Molecular Studies of SARM1, an Axonal Self-Destruction Switch

Gerdts, Josiah Washington University in St. Louis 2016 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Neurosciences Washington University in St. Louis, 2016 Professor Jeffrey Milbrandt, Chairperson Axons are neuronal extensions that make up the wiring of the nervous system. Recent studies have revealed that axons possess a unique and largely uncharacterized self-destruction program that may be a therapeutic target for diseases in which axon loss contributes to disability. We have developed a scalable assay of axon degeneration that allows quantitative screening for agents that alter axon degeneration. From a library of bioactive compounds, we have identified and characterized several novel axon-protective compounds. Using this system we screened a genome-scale lentiviral shRNA library to identify proteins that promote axon degeneration following injury. A top hit in this screen was the protein SARM1, and validation experiments and the work of others confirm that SARM1 is a central regulator of axon destruction. Combining structure-function and biochemical studies, we have developed a working model of SARM1-mediated axon destruction: SARM1 complexes are present within axons and are held inactive by an auto-inhibitory N-terminus. Following injury, SARM1 activation leads to dimerization of the SARM1 Toll-Interleukin Receptor (TIR) domain, which is sufficient to trigger a program of rapid NAD+ breakdown leading to axon destruction.

The role of the clock gene Bmal1 in female fertility and parturition

Ratajczak, Christine Kathleen Washington University in St. Louis 2010 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Successful reproduction is essential for survival of species. For humans, reproductive problems can cause large emotional and medical burdens. Evidence in both humans and rodents indicates that circadian rhythmicity is important for supporting reproductive function. A molecular clock orchestrates circadian rhythmicity. Impairment in fertility and parturition are observed in female mice expressing a mutant form of Clock, suggesting critical roles for clock genes in reproduction. Since the clock gene Bmal1, but not Clock, is necessary for the generation of circadian rhythmicity, exploring the role of Bmal1 in reproduction may yield a better understanding of the importance of the molecular clock in this process. We characterized the reproductive phenotype of Bmal1-/- females. These females were infertile, and our data suggests that implantation failure due to impaired steroidogenesis is a major contributor to this infertility. Expression of Bmal1 and other clock genes has been demonstrated in such tissues important for the early stages of the reproductive process as the ovary, oviduct, GnRH neurons, and non-gravid uterus. However, the expression of clock genes in tissues involved in the later stages of gestation has been largely unexplored. We determined that Bmal1 and other core clock genes are expressed in the gravid uterus, placenta, and fetal membranes of wild-type mice during the last third of gestation. Many of these genes were expressed in a rhythmic fashion throughout the circadian day suggesting the presence of operating peripheral molecular clocks in these tissues. The study of the role of Bmal1 in the late stages of the reproductive process is complicated by the infertility experienced by Bmal1-/- mice. To circumvent the problem of infertility and to examine the role of peripheral tissue Bmal1 expression in late gestation, we generated conditional Bmal1 knockout mice. These mice were used in concert with Telokin-Cre mice to disrupt Bmal1 specifically in the myometrium, the muscle portion of the uterus. Myometrial Bmal1 disruption was found to result in a disregulation of labor timing and altered expression of contractile-associated proteins. Results presented here demonstrate an importance of the core clock gene Bmal1 in both early and late stages of the reproductive process. These studies may lead to a better understanding of the roles of clock genes in supporting normal reproductive function in women.

The role of limbic glucocorticoid receptors and corticotropin-releasing hormone in the modulation of normal and pathological stress adaptation

Kolber, Benedict James Washington University in St. Louis 2008 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

