The human adenovirus 5 L4 promoter is activated by cellular stress response protein p53

During adenovirus infection, the emphasis of gene expression switches from early genes to late genes in a highly regulated manner. Two gene products, L4-22K and L4-33K, contribute to this switch by activating the Major Late Transcription Unit (MLTU) and regulating the splicing of its transcript. L4-22K and L4-33K expression is driven initially by a recently described L4 promoter (L4P) embedded within the MLTU that is activated by early and intermediate viral factors: E1A, E4 Orf3 and IVa2. Here we show that this promoter is also significantly activated by the cellular stress response regulator, p53. Exogenous expression of p53 activated L4P in reporter assays whilst depletion of endogenous p53 inhibited the induction of L4P by viral activators. Chromatin immunoprecipitation studies showed that p53 associates with L4P and that during adenovirus type 5 (Ad5) infection this association peaks at 12 h.p.i., coinciding with the phase of the infectious cycle when L4P is active, and is then lost as MLP activation commences. P53 activation of L4P is significant during Ad5 infection since depletion of p53 prior to infection of either immortalised or normal cells led to severely reduced late gene expression. The association of p53 with L4P is transient due to the action of products of L4P activity (L4-22K/33K), which establish a negative feedback loop that ensures the transient activity of L4P at the start of the late phase and contributes to an efficient switch from early to late phase virus gene expression

THE GEOLOGY AND GEOCHEMISTRY OF THE WESTERN IRON ORE GROUP GREENSTONE BELT, SINGHBHUM CRATON, EASTERN INDIA

This thesis presents new data on the origin of the Western Iron Ore Group (W-IOG), a Paleoarchean succession deposited on sialic rocks of the Singhbhum Craton. U-Pb ages from zircons from a tuff overlying the Lower Shales just below a banded iron formation (BIF) pin the age of the section to 3.39 Ga. Unconformably overlying the whole IOG section is the Darjing Group, which contains the Birtola Sandstone (on the basis of this work, now estimated to be 2.35 Ga). The lack of penetrative deformation and low-grade metamorphism makes these rocks excellent subjects for combined radiogenic and stable isotope studies. Data on the W-IOG constrain the development of earth systems operating during the Archean into the Proterozoic (4.0-2.4 Gyr). 143Nd/144Nd isotopes for basal Lower Lava greenstones suggest a whole-rock isochron with an apparent age of 3.42 ± 0.14 Ga exhibiting perhaps the highest positive initial εNd (εNd = +5.7 ± 2.5) observed thus far for an Archean greenstone, and suggesting it is a possible remnant of early Earth differentiation. The initial εNd of 2.7 at 2.65 Ga for the Upper Lava (i.e., 3 εNd units lower than the greenstones of the Lower Lava) might reflect the cumulative effect of recycling of less Light Rare Earth Element (LREE) depleted sediments into the mantle by subduction processes. The Lower Lava and the Lower Shale plot on a common whole-rock 143Nd/144Nd versus147Sm/144Nd line. When combined with REE data, this indicates that the provenance for the Lower Shale is from mafic rocks similar to the underlying Lower Lava, perhaps a subaerial constructional pile of mafic rocks (i.e., the Lower Lava). Lower Shale rocks are rich in iron (avg. 18 Fe2O3 wt.%, n = 7) and with diachronous sedimentation could be the proto-ore for the BIF. The spread in iron isotope ratios (-0.693 ≤ δ56Fe ≤ 1.735 ‰) suggests the oxidation of iron may have been biologically mediated 3.4 billion-years ago. The Upper Shales share a common line with Singhbhum’s granitic basement (with an apparent age of 3.36 ± 0.09), implying that these iron-poor shales formed primarily from the unroofing of subvolcanic granitic basement. New zircon U-Pb, Lu/Hf and 18O/16O along with the d18O of host rock sandstones of the Birtola Formation demonstrates that mantle-like zircon d18O values are consistent with source regions that have been affected by the addition of subducted recycled crustal Hf. If so, the additions would have been in proposrtions implying significant refertilization of the MASH source regions that are the presumed igneous source for the Birtola Sandstone. Using the D18O quartz-zircon, forward modelling of the d18O values of the quartz from the zircons and zircons from the quartz distributions are consistent with temperatures of silicic extrusive rock magmatic temperatures. Some low 18O Archean zircons (d18O18O of the IOG greenstone succession requires surface temperatures more consistent with the faint early Sun. The climate implications and the evidence of recycling down into the mantle all suggest plate tectonics in the early Earth

Rescued Secretion of Misfolded Mutant Proinsulin.

