Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 96-116). Available also in a digital version from Dissertation Abstracts.

Thesis--Wisconsin. / Vita. Includes bibliographical references.

Thesis (Ph. D.)--University of Texas at Austin, 2005. / Supervisor: Kenneth A. Johnson. Vita. Includes bibliographical references.

Thesis (Ph.D.)--University of Delaware, 2005. / Principal faculty advisor: Simmons, Daniel T., Dept. of Biological Sciences. Includes bibliographical references.

During DNA replication the DNA has to be unpacked, duplicated and repacked into chromatin, which is comprised of DNA and histone proteins (Nicholson and Muller, 2008b). The coordinated replication of DNA and histone protein synthesis is vital for the correct chromatin formation (Marzluff et al., 2008). The mechanism controlling the histone gene expression is only partially understood, especially the mechanism controlling any disturbances in the coordination of DNA replication and histone protein synthesis. Previous experiments suggested that the regulation mechanism of histone balance could involve regulation by a free histone protein pool (Takami and Nakayama, 1997b; Takami and Nakayama, 1997a; Dominski et al., 2005; Kroeger et al., 1995). A mathematical model was produced by Dr Hameister (Hameister, 2012) to describe the control of histone production during S phase. The parameters used were taken from literature. Modifications were made using experimentally measured data to replace literature values (Harris et al., 1991; Clark, 2006; Strachen and Read, 2003). The aim of the project was to both optimise the model by defining parameters to reflect what occurs naturally in the cell, as well as trying to validate the model. The DNA replication rate was measured by FACS analysis and was input into the model. Histone RNA levels during S phase were measured by Northern blots and histone RNA degradation rates were analysed. To confirm that the modified DNA replication rate was producing accurate simulations, the curve produced for the mRNA levels could be compared to the experimentally measured mRNA values (Figure 4.3.1). The over expression of an H2B gene was verified using Western and Northern analysis. The validation of the model that the histone gene balance was being regulated by a free histone pool was not absolutely confirmed. However, results seen in Figure 3.6.1, showed an increase in H2B RNA degradation in the sample with the additional gene due to the additional histone proteins. Further work is required for confirmation of the regulation mechanism.

Bunyamwera (BUN) virus multiplied readily in mosquitoes following intrathoracic injection and also after imbibing'-an infective blood meal. This agent also multiplied following inoculation of human and avian cells in tissue cultures, with production of cytopathic effects. Both in arthropod and vertebrate tissues, enveloped virions 84 nm diameter were visualized by electron microscopic observation of tissues collected after maximum viral proliferation was attained. Following intrathroacic injection of 10 ²•² mouse LD₅₀ of BUN virus into groups of wild-caught mosquitoes comprising both Aedes canadensis and A. vexans, increments of infectivity were first detected in salivary glands and gut at 3 days, and maximum titres of 10 ⁵•² mouse LD₅₀ per organ were attained in salivary glands at 10 days. However, 50 the virus content of legs, which provided a convenient means of sampling hemocelic fluid, increased at 2 days. Virus transmission by biting mice was demonstrated with mosquitoes injected 10 days previously, but not after shorter intervals. No virus replication was demonstrated following ingestion of 10⁴•⁰ mouse LD₅₀ of BUN virus in a blood meal. Aedes aegypti mosquitoes readily supported the replication of BUN virus following injection with 10 ³•³ mouse LD₅₀ or imbibing of 10 ⁴•⁶ mouse LD₅₀. After injection, virus titres of whole mosquitoes declined to 10 ¹•⁷ mouse LD₅₀ at 12 hours, followed by an increase to a peak amount of 10 ⁵•⁰ mouse LD₅₀ at 2 days. After feeding, virus was first detected in legs and salivary glands at 4 days, and attained maximum titres of 10 ⁵•⁰ mouse LD₅₀ in salivary glands at 10 days. Transmission of virus to mice was effected by A. aegypti following feeding and injection 10 days previously, but not at earlier intervals. Following exposure of whole gut cultures of adult A. aegypti mosquitoes to 10 ³•⁷mouse LD₅₀ maximum yields of 10 ⁶•⁰ mouse LD₅₀ per ml. were observed after k days incubation at 29°C, after an initial decline of infectivity to 10 ¹•⁸ mouse LD₅₀ at 12 hours. Enveloped virions with cores 45 nm diameter and total diameters 80 to 100 nm were observed within vacuoles and lining vacuolar membranes of salivary glands and gut cells of A. aegypti mosquitoes 10 days or more after infection with BUN virus. No particles were observed earlier, despite high virus titres 4 days or more after injection. After inoculation of continuous live tissue cultures of human epidermoid corcinoma cells (H.Ep. 2) with 10 ⁶•⁵ mouse LD₅₀, the highest amount of virus produced was 10 ⁷•⁰ mouse LD₅₀ per ml. cell suspension after 24 hours incubation at 37°C. Maximum yields of BUN virus (10 ⁶•² mouse LD₅₀ per ml. cell suspension) were attained 24 hours after inoculation of primary chick embryo fibroblast monolayers with 10 ⁵•² mouse LD₅₀ following incubation at 37 C . However, a peak titre of 10 ⁵•⁰ mouse LD₅₀ was attained 3 days after inoculation with 10 ³•⁷mouse LD₅₀ in cultures incubated at 29°C. Before an increment of virus titre was observed infectivity declined to zero during the initial 4 hours after inoculation of cultures incubated at 37°C, and a tenfold decline of infectivity was noted in cultures incubated at 29°C. Enveloped virions with total diameter 84 nm which contained electron-dense nucleoids 44 nm diameter were observed extracellularly in thin sections of chick embryo fibroblasts infected 12 hours previously with BUN virus. These particles were released by budding. Precursor particles 41 nm diameter were associated with intracellular membranes in occasional cells sectioned at 4 hours. Extracellular virions released one day after inoculation of H.Ep. 2 cultures were tagged by ferritin-labelled anti-BUN antibody. Enveloped virions with mean diameters 100 nm were observed in suspensions of suckling mouse brain infected with BUN virus and stained negatively with phosphotungstic acid. These results show clearly that BUN virus exhibits the essential biological and morphological characteristics of a mosquito-borne arbovirus. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

