Functional Roles of Nucleases in DNA Metabolism and Genome Stability

The work outlined in this dissertation focuses on two distinct areas that are important for genome stability. Both areas focus on DNA repair pathways that require the action of nucleases, specifically Exonuclease 5 and Ribonuclease H2. First, I describe the biochemical and molecular characterization of the novel Exonuclease 5 family of enzymes from S. cerevisiae, S. pombe, and humans. The Exo5 family consists of bi-directional single-strand DNA specific exonucleases that all contain an iron-sulfur cluster as a structural motif and all have various roles in DNA metabolism. In the Saccharomycetales order that includes the budding yeast, S. cerevisiae, Exo5 is a mitochondrial protein that is essential for mitochondrial genome maintenance. In an unrelated yeast species, Schizosaccharomyes pombe, Exo5 is important for both nuclear and mitochondrial DNA metabolism. The human ortholog is important for nuclear genome stability, and for DNA repair. The work outlined in Chapter II of this Dissertation establishes Exo5 as a protein that is important for DNA metabolism. The second area of study outlined in Chapters III and IV is related to the phenomenon of ribonucleotide incorporation into the genome by replicative polymerases, and these chapters focus on the enzymes that remove these noncanonical nucleotides. Ribonucleotides are incorporated into DNA by the replicative DNA polymerases at frequencies of about 2 per kb, which makes them by far the most abundant form of potential DNA damage in the cell. Their removal is essential for restoring a stable intact chromosome. In Chapter III, I present a complete biochemical reconstitution of the ribonucleotide excision repair (RER) pathway with enzymes purified from Saccharomyces cerevisiae. I highlight the requirement for RNase H2 in the process of RER and investigate the redundancies at different steps of repair. Also outlined in this dissertation is the dissection of the different functions of RNase H2 in RER and in the removal of RNA-loops in DNA, and implications for genome instability in human diseases that are affected for these activities. Chapter IV of this dissertation discusses work on an alternative pathway for ribonucleotide removal from the genome by Topoisomerase I. In S. cerevisiae, deletion of rnh201, the catalytic subunit of RNase H2, results in the persistence of ribonucleotides remain in the genome, which leads to ~100-fold increase in the frequency of 2-5 bp deletions at di-nucleotide repeat sequences. These deletions are dependent on topoisomerase I (Top1) activity. Here we present an in vitro reconstitution of the mechanism of Top1-dependent deletions at di-nucleotide repeat sequences and a mechanism for Top1-initiated removal of ribonucleotides outside of the context of these repeat sequences in S. cerevisiae. Top1 attack at a ribonucleotide leads to the formation of a 2\u27, 3\u27 cyclic phosphate terminated ssDNA nick, followed by subsequent formation of a Top1-cleavage complex (Top1-cc) upstream of the 2\u27, 3\u27 cyclic phosphate. If the ribonucleotide is in the context of a di-nucleotide repeat, there can be realignment of the DNA allowing for religation and release of Top1, leading to a 2-nucleotide deletion. If the ribonucleotide resides outside a repeat sequence, the realignment is not possible and a different pathway must repair the Top1-cc. Tdp1-dependent repair of Top1-cc requires prior proteolytic processing of the Top1-cc before it can be removed leaving a 3\u27-phosphate that can be removed by Tpp1, Apn1, or Apn2 forming a substrate suitable for repair by DNA polymerase δ, FEN1 and DNA ligase

Finite element model of SCOLE laboratory configuration

The Spacecraft Control Laboratory Experiment (SCOLE) is defined by element properties: material constants; mast, reflector, rigid links as beam elements; cable as bar element; and space shuttle as very stiff beam. Two boundary conditions are modeled: suspended (6 degrees of freedom for all joints except the top of the cable) and cantilevered cables (shuttle platform fixed in all degrees of freedon). Calculations include stiffness and mass matrices, initial stresses, static displacements and reactions, and eigensolutions

