Corrosion on bridges is a common issue since it can be caused by multiple agents such as marine environments or deicing chemicals. The damages caused by these agents, if left unmitigated, may lead to failure of the superstructures. If corrosion is present in the end regions of the beams, failure of the girders will likely be in shear, which is a sudden failure mode and not the preferred limit state. Therefore, it is beneficial to study repair practices and their advantages, as repairs are often more cost-effective than building entirely new structures. Repairing prestressed girders for shear is not common practice, but with the number of superstructures considered structurally deficient in the United States, additional consideration should be given to repair methods. In this study, two beams were extracted from two decommissioned bridges and were repaired using Carbon Fiber Reinforced Polymer (CFRP) to investigate how well proposed repairs functioned. One of the beams is an American Association of State Highway and Transportation Officials (AASHTO) Type II beam, while the second one is an adjacent box beam. Before repairs were done, the beams had their ends further damaged with accelerated corrosion induced via electrolysis to ensure that the beams would have enough deterioration in their shear span to simulate the worst-case scenario found in the field. Afterwards, the girders had their damage and residual strength estimated, and repairs were designed using guidelines from the American Concrete Institute (ACI) and AASHTO for CFRP repairs. Since the adjacent box beam could only be repaired in flexure, it failed in shear with a load similar to previous studies done in beams from the same bridge, which indicates that repairs for box beams need further investigation. The repairs on the AASHTO Type II beam worked well for shear, and both ends failed in flexure, which is an improved failure mode since failing in flexure is more ductile and predictable than shear failures. One end of the AASHTO Type II beam failed by concrete crushing and CFRP rupturing, and the other end failed by strand rupturing, which shows that the accelerated corrosion worked as was predicted. / Master of Science / Corrosion on bridges is a major problem across the United States, especially in marine environments and in cold areas where deicing chemicals are needed to ensure the safety of the drivers. These external agents typically accelerate the deterioration of bridges and lead to expensive repairs and sometimes total replacement of structures. In order to study repair methods for bridges that have been exposed to corrosive agents, bridge girders were extracted from two decommissioned bridges in Virginia. The level of damage in these girders was assessed and it was determined that more corrosion had to be induced into the girder to ensure repairs were needed. After that process, the strength of the girders was estimated using guidelines from the American Concrete Institute (ACI) and American Association of State Highway and Transportation Officials (AASHTO), and the girders were repaired for shear using sheets of Carbon Fiber Reinforced Polymers (CFRP). These repairs were done by using two different wrapping schemes, one that enveloped the sides of the girder, and one that was only at the bottom of the girder. The girders then had their ends tested under three-point bending to evaluate the effectiveness of the repairs. The girder that had the sides repaired as well performed better since the failure mode of it changed from shear to flexure. The girder that only had its bottom repaired did not do as well since its failure mode was still shear.

DNA repair is not a single mechanism found within cells. There exists numerous different DNA repair mechanisms that function within every type of cell. The majority of these mechanisms risk accumulating mutations. However, there are a few repair mechanisms that are known to be error-free and one of these is direct reversal repair. This study focused on two proteins highly involved in direct reversal DNA repair--ALKBH2 and ALKBH3. Previous studies have shown that in mice, these two proteins play a significant role in preventing and repairing DNA damage due to methylation as well as decreasing the frequency of mutagenic alkyl adducts. The goal of this study was to characterize the roles of the direct reversal repair proteins in human cells. We expected to see a similar phenotype to that of the Alkbh2 and Alkbh3-deficient mice. Telomerase immortalized human skin fibroblasts were targeted for the ALKBH2 and ALKBH3 alleles using a RNA-guided CRISPR-Cas9 construct that was designed to induce double stranded DNA breaks within the exons and disrupt the open reading frame, eliminating protein activity. Isolated clones were analyzed using fragment analysis and DNA sequencing to characterize any alterations in the open reading frame of the genes. Through sequencing analysis, results showed that one clone was successfully targeted for one of the ALKBH3 alleles with a single nucleotide insertion in its sequence, causing a disruption of the open reading frame. Though the ultimate goal of the experiment was not attained, we concluded that HTERTG fibroblasts can be expanded to serve as a model in which to construct targeted human cell lines that have near normal karyotypes.