The amygdala and hippocampus are key limbic areas involved in the proper adaptation to stress. During stressful conditions, these areas are modulated by glucocorticoids controlled by the hypothalamic pituitary adrenal (HPA) axis. Alterations in HPA axis-regulated molecules such as the glucocorticoid receptor (GR) and the peptide corticotropin-releasing hormone (CRH) are hypothesized to be involved in the pathogenesis of a variety of mood disorders, including major depressive disorder. In this dissertation we took two approaches to address the role of the HPA axis in modulating stress related behavior and physiology. First, we investigated the role of GR in the formation of fear responses and Pavlovian conditioning. To gain insights into the molecular mechanisms and sub-regional specificity of fear conditioning, we disrupted GRs in the central nucleus of the amygdala (CeA) by delivering lentiviral vectors containing Cre-recombinase into floxed-GR mice. GR deletion in the CeA (CeAGRKO mice) prevented conditioned fear behavior. The conditioned fear deficit in CeAGRKO mice was associated with decreases in cFos and corticotropin-releasing hormone (CRH) expression. Moreover, intracerebroventricular delivery of CRH rescued the conditioned fear deficit in CeAGRKO mice. As a second approach, we studied the role of early CRH exposure in the long-term organization of the stress adaptation system. We overexpressed CRH in the forebrain of developing mice and then measured behavioral and HPA axis changes in adult animals. CRH overexpressing mice (FBCRHOE) exposed to CRH until weaning at postnatal day 21 demonstrate increases in anxiety-like and despair-like behavior. These behavioral changes were not associated with adult HPA axis changes but were reversed with antidepressant treatment. From these studies, we have established an important role for GR activation and CRH signaling in the behavioral response and long-term modulation of the HPA axis to acute stress.

Leadership acceptance: Context, cognitive style and the background of IT professionals as determinates of IT leadership

Williamson, John A Washington University in St. Louis 2007 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

This study is about the leadership of Information Technology (IT) within organizations. It is framed in the notion that effective IT leadership results from followership: that leaders cannot lead without the willingness of followers to follow. Thus, it will focus on the IT staff professionals' views of effective IT leadership and how it is influenced by organizational context and their own background and cognitive thinking style. A 4-part questionnaire was administered to 68 subjects from Masters level classes at a Midwestern university. The information was then analyzed to determine how context (operational vs. strategic), cognitive style (analytical vs. intuitive) and background affected the selection of desirable CIO attributes. The study indicates that individuals with technical backgrounds prefer technical leadership. Context has influence when examined in concert with an IT professionals' experiential background. It was found that technical IT professionals, and in particular younger professionals, consider technical CIO attributes important in strategic scenarios over operational scenarios.

Modern Assessment of the High-Energy Background Environment at Small Atmospheric Depths Using the X-Calibur X-Ray Polarimeter and Its Implications

Amini, Rashied Baradaran Washington University in St. Louis 2016 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Since the discovery of cosmic rays, Earth's upper atmosphere at depths of 1-10 g cm--2 has been used for balloon-borne observations probing the high-energy universe. However, the interaction of cosmic rays with the earth atmosphere generates numerous particles, each with their own flux and interaction physics that contribute to instrument background. In Fall 2014, the X-Calibur X-ray polarimeter designed and built at Washington University in St. Louis was launched from Ft. Sumner, New Mexico. While no astrophysical observation was performed as a result of a failure in telescope mechanisms, X-Calibur was able to record hours of instrument background with an energy resolution of sigmaE ~ keV at a depth of 3.45 g cm--2. Using the 2014 X-Calibur data, existing observations of atmospheric gamma-rays, hadrons, and leptons, and the MEGAlib/GEANT particle transport simulation environments, I develop and perform preliminarily validation of detailed background models in the regime of 1 keV-100 GeV. The models are generally constructed by involving experimental data with atmospheric mass and composition models rather than integration of source/emission functions or complete Monte Carlo simulation of cosmic rays entering at the top of the atmosphere. Notably, these models are derived as functions of energy, off-zenith angle to degree precision, geomagnetic latitude, and solar modulation factor enabling predictions of instrument background for future X-Calibur flights. These background models are also used to optimize the shielding conguration through additional passive material or active components as constrained by the existing active shield.