Disordered Eating in Non-Elite Marathon Runners

The primary purpose of this study was to evaluate prevalence of disordered eating in non-elite marathon runners. Another purpose for this study was to analyze the difference in disordered eating prevalence between males and females, as well as, to evaluate the relationship of BMI categories and education levels with disordered eating.The participants for this study were recruited through running club directors throughout the states of Oklahoma, Arkansas, and Texas via email. Running club directors sent an email containing the information sheet on the study procedures and a hyperlink to the Qualtrics survey. The participants were required to fill out a two-part survey that included anthropometrics, demographics, marathon information, and the EAT-26 questionnaire.A total of 98 non-elite marathon runners participated in this study. There were 49 males and 49 females. Disordered eating risk was exhibited by 23 (23.5%) of the participants (EAT-26 ≥ 20). There was no significant difference in disordered eating risk (EAT-26 ≥ 20) between genders (p = .057) There was also no significant difference found in the EAT-26 subscales Dieting (D) (p = .221), Food Preoccupation (FP) (p = .133), and Oral Control (OC) (p = .062) between males and females. There was no significant difference found in EAT-26 total scores between the BMI categories, normal and overweight (p = .858). There was a significant relationship between the BMI categories and the subscale OC (p = .038). There was a significant negative correlation between BMI and subscale OC (r = -.228, p = .024). There was no significant difference found in EAT-26 total scores or subscales between the education levels (total p = .620; D p = .842, FP p = .227, and OC p = .600).Non-elite marathon runners are a population that could benefit from dietary education, specifically education on binge eating. Further research should explore BMI and the subscale Oral Control.Nutritional Science

Change Blindness, Attention, and Driving Performance

Concern over older driver high traffic fatality rates has resulted in an effort to identify risk factors and to develop methods of assessment. This study examines two attention-related tasks, Useful Field of View (UFOV) and Change Blindness (CB), in relation to vision and cognitive test batteries and driving performance measures collected using a simulator and an instrumented vehicle. Eight older adults with Alzheimer’s disease and nine comparison subjects between 64 and 81 participated. Factor analysis results indicate that UFOV and CB relate to different factors. While UFOV relates to memory, decision-making, attention, and visual spatial ability, CB relates to vision and attention. The type of images used on a CB task influence how the task relates to driving performance measures. Researchers should be thoughtful when selecting images to include in CB tasks to maximize insight into real-world drivin

A Method for Measuring the Bias of High-Redshift Galaxies from Cosmic Variance

As deeper observations discover increasingly distant galaxies, characterizing the properties of high-redshift galaxy populations will become increasingly challenging and paramount. We present a method for measuring the clustering bias of high-redshift galaxies from the field-to-field scatter in their number densities induced by cosmic variance. Multiple widely-separated fields are observed to provide a large number of statistically-independent samples of the high-redshift galaxy population. The expected Poisson uncertainty is removed from the measured dispersion in the distribution of galaxy number counts observed across these many fields, leaving, on average, only the contribution to the scatter expected from cosmic variance. With knowledge of the Lambda Cold Dark Matter power spectrum, the galaxy bias is then calculated from the measured cosmic variance. The results of cosmological N-body simulations can then be used to estimate the halo mass associated with the measured bias. We use Monte Carlo simulations to demonstrate that Hubble Space Telescope pure parallel programs will be able to determine galaxy bias at z>~6 using this method, complementing future measurements from correlation functions.Comment: Accepted by ApJ Letter

Thermal properties of vesicular rhyolite

Thermal diffusivity of rhyolite melt and rhyolite foam (70–80% porosity) has been measured using the radial heat transfer method. Cylindrical samples (length 50–55 mm, diameter 22 mm) of rhyolite melt and foam have been derived by heating samples of Little Glass Mountain obsidian. Using available data on heat capacity and density of rhyolite melt, the thermal conductivity of samples has been determined. The difference in thermal conductivity between rhyolite melt and foam at igneous temperatures ( 1000°C) is about one order of magnitude. The effect of thermal insulation of magmas due to vesiculation and foaming of the top layer is discussed in terms of the data obtained using a simple illustrative model of magma chamber convection

Violence Exposure and Sexual Risk Behaviors for African American Adolescent Girls: The Protective Role of Natural Mentorship and Organizational Religious Involvement

African American adolescent girls are at increased risk of being exposed to community violence and being diagnosed with a sexually transmitted infection. Fewer studies, however, have examined the protective roles of natural mentorship and organizational religious involvement as potential moderators that could lessen the effects of violence exposure on health risk behavior. Data from 273 African American ninth grade girls were used to test hypothesized independent and moderatedâ moderation models. Results suggest that natural mentorship and religious involvement were protective for girls who reported at least one mentor and moderate to high levels of religious involvement. Our findings may be relevant for community stakeholders and organizations that directly interact with religious institutions and community programs that focus on outreach to African American adolescent girls.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/151908/1/ajcp12341_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/151908/2/ajcp12341.pd