In pancreatic beta cells, the insulin precursor proinsulin is folded in the endoplasmic reticulum (ER), forming three critical intramolecular disulfide bonds. After homo-dimerizing, native proinsulin exits the ER en route to secretory vesicles, where it forms hexamers, is endoproteolytically cleaved to mature insulin, and is stored until it is secreted in response to elevated blood glucose. In Mutant Ins-gene induced Diabetes of Youth (MIDY), misfolded mutant proinsulin is retained in the ER and acts in a dominant-negative manner to impair maturation of wild-type (wt) proinsulin, leading to decreased insulin release and eventual ER stress-induced beta cell death. Using cell culture and mouse models, I have investigated two potential mechanisms to improve secretion of misfolded mutant proinsulin. First, I found that intermolecular interactions between proinsulin molecules impact strongly on the fate of those molecules. Misfolded mutant proinsulin molecules dimerize with and impair secretion of co- expressed wt molecules. Interestingly, the opposite is also true; wt proinsulin molecules also stabilize and enhance secretion of mutant molecules. Thus, there is a dynamic bidirectional interaction between dimerization partners, which we hope to exploit pharmacologically to improve clearance of misfolded proteins from the ER and alleviate ER stress-induced cell death. In the second half of my project, I investigated how manipulating the oxidative environment of the ER may impact proinsulin secretion and beta cell health in cells expressing mutant proinsulin. ER Oxidoreductin-1 (Ero1), the best-studied ER oxidant, contributes to oxidative folding of secretory proteins by coupling generation of de novo disulfide bonds with reduction of molecular oxygen. Due to its generation of hydrogen peroxide as a byproduct, Ero1 hyperactivity has been speculated to contribute to cell death in stressed beta cells. Surprisingly, I found the opposite to be true. Overexpression of Ero1 rescued secretion of wt proinsulin in the presence of mutant proinsulin. Furthermore, Ero1 directly rescued a subset of MIDY mutant proinsulins by improving their oxidative folding, resulting in a decrease in mutant proinsulin-induced ER stress response. These findings improve our understanding of proinsulin maturation in beta cells, and may contribute to novel therapeutic approaches in this and other secretory protein conformational diseases.PHDMolecular and Integrative PhysiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/111355/1/jordwrig_1.pd

The role of PML proteins in adenovirus type 5 infection and the type I interferon response

Promyelocytic leukaemia (PML) proteins consist of a collection of related isoforms that are the nucleating components of sub-nuclear structures termed PML nuclear bodies (PML NBs). Numerous functions are attributed to PML and PML NBs, including a role in antiviral responses. Adenovirus type 5 (Ad5) has previously been shown both to disrupt PML NBs and to cause the appearance of a novel PML protein species, termed PML-A, in a manner dependent on the viral E4 Orf3 protein. Here, the interactions between Ad5 and PML proteins were further investigated. The E4 Orf3-dependent species of PML (PML-A) was found to be related to the full-length PML II isoform. In addition, two previously unknown infection-specific species of PML were detected (PML-B and PML-C). Both PML-B and PML-C were produced independently of Ad5 E4 Orf3, though their appearance required viral DNA replication. PML-C was found to be localised within the cytoplasm of infected cells and to be related to PML I. To test the hypothesis that these changes in PML were related to virus effects on antiviral responses, the roles of individual PML proteins in type I interferon (IFN) signalling were investigated. Depletion of PML II by siRNA resulted in a 50% decrease in activity of an IFNβ promoter reporter plasmid during stimulation by poly (I:C). Exogenous expression of a ΔRBCC truncated mutant, but not full-length, PML II resulted in enhanced IFNβ promoter activity, suggesting a role of a PML II-related species in type I IFN signalling. Moreover, expression of the Ad5 E4 Orf3 protein was sufficient to inhibit the activity of the IFNβ promoter, an activity that correlated with Orf3’s ability to bind PML II. Collectively, these data indicate that a PML II-related molecule is involved in the interferon response and that the E4 Orf3- PML II interaction may therefore facilitate Ad5 in mitigating this activity

PROJECT ADMIRAL AND SINKING SHIP: A DETAILED ANALYSIS FROM DEVELOPING TWO RAPID GAME PROTOTYPES

The purpose of this research is to study and document the process of creating educational procurement games, with the goal being to expand the lexicon of knowledge for Air Force (AF) contracting. We advise two game development teams through the process of integrating contracting learning objectives into virtual games. Working alongside North Carolina State University (NC State), we help develop a tower defense game (Project Admiral) and a digital escape room game (Sinking Ship). We meticulously document our experiences advising student game developers on which contracting elements to employ and how to properly design the game. We also document what we discover about the game development process to inform future research. Our documentation utilizes engaged scholarship methods, treating these games as case studies to provide insights into the process of developing educational procurement games. We evaluate our successes, failures, and lessons learned to inform future educational game development projects. Ultimately, we aim to provide useful guidance for educators and researchers interested in developing their own educational procurement games. Our findings are beneficial both to those newly introduced to the field as well as experienced professionals who desire an update on the current state of contracting game development.Outstanding ThesisCaptain, United States Air ForceCaptain, United States Air ForceApproved for public release. Distribution is unlimited