The Regulation of Glucose Metabolism during Osteoblast Differentiation

Esen, Emel Washington University in St. Louis 2014 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Differentiation and cell-specific functions are coupled with metabolic alterations to meet the needs of the cell. In this thesis, I have investigated the alterations of cellular metabolism in osteoblast-lineage cells in response to two different bone anabolic signals, WNT and PTH. I have further elucidated the mechanism underlying the metabolic changes, and have explored the functional importance of such changes for bone anabolism. Osteoblasts, the principal bone-forming cells, are differentiated from mesenchymal progenitor cells through sequential stages. These stages are identifiable by molecular markers, cell morphology and location. Transcription factors and developmental signals important for osteoblast differentiation have been studied in detail. One such developmental signal is the WNT family of proteins. WNTs are a large family of glycoproteins that activate beta-catenin-dependent or -independent intracellular pathways, both of which are involved in bone formation. However, the mechanism through which WNT signaling stimulates osteoblast differentiation is not well understood. Early studies demonstrated that bone cells consume a large amount of glucose, producing lactate as the major end product even in aerobic conditions, a phenomenon known as aerobic glycolysis. However, the significance of increased aerobic glycolysis for bone formation was not known. Based on the link between metabolic abnormalities and the genetic mutations in WNT pathway components, I hypothesized that WNT regulates cellular metabolism and that such regulation contributes to osteoblast differentiation. I tested this hypothesis in vitro by using ST2 cells, and showed that WNT signaling increased glucose utilization, stimulated aerobic glycolysis via induction of glycolytic enzymes, and suppressed glucose entry to TCA cycle. This process was mostly regulated by a signaling cascade dependent on Lrp5-Rac1-mTORC2 and independent of beta-catenin. Increased glycolysis was important for in vitro osteoblast differentiation and correlated with increased bone formation in WNT hyperactivation mouse models. I tested the functional importance of enhanced aerobic glycolysis in vivo by two different models. First, I showed that pharmacological enhancement of pyruvate entering the TCA cycle attenuated the high-bone mass phenotype caused by hyperactive WNT signaling in the mouse. Second, I showed that genetic deletion of LDHA, the enzyme catalyzing the last step of glycolysis, from osteoblast-lineage cells suppressed normal postnatal bone accrual due to reduced osteoblast number and function. Thus, WNT signaling reprograms glucose metabolism, and WNT-induced metabolic reprogramming contributes to osteoblast differentiation both in vitro and in vivo. Moreover, LDHA is required for optimal bone formation in postnatal mice. Parathyroid hormone (PTH) has been an effective bone anabolic drug in the clinic by targeting osteoblasts and stimulating bone formation. However, it is not well understood how PTH signaling stimulates bone formation. In early studies, PTH was shown to alter cellular metabolism towards lactate production. In light of the role of metabolic regulation in WNT-induced bone formation, I examined the potential role of metabolic alterations in mediating the anabolic effect of PTH. In MC3T3-E1 cells and neonatal calvarial cells, I showed that PTH enhanced glucose uptake and aerobic glycolysis, activated pentose phosphate pathway but reduced contribution of glucose to TCA cycle. PTH-induced glucose utilization required IGF-PI3K-SGK1 signaling. Importantly, pharmacological enhancement of pyruvate entering the TCA cycle attenuated the bone anabolic effect by PTH. Thus, changes in cellular glucose metabolism may be an important mechanism mediating the anabolic effect of PTH. This thesis confirms the earlier findings that lactate-producing glycolysis is an important feature of osteoblasts, and further characterizes the alterations of cellular metabolism during osteoblast differentiation in response to both WNT and PTH pathways. More importantly, this thesis shows for the first time that metabolic alterations are functionally important for the differentiation process.

Discovery and characterization of WU polyomavirus, a novel virus detected in pediatric respiratory tract infections

