (RD) were derived from Kaplan-Meier survival curves. CTS PS and IV results were compared with conventional PS-matched estimates. Oxaliplatin use increased considerably during the study timeframe, with 8% receipt in the first time period vs. 52% in the last (N=2800). Channeling by comorbidities, income, and age appeared to change over time. The CTS PS improved covariate balance within calendar time strata and yielded an attenuated estimated benefit of oxaliplatin (HR=0.75) compared with the conventional PS (HR=0.69). The calendar time instrument resulted in 54% compliance (N=2881). The 3-year IV RD (95% confidence interval) was -0.09 (-0.15,-0.03) favoring oxaliplatin; PS-adjusted RD was -0.04 (-0.08,-0.01). All analyses indicated better survival among oxaliplatin-treated patients. These consistent results based on differing assumptions lend plausibility to the conclusion that oxaliplatin retains effectiveness among older stage III patients. In nonexperimental CER of emerging therapies, calendar time's role as a confounder or instrument should be carefully considered.Doctor of Philosoph

    The role of human somatosensory cortex in tactile stimulus processing: fMRI responses to microstimulation of individual tactile afferents

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    Mounting evidence shows that primary somatosensory cortex, particularly SI, is actively engaged in dynamic processing of sensory information evoked by skin stimulation. SI plays both active and modulating roles in amplitude and frequency discrimination as well as in the processing of noxious mechanical and thermal stimuli. SI is one cortical region in which interactions between cortical activity evoked by low frequency flutter and high frequency vibration are spatially and temporally integrated. In this dissertation, functional magnetic resonance imaging was used to image cortical responses to microstimulation of individual, functionally identified skin mechanoreceptive afferents which are, under natural stimulus conditions, effectively activated by very selective ranges of stimulation frequency. Microstimulation of mechanoreceptive and nociceptive afferents evoked significant activation in multiple cortical areas. Peak magnitudes of hemodynamic responses in SI and SII did not differ significantly when rapidly adapting afferent microstimulation frequency was increased from 30 to 200Hz. Thus, rapidly adapting afferent activation alone cannot account for the suppression of SI cortical activity that is observed when high frequency vibrotactile stimuli are applied to the skin; this leads to the hypothesis that PC microstimulation causes suppression of SI cortical activity regardless of stimulus frequency. Images of activity evoked by microstimulation of PC afferents support this hypothesis. Additionally, microstimulation of nociceptive afferents evoked significant activation in multiple cortical areas consistent with animal studies utilizing invasive data collection methods. Future studies of hemodynamic responses to vibrotactile stimulation and noxious stimulation will clarify human cortical mechanisms responsible for the spatial-temporal integration evoked by different modalities of skin stimulation. The construction and utilization of an MRI-compatible vibrotactile stimulator that can be used in such future studies is also described in this manuscript

    Hydrostatic skeletons in the Crustacea: support during molting in an aquatic and a terrestrial crab

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    All animals require a skeletal support system for posture and locomotion. Arthropods, however, repeatedly shed their rigid exoskeleton in order to grow, yet they maintain shape and mobility during these periods. My research focuses on this apparent paradox and suggests that crabs, and possibly all arthropods, alternate between a rigid and a hydrostatic (fluid-based) skeleton in order to remain functional during molting. I tested for the use of hydrostatic skeletal support in blue crabs, Callinectes sapidus, by simultaneously measuring internal hydrostatic pressure and force of claw adduction. I found a strong correlation between force and hydrostatic pressure in soft-shell crabs, but not in hard-shell crabs, which is consistent with the use of hydrostatic support during molting. Switching skeletons requires a change in function of the cuticle, from resisting primarily bending, compression, and torsion, to resisting tension. This change in function implies correlated changes in the properties of the cuticle. I tested the mechanical properties of the cuticle throughout the molt cycle of C. sapidus and found that the flexural and tensile stiffness is greater in hard cuticle than soft cuticle, but the tensile strength is the same. The blackback land crab, Gecarcinus lateralis, does not molt in water and inflates its gut with air during molting, which may serve as a support mechanism. I simultaneously measured the force of claw flexion, hydrostatic pressure within the claw, and gas pressure within the gut. I obtained a strong correlation between all three measurements, which suggests that the gas helps maintain turgidity throughout the body, and thus acts as a critical component of the skeleton. Rigid and hydrostatic skeletons operate according to different principles and each is likely to be influenced by scale in distinct ways. Using morphological techniques, I found that cuticle thickness scales isometrically for rigid skeletons but allometrically for hydrostatic skeletons, suggesting that both play a role in determining growth to maximum size in crabs. This research provides novel insights into how skeletal support systems influence the way in which animals are built, develop, and function