Dijksterhuis and van Knippenberg (1998) reported that participants primed with a category associated with intelligence (“professor”) subsequently performed 13% better on a trivia test than participants primed with a category associated with a lack of intelligence (“soccer hooligans”). In two unpublished replications of this study designed to verify the appropriate testing procedures, Dijksterhuis, van Knippenberg, and Holland observed a smaller difference between conditions (2%–3%) as well as a gender difference: Men showed the effect (9.3% and 7.6%), but women did not (0.3% and −0.3%). The procedure used in those replications served as the basis for this multilab Registered Replication Report. A total of 40 laboratories collected data for this project, and 23 of these laboratories met all inclusion criteria. Here we report the meta-analytic results for those 23 direct replications (total N = 4,493), which tested whether performance on a 30-item general-knowledge trivia task differed between these two priming conditions (results of supplementary analyses of the data from all 40 labs, N = 6,454, are also reported). We observed no overall difference in trivia performance between participants primed with the “professor” category and those primed with the “hooligan” category (0.14%) and no moderation by gender.

Viruses are obligate intracellular parasites that replicate utilizing the resources of host cells. The replication of positive sense RNA viruses is coupled with alterations to host cell membranes. These viruses are believed to replicate efficiently by using co-opted membrane structures assembled from viral and host cell proteins and lipids. Poliovirus is a prototypical positive-sense RNA virus, however the topological details of viral RNA replication are not well understood. In this work we use electron cryotomography, among other methods, to examine the ultrastructure of fractionated poliovirus RNA replication factories that were formed within infected cells, and to investigate oligomeric interactions within a three dimensional crystal formed by a poliovirus polymerase point mutant. Investigation of the ultrastructure of isolated viral RNA replication factories shows that the low resolution features of cryopreserved membrane structures are essentially identical to previously observed structures within plastic sections of infected cells. Furthermore, greater detail visible using electron cryotomography reveals pore-like structures and other high energy membrane conformations within the replication factories. We see a mix of single, double, and multi-membrane structures that are arranged with openings that connect their interior lumenal space to the exterior environment. The lumen of some of these membranous structures contains a linear polymeric density thought to be RNA. We conclude that the RNA replication of poliovirus may occur on the lumenal surface of vesicular membranes with an opening to the cytoplasm for metabolite and product exchange. Within the poliovirus replication machinery, the principal component is the RNA polymerase 3Dpol. This prototypical RNA-dependent RNA-polymerase forms homo-oligomeric interactions that are key to its functions. To investigate these interactions, previous studies focused on hollow helical structures formed by wild-type polymerase. Here, we investigate the structure of small three-dimensional crystals formed by 3Dpol with a mutation of a single residue, lysine 314, to alanine. Using electron cryotomography and volume averaging, we demonstrate that the crystal packing within this point mutant does not include physiological polymerase-polymerase interactions. Elucidation of the topology of poliovirus replication machinery provides a basis for future development of antiviral therapeutics.