Gaynor, Anne Margaret Washington University in St. Louis 2010 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Respiratory tract infections are responsible for greater than 4 million deaths per year, making them the leading cause of mortality due to an infectious disease. Despite the availability of improved diagnostic methods a known pathogen cannot be identified in up to 30% of respiratory infections. This dissertation is a direct result of our laboratory's hypothesis that a fraction of these respiratory cases of unknown cause are the result of as yet-to-be identified viral agents. As part of our laboratory's efforts to identify unknown viral agents I have focused on analyzing samples from patients with respiratory illnesses of unknown etiology. A high throughput sequencing method was utilized in the identification of WU Polyomavirus (WUV), a novel polyomavirus detected in the respiratory tract of a child with pneumonia of unknown etiology. I subsequently sequenced the entire virus genome of 5229bp and found it to have genomic features characteristic of the family Polyomaviridae. Phylogenetic analysis clearly revealed that WUV was divergent from all known polyomaviruses. Following this discovery, I hypothesized that WUV is likely a human pathogen. The goal of my dissertation work was to systematically address questions that would provide insight about the biology of WUV and shed light on the role of WUV in human disease. The first question that I attempted to address was, "Is WUV associated with respiratory disease?" In two separate studies WUV was detected in a total of 114/4772 (2.4%) samples from patients with acute respiratory tract infections in St. Louis, United States and Brisbane, Australia. WUV was detected predominantly in children, but the eldest patients (12-51yrs) had compromised immune systems reflecting the paradigms for the related pathogenic human polyomaviruses, BKV and JCV. The second question to address was: "Which cells, tissues, and organs in the human body support replication of WUV?" This is a very broad question with critical importance to understanding the biology of WUV. However, given the expansive scope I focused on identifying cell lines permissive for WUV replication using a full length genomic clone (cWUV-wt). Co-transfection of cWUV-wt and a plasmid containing the WUV early region (pER) into Vero cells was sufficient to recapitulate viral replication, late protein expression and generation of viral like particles of the appropriate size and morphology for a member of the Polyomaviridae family. I have identified a novel human virus in respiratory secretions that may be able to explain a portion of the cases of respiratory infection of unknown cause. The discovery, characterization and development of tools to study WU polyomavirus lay the foundation for further studies to investigate its role in human disease.

Probing Intergalactic Magnetic Fields from gamma-Ray Observations

Chen, Wenlei Washington University in St. Louis 2016 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

The large-scaled magnetic fields in intergalactic space may contain detailed information of the primordial magnetic fields produced in the early Universe. The generation of the cosmological matter-antimatter asymmetry may produce helical magnetic fields via CP (charge conjugation and parity) violating interactions during matter-genesis. Thus, the study of the intergalactic magnetic field (IGMF) could provide a new window on early-Universe cosmology. IGMFs deflect the electron-positron pairs produced by TeV gamma-rays from blazars, resulting in broadened beams of cascade GeV gamma-rays known as pair halos. In the presence of helical IGMFs, cascade GeV gamma-rays at different energies from a distant source fall into patterns in the observation plane in the GeV gamma-ray sky that have a similar chirality. These signatures of the IGMF on the gamma-ray emission from extragalactic sources, i.e. pair halos and helical patterns, can be observed using data from the Fermi Large Area Telescope (Fermi-LAT). In this dissertation, I describe methods for probing the IGMF using gamma-ray observations. I report on the results of a search for gamma-ray pair halos with a stacking analysis of low-redshift blazars using data from Fermi-LAT. For this analysis we used a number of a priori selection criteria, including the spatial and spectral properties of the Fermi sources. The angular distribution of ∼ 1 GeV photons around 24 stacked isolated high-synchrotron-peaked BL Lacs with redshift z < 0.5 shows an excess over that of point-like sources. A frequentist test yields a p-value of p ∼ 0.01 for the extended emission against the point-source hypothesis. A Bayesian estimation provides the common logarithm of Bayes factors > 2, consistent with expectations for pair halos produced in the IGMF with strength of ∼ 10-17 to 10-15 Gauss. In addition, I present a search for parity violating signatures of helical IGMFs in the GeV gamma-ray sky. In this study, the IGMF helicity is examined by evaluating a parity-odd statistic using gamma-ray data obtained from Fermi-LAT observations at high galactic latitudes. The resulting negative values of the parity-odd statistic imply that there is an excess of left-handed spirals in the gamma-ray sky, indicating a left-handed helicity consistent with a helical magnetic field with strength of ∼ 10 -14 Gauss on ∼ 10 Mpc scales.