Golf Club Prototyping and Design for Spin Rate Tuning

The aim of this project was to design a golf wedge capable of increasing backspin for the amateur golfer. This was accomplished by embedding a metal lattice structure behind the clubface to allow the face to elastically deform slightly upon impact. This would increase contact time between the club and ball. The mechanism of spin generation was discussed and the relationship between contact time and spin rate was established. The design was enabled by using additive manufacturing, which allowed for the generation of a metal lattice structure. An appropriate control and prototype were designed to minimize run time and material usage due to limited machine capacity. Various lattice topologies were generated and analyzed with finite element analysis. Design validation build in plastic revealed that these were not feasible due to support material generation, so X topology was used instead. After printing, player testing was conducted. The prototype design underwent plastic deformation during testing, and resulted in a significantly lower spin rate than the control. The design outlined in the report is not recommended unless changes to prevent plastic deformation are made and more testing is performed. Economic justification for the production of additive manufacturing golf club designs is made in case future designs prove viable. Future work involves earlier consideration of design for manufacturability given the constraints of the selective laser melting (SLM) machine and better testing using an automated process such as a golf swing robot

Mathematical Modeling of COVID-19 and the Potential Effects on Eastern Washington University Student Population

Coronaviruses are a large family of viruses commonly found in people and animals and can sometimes spread from animals to humans. COVID-19 primarily spreads from person to person through contact with the mucous membranes of the eye and nose and is also found in saliva. These shared droplets are able to spread a considerable distance and can remain alive on surfaces. Currently, there is no supported treatment or cure for COVID-19. This project’s objective is to find the intervention threshold in which the spread is reduced below an effective reproductive number of 1 thereby constituting no additional spread. To test our hypothesis, we will be using a stochastic (randomly generated) agent-based simulator designed to be used for COVID-19 epidemic analyses. This model uses the SIR model in conjunction with networking groups to determine the likely course of spread on the population given parameters. The results will give us insight into the efficacy and feasibility of reopening campus for the Fall with interventions in place given the possibility of an outbreak

The role of PML proteins in adenovirus type 5 infection and the type I interferon response

Promyelocytic leukaemia (PML) proteins consist of a collection of related isoforms that are the nucleating components of sub-nuclear structures termed PML nuclear bodies (PML NBs). Numerous functions are attributed to PML and PML NBs, including a role in antiviral responses. Adenovirus type 5 (Ad5) has previously been shown both to disrupt PML NBs and to cause the appearance of a novel PML protein species, termed PML-A, in a manner dependent on the viral E4 Orf3 protein. Here, the interactions between Ad5 and PML proteins were further investigated. The E4 Orf3-dependent species of PML (PML-A) was found to be related to the full-length PML II isoform. In addition, two previously unknown infection-specific species of PML were detected (PML-B and PML-C). Both PML-B and PML-C were produced independently of Ad5 E4 Orf3, though their appearance required viral DNA replication. PML-C was found to be localised within the cytoplasm of infected cells and to be related to PML I. To test the hypothesis that these changes in PML were related to virus effects on antiviral responses, the roles of individual PML proteins in type I interferon (IFN) signalling were investigated. Depletion of PML II by siRNA resulted in a 50% decrease in activity of an IFNβ promoter reporter plasmid during stimulation by poly (I:C). Exogenous expression of a ΔRBCC truncated mutant, but not full-length, PML II resulted in enhanced IFNβ promoter activity, suggesting a role of a PML II-related species in type I IFN signalling. Moreover, expression of the Ad5 E4 Orf3 protein was sufficient to inhibit the activity of the IFNβ promoter, an activity that correlated with Orf3’s ability to bind PML II. Collectively, these data indicate that a PML II-related molecule is involved in the interferon response and that the E4 Orf3- PML II interaction may therefore facilitate Ad5 in mitigating this activity.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Impaired Cleavage of Preproinsulin Signal Peptide Linked to Autosomal-Dominant Diabetes

Recently, missense mutations upstream of preproinsulin’s signal peptide (SP) cleavage site were reported to cause mutant INS gene-induced diabetes of youth (MIDY). Our objective was to understand the molecular pathogenesis using metabolic labeling and assays of proinsulin export and insulin and C-peptide production to examine the earliest events of insulin biosynthesis, highlighting molecular mechanisms underlying β-cell failure plus a novel strategy that might ameliorate the MIDY syndrome. We find that whereas preproinsulin-A(SP23)S is efficiently cleaved, producing authentic proinsulin and insulin, preproinsulin-A(SP24)D is inefficiently cleaved at an improper site, producing two subpopulations of molecules. Both show impaired oxidative folding and are retained in the endoplasmic reticulum (ER). Preproinsulin-A(SP24)D also blocks ER exit of coexpressed wild-type proinsulin, accounting for its dominant-negative behavior. Upon increased expression of ER–oxidoreductin-1, preproinsulin-A(SP24)D remains blocked but oxidative folding of wild-type proinsulin improves, accelerating its ER export and increasing wild-type insulin production. We conclude that the efficiency of SP cleavage is linked to the oxidation of (pre)proinsulin. In turn, impaired (pre)proinsulin oxidation affects ER export of the mutant as well as that of coexpressed wild-type proinsulin. Improving oxidative folding of wild-type proinsulin may provide a feasible way to rescue insulin production in patients with MIDY