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published_or_final_version / Applied Statistics / Master / Master of Social Sciences

The budding yeast, Saccharomyces cerevisiae, serves as an excellent model for identifying fundamental mechanisms of DNA repair. A Local Coherence Detection (LCD) algorithm that uses biclustering to assign genes to multiple functional sub-groups was applied on the chromosome E-MAP containing genetic interactions among genes involved in nuclear processes. Using this method, we found that Asf1 and Rtt109, genes that are together required for histone H3K56 acetylation, cluster together with Ctf4, Ctf18, Ctf8 and Dcc1, genes important for efficient sister chromatid cohesion. It is known that H3K56 acetylation is required for post-repair chromatin reassembly at sites of DNA double-strand breaks (DSBs). The cohesion genes were previously implicated in the repair of some DNA DSBs, but the nature of their involvement has not been reported. The experimental data in my thesis work suggest that Ctf4, Ctf8, Ctf18 and Dcc1 function in the post-repair chromatin reassembly pathway.

Loss of genomic stability is associated with a variety of diseases, particularly cancer. Of the many proteins which maintain genomic integrity, two of the most important are MLH1 and MSH2, which participate in DNA mismatch repair. Previous work established derivatives of the CaCo2 human colorectal cancer cell line with siRNA-mediated knockdown of these proteins. When xenografted into mice, tumors with reduced MLH1 or MSH2 expression grew faster than controls. Following growth in vivo, clonal cell lines were established from the tumors and used to examine the effects that knockdown of MSH2 had on other members of the DNA mismatch repair system. Clonal survival following exposure to 5-fluorouracil was also evaluated, and those cells with reduced MLH1 and MSH2 levels were found to be resistant. This study has implications for the importance of knowing the MMR status of a given tumor when deciding on a course of treatment, and of the compounding effects of the loss of one MMR protein on others in the family. / Canadian Cancer Society Research Institute

The budding yeast, Saccharomyces cerevisiae, serves as an excellent model for identifying fundamental mechanisms of DNA repair. A Local Coherence Detection (LCD) algorithm that uses biclustering to assign genes to multiple functional sub-groups was applied on the chromosome E-MAP containing genetic interactions among genes involved in nuclear processes. Using this method, we found that Asf1 and Rtt109, genes that are together required for histone H3K56 acetylation, cluster together with Ctf4, Ctf18, Ctf8 and Dcc1, genes important for efficient sister chromatid cohesion. It is known that H3K56 acetylation is required for post-repair chromatin reassembly at sites of DNA double-strand breaks (DSBs). The cohesion genes were previously implicated in the repair of some DNA DSBs, but the nature of their involvement has not been reported. The experimental data in my thesis work suggest that Ctf4, Ctf8, Ctf18 and Dcc1 function in the post-repair chromatin reassembly pathway.

Indiana University-Purdue University Indianapolis (IUPUI) / DNA double strand breaks (DSB), particularly those induced by ionizing radiation (IR) are complex lesions and if not repaired, these breaks can lead to genomic instability, chromosomal abnormalities and cell death. IR-induced DSB often have DNA termini modifications including thymine glycols, ring fragmentation, 3' phosphoglycolates, 5' hydroxyl groups and abasic sites. Non-homologous end joining (NHEJ) is a major pathway responsible for the repair of these complex breaks. Proteins involved in NHEJ include the Ku 70/80 heterodimer, DNA-PKcs, processing proteins including Artemis and DNA polymerases µ and λ, XRCC4, DNA ligase IV and XLF. The precise molecular mechanism of DNA-PK activation and Artemis processing at the site of a DNA DSB has yet to be elucidated. We have investigated the effect of DNA sequence and structure on DNA-PK activation and results suggest a model where the 3' strand of a DNA terminus is responsible for annealing and the 5' strand is involved in activation of DNA-PK. These results demonstrate the influence of DNA structure and orientation on DNA-PK activation and provide a molecular mechanism of activation resulting from compatible termini, an essential step in microhomology-mediated NHEJ. Artemis, a nuclease implicated in processing of DNA termini at a DSB during NHEJ, has been demonstrated to have both DNA-PK independent 5'-3' exonuclease activities and DNA-PK dependent endonuclease activity. Evidence suggests that either the enzyme contains two different active sites for each of these distinct processing activities, or the exonuclease activity is not intrinsic to the Artemis polypeptide. To distinguish between these possibilities, we sought to determine if it was possible to biochemically separate Artemis endonuclease activity from exonuclease activity. An exonuclease-free fraction of Artemis was obtained that retained DNA-PK dependent endonuclease activity, was phosphorylated by DNA-PK and reacted with an Artemis specific antibody. These data demonstrate that the exonuclease activity thought to be intrinsic to Artemis can be biochemically separated from the Artemis endonuclease. These results reveal novel mechanisms of two critical NHEJ proteins, and further enhance our understanding of DNA-PK and Artemis activity and their role in NHEJ.

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No description available.