Identification and characterization of novel astroviruses

Finkbeiner, Stacy Renee Washington University in St. Louis 2009 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Approximately 1.8 million children die from diarrhea annually, and millions more suffer multiple episodes of nonfatal diarrhea. Despite the availability of improved molecular diagnostics to detect the known viral agents, the etiology of a large proportion of diarrheal cases is unknown. In fact, it is estimated that no causative agent can be identified in up to 40% of sporadic cases or in gastroenteritis outbreaks. Detection of novel or unexpected viruses is the first step in identifying agents that could potentially close the diagnostic gap and pave the way for the development of more comprehensive preventative measures and better treatments. This dissertation encompasses the first application of cutting edge mass sequencing approaches to the analysis of viruses present in fecal specimens from patients with diarrhea. Known enteric viruses as well as multiple sequences (with only limited sequence similarity to viruses in GenBank) from putatively novel viruses were detected in pediatric sporadic diarrhea specimens. One virus, Astrovirus MLB1 (AstV-MLB1), was fully sequenced and determined to be a highly divergent, novel astrovirus based on phylogenetic analysis. AstV-MLB1 was further detected by RT-PCR in 4/254 fecal specimens collected at the St. Louis Children's hospital in 2008, indicating that AstV-MLB1 is currently circulating in North America. A second highly divergent, novel astrovirus, Astrovirus VA1 (AstV-VA1), was identified in two specimens from a gastroenteritis outbreak at a child care center. Mass sequencing yielded nearly the entire genome of AstV-VA1 which appears to be most closely related to astroviruses found in mink and sheep. One additional sample also tested positive for AstV-VA1 by RT-PCR, resulting in detection of the virus in 3/5 specimens collected from the outbreak. This presents the possibility that further investigations might reveal that AstV-VA1 is a causative agent of gastroenteritis outbreaks. The identification of two novel astroviruses in fecal specimens from children with diarrhea suggests that astroviruses may cause a larger fraction of diarrhea cases than previously recognized. Furthermore, the identification and characterization of novel astroviruses MLB1 and VA1 lays the foundation for future investigations into their potential roles as etiologic agents of diarrhea.

Genetic influences on preterm birth

Plunkett, Jevon Anastasia Washington University in St. Louis 2010 해외박사(DDOD)

소속기관이 구독 중이 아닌 경우 오후 4시부터 익일 오전 9시까지 원문보기가 가능합니다.

Preterm birth (PTB) is a leading cause of mortality and disease burden globally; however, determinants of human parturition remain largely uncharacterized, making prediction and prevention of difficult. Genetic studies are one way in which we can attempt to better understand this disorder. We first sought to develop a model for the genetic influences on PTB to facilitate gene discovery. Study of standard measures of familial aggregation, the sibling risk ratio and the sibling-sibling odds ratio, and segregation analyses of gestational age, a quantitative proxy for preterm birth, lend support to a genetic component contributing to birth timing, since preterm deliveries cluster in families and models in which environmental factors alone contribute to gestational age are strongly rejected. Analyses of gestational age attributed to the infant support a model in which mother's genome and/or maternally-inherited genes acting in the fetus are largely responsible for birth timing. We also aimed to discover specific genes associated with PTB by screening genes selected based on an evolutionary-motivated filter, rather than known parturition physiology. Because humans are born developmentally less mature than other mammals, birth timing mechanisms may differ between humans and model organisms that have been typically studied; as a result, we screened 150 genes, selected because of their rapid evolution along the human lineage. A screen of over 8000 SNPs in 165 Finnish preterm and 163 control mothers identified an enrichment of variants in FSHR associated with PTB and prompted further study of the gene. Additionally, PLA2G4C, identified as the gene with the most statistically significant evidence for rapid evolution that was also included in a list of preterm birth candidate genes, was examined further. Three SNPs in PLA2G4C and one SNP in FSHR were statistically significant across populations after multiple testing corrections. Additional work to identify variants in these genes with functional effects was also initiated, including comparisons of prostaglandin metabolite levels among genotype classes for significantly associated SNPs in PLA2G4C and sequencing of FSHR to identify functional coding variants. Together, these experiments better characterize the nature of genetic influences on PTB and support the role of PLA2G4C and FSHR in PTB.