    On cosmos and concepts

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    Priority monists maintain that the most fundamental concrete thing is the entire cosmos. However, in contrast with existence monists, priority monists hold that other objects, including medium-sized objects such as tables, exist in exactly the same way as the cosmos. They hold the metametaphysical view that the job of metaphysics is to organize the ontology of the world into hierarchical relations; they are neo-Aristotelians. And, on the most natural reading of priority monism, wholes are more fundamental than parts. Here, I attempt to show that these commitments lead the priority monist into an inescapable trilemma. My argument will focus upon the fact that a priority monist must make room in her ontology for a certain kind of structured mental object, i.e. concepts. But, I shall argue, because of the nature of concepts, every way a priority monist can possibly fit them into her hierarchical ontology leads her to untenable conclusions

    Characterization of a Novel Mycobacterium Tuberculosis-Simian Immunodeficiency Virus Vaccine to Prevent Oral Pediatric HIV Transmission

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    Over 3.3 million children are living with HIV, infected primarily by mother-to-child transmission (MTCT). Breast milk exposure of HIV accounts for up to 44% of MTCT events. Despite serious efforts to prevent vertical HIV transmission, infant testing is often delayed and access to antiretroviral therapies is still limited. Compared to adults, HIV-infected infants experience enhanced disease progression and more severe co-morbidities with pathogens like Mycobacterium tuberculosis (Mtb). The live attenuated BCG vaccine is the only licensed tuberculosis vaccine but BCG can disseminate in immunosuppressed, HIV-infected infants. Considering the significant geographical overlap of the HIV and TB epidemics and the high vulnerability of infants to both pathogens, a pediatric vaccine to safely protect against HIV and Mtb is urgently needed. We hypothesized that human-adapted attenuated Mtb strains engineered to co-express HIV genes (rAMtb-HIV) would safely induce the development of dually-immunogenic HIV- and Mtb-specific cellular and humoral responses. Three distinct attenuated Mtb-SIV strains were evaluated in the neonatal rhesus macaque model. Oral priming with strain mc26435 plus systemic MVA-SIV boosts successfully induced the development of Mtb- and SIV-specific cellular and humoral responses and was identified to be safe in healthy and immunosuppressed, SIV-infected neonatal macaques. However, despite vaccine-induced immunity, vaccination did not protect infants against low-dose oral SIV challenges designed to mimic oral MTCT during breastfeeding. Two important results emerged from the challenge study. First, higher Env-specific mucosal IgA activities and plasma IgG avidities positively correlated with controlled viremia in a subset of vaccinated animals. These animals also maintained peripheral CD4+ T cell populations and IL-17-expressing lymphocytes in the intestinal mucosa. However, the majority of vaccinated animals required fewer low-dose SIV exposures to become infected than unvaccinated animals. Enhanced viral acquisition was associated with vaccine-induced persistent immune activation. At the time of challenge, CCR5-expressing CD4+ T cells were observed with greater frequencies in blood, oral and intestinal tissues in vaccinated animals only, providing increased frequencies of SIV target cells. Due to the potential impact of these data on BCG vaccine safety and pediatric HIV and TB vaccine development, additional studies are required to confirm these complex and intriguing results.Doctor of Philosoph