Indiana University-Purdue University Indianapolis (IUPUI) / Papillomaviruses (PVs) are non-enveloped DNA viruses that are the primary etiological agents of cervical and oropharyngeal cancers. Vaccines for H(human)PV have proven to be effective prophylactic treatments; however, there is no treatment available for those currently infected. To develop new therapies, we require a clear understanding of viral pathogenesis and regulation. The Papillomavirus E2 protein is a sequence specific DNA binding protein that recruits cellular factors to its genome in infected epithelial cells. E2 also binds to and loads the viral E1 DNA helicase at the origin of replication. Post-translational modifications of PV E2 have been identified as potential regulators of E2 functions. We recently reported lysine (K) 111 as a target of p300 acetylation in B(bovine)PV that is involved in the regulation of viral transcription. K111 is conserved in most papillomaviruses, so we pursued a mutational approach to query the functional significance of lysine in HPV E2. Amino acid substitutions that prevent acetylation, including arginine, were unable to stimulate transcription and E1 mediated DNA replication. The arginine K111 mutant retained E2 transcriptional repression, nuclear localization, DNA and chromatin binding, and association with E2 binding partners involved in PV transcription and replication. When directly investigating origin unwinding, the replication defective E2 K111R mutant recruited E1 to the viral replication origin, but surprisingly, unwinding of the duplex DNA did not occur. In contrast, the glutamine K111 mutant increased origin melting and stimulated replication compared to wild type E2. We have identified Topoisomerase I as a key host factor involved in viral replication whose recruitment is dependent on K111 acetylation, and propose a new model for viral origin dynamics during replication initiation. This work reveals a novel activity of E2 necessary for denaturing the viral origin that likely depends on acetylation of highly conserved lysine 111.

Decision making is heavily influenced by the environment around us. Berry et al. (2014, 2015, 2019) showed that viewing images of natural environments during the delay discounting task resulted in lower impulsive choice, as compared to viewing images of built environments or geometric figures. Berry et al. proposed that attentional factors could explain this effect, however, recent attempts to reproduce Berry et al.’s findings in a different laboratory have been unsuccessful (Johnson 2017, 2018, 2019). The present study tested if manipulating the participants’ observing responses towards different types of images (natural, built, and no images) modulates the effect reported by Berry et al. Eighty-seven college students were exposed to a matching-to-sample task aimed at increasing observation responses to the images (attentional manipulation) throughout the same delay discounting task implemented by Berry et al. (2014). It was expected that increasing the participants interaction with the images via the matching task would increase the magnitude of the effect reported by Berry et al. (2015); namely, further reduction of impulsive responses after being exposed to images of natural environments and increase of impulsive choice when exposed to built environments. Results indicated that participants who engaged in matching images of built environments had a higher rate of discounting than the group that replicated Berry et al. (2014). Matching images of natural environments did not seem to reduce impulsive choice, as predicted based on Berry et al.’s findings. Furthermore, none of the groups of the present study reproduced the rates of delay discounting originally reported by Berry et al. Participants in Berry et al., (2014) overall discounted less steeply when compared to participants in the present study. This finding resembles the results reported by Johnson et al. (2017, 2018, 2019) in their replica attempts. Lastly, participants’ self-reports regarding time spent in natural and built environments did not correlate with rate of discounting. Future research should use the number of correct responses as a measure of procedural integrity. Also, it is possible that perhaps participants failed to match more often in the built condition, and such aversive condition could have increased the impulsive choice for that group (Flora et al. 1992, 2003).