    Exploring RNA and protein 3D structures by geometric algorithms

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    Many problems in RNA and protein structures are related with their specific geometric properties. Geometric algorithms can be used to explore the possible solutions of these problems. This dissertation investigates the geometric properties of RNA and protein structures and explores three different ways that geometric algorithms can help to the study of the structures. Determine accurate structures. Accurate details in RNA structures are important for understanding RNA function, but the backbone conformation is difficult to determine and most existing RNA structures show serious steric clashes (greater than or equal to 0.4 A overlap). I developed a program called RNABC (RNA Backbone Correction) that searches for alternative clash-free conformations with acceptable geometry. It rebuilds a suite (unit from sugar to sugar) by anchoring phosphorus and base positions, which are clearest in crystallographic electron density, and reconstructing other atoms using forward kinematics and conjugate gradient methods. Two tests show that RNABC improves backbone conformations for most problem suites in S-motifs and for many of the worst problem suites identified by members of the Richardson lab. Display structure commonalities. Structure alignment commonly uses root mean squared distance (RMSD) to measure the structural similarity. I first extend RMSD to weighted RMSD (wRMSD) for multiple structures and show that using wRMSD with multiplicative weights implies the average is a consensus structure. Although I show that finding the optimal translations and rotations for minimizing wRMSD cannot be decoupled for multiple structures, I develop a near-linear iterative algorithm to converge to a local minimum of wRMSD. Finally I propose a heuristic algorithm to iteratively reassign weights to reduce the effect of outliers and find well-aligned positions that determine structurally conserved regions. Distinguish local structural features. Identifying common motifs (fragments of structures common to a group of molecules) is one way to further our understanding of the structure and function of molecules. I apply a graph database mining technique to identify RNA tertiary motifs. I abstract RNA molecules as labeled graphs, use a frequent subgraph mining algorithm to derive tertiary motifs, and present an iterative structure alignment algorithm to classify tertiary motifs and generate consensus motifs. Tests on ribosomal and transfer RNA families show that this method can identify most known RNA tertiary motifs in these families and suggest candidates for novel tertiary motifs

    Noble simplicity and quiet grandeur: Franz Schubert's settings of Johann Mayrhofer's neoclassical poems

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    Historians, artists, architects, linguists, and politicians in nineteenth-century Germany and Austria were fascinated with Greco-Roman antiquity. The neoclassical movement in German and Austrian art, which was largely inspired by Johann Joachim Winckelmann, found its way into music through poetry. Franz Peter Schubert, the preeminent Austrian art song composer of the early nineteenth-century, composed several Lieder that illustrate the prominent place that Greek classicism had in the arts during his lifetime. Of Schubert's many neoclassical settings, those based on poems by Johann Baptist Mayrhofer, taken collectively, most closely embody the spirit of Greek classicism as it was understood at the time. Following an examination of the neoclassical movement within Germany and the life of Mayrhofer, I discuss four of Schubert's songs that demonstrate, in varying degrees, the classical Greek ideal developed by Winckelmann: Memnon, Philoktet, Iphigenia, and Der zürnenden Diana

    Religious Transgression and Monarchy in Herodotus’ Histories

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    This thesis investigates the role of transgressions of a religious nature in Herodotus’ Histories, beginning with a consideration of modern and ancient terminology, Herodotus’ cultural relativism, and the complicated interplay of fate and the gods within human affairs. The examination of episodes involving such transgressions reveals that these acts are particularly associated with individuals wielding power and authority, and that a transgressive relationship with religion is a symptom of monarchy, rather than of ethnicity or culture. I analyze Herodotus’ depiction of several monarchs who commit, or avoid committing, religious transgressions. These figures provide several interpretative options for readers regarding the singular character of Xerxes, and thus contribute to Herodotus’ nuanced presentation of this last Persian king, and his motives for engagement against the Greeks.Master of Art
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