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The impacts of the paternal genome and proteins transferred to the oocyte through spermatozoa cannot be neglected during mammalian embryonic development. Studies over the past 40 years suggest that sperm chromatin alterations (such as DNA fragmentation induced by either chromatin condensation errors, apoptosis and/or oxidative stress) might be negatively associated with fertilization and early embryonic development [1, 2], [3] [4].However, precise molecular mechanisms by which sperm chromatin integrity and sperm proteins impact early embryonic development still remain unclear. Therefore, the objectives of this study were 1) determine DNA fragmentation induced by apoptosis its relationship with male fertility in spermatozoa from bulls with varying fertility, and 2) identify expression dynamics of Protamine 1 and examine chromatin structure in spermatozoa from bulls with varying fertility. To accomplish our goals we determined 1) the DNA damage, phosphatidylserine (PS) translocation, and expression of pro- and anti-apoptotic proteins (BAX and BCL-2) as well as 2) the expression and localization of Protamine 1 (PRM1) with chromatin condensation and protamination in sperm from bulls with varying fertility. Our results demonstrated that the most relevant fertility markers might be the percentage of necrotic spermatozoa detected by flow cytometry and live spermatozoa determined via eosin-nigrosin staining and that there was no relationship between apoptosis and male fertility. While BCL-2 was not expressed, BAX was identified in bovine spermatozoa. However, the expression of BAX did not differ among groups. In addition, defective chromatin condensation and protamination errors were significantly increased in sperm from low fertility bulls, while the expression of PRM1 was significantly abundant in high fertility bulls. Bull fertility was negatively correlated with protamination errors and defective chromatin condensation, and it was positively correlated with the expression of PRM1. We concluded that defective sperm DNA condensation, not abortive apoptosis, might be the major reason of male infertility in bulls and that sperm chromatin stability differs among bulls with varying fertility. Improper chromatin packaging during spermatogenesis might be caused by the limited expression and/or mislocalization of PRM1. Thus, inadequate chromatin dynamics were associated with bull infertility, which might lead improper fertilization.

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Adverse outcome pathways (AOPs) provide a framework to organize and weigh the evidence linking a toxicant’s initial interactions with molecules in the cell to adverse outcomes of regulatory concern. AOPs are constructed in modules that include key events (KEs) and key event relationships (KERs). Quantitative understanding of the KERs is critical for the development of predictive toxicological models. The objective of this project was to investigate the ability to define the quantitative associations of the KERs upstream, and contained in, an existing AOP (#296): “Oxidative DNA Damage Leading to Chromosomal Aberrations and Mutations”. The data supporting quantitative associations between these KERs was gathered through literature review and experimental methods. I first used systematic literature review tools to develop and apply a pragmatic and transparent method to search the literature for AOP evidence. A broad search, covering all of the KERs of interest, was initially conducted. This search, which retrieved more than 230 thousand articles, demonstrates the data-rich nature of the AOP. An artificial intelligence informed prioritization of the top 100 articles were then examined in detail. This approach identified 39 articles containing qualitative empirical support for the AOP, but limited quantitative evidence of the KERs. A second search was conducted to address the need for quantitative evidence as well as the lack of evidence for the KER between and increase in reactive oxygen species (ROS) and oxidative DNA damage. The second search retrieved 12 articles that could be used to define a quantitative relationship between cellular ROS and oxidative DNA damage. To begin to address gaps in quantitative understanding, I then conducted experiments in the laboratory to measure oxidative DNA damage, DNA strand breaks, chromosomal aberrations, and mutations in TK6 cells after exposure to a range of concentrations of 4-Nitroquinoline 1-oxide (4NQO: a prototype ROS producing agent). An increase in both oxidative DNA damage and DNA strand breaks was observed after 2, 4, and 6 h exposures with the high throughput comet assay (CometChip). An increase in the incidence of micronuclei was observed after a 24 h exposure to a low concentration of 4NQO, as measured with the flow cytometry micronucleus assay, while high cytotoxicity was found at higher concentrations. Lastly an increase in mutation frequency was observed with Duplex Sequencing, an error-corrected sequencing technology. Additionally, an increase in the proportion of C>A transversions was observed, consistent with the expected mutations following oxidative DNA lesions. Overall, my work contributes to the quantitative understanding of AOP #296 and this project serves as a key example of AOP-informed study design, highlighting notable challenges in characterizing quantitative relationships.

Translesion DNA Synthesis (TLS) is a mechanism that promotes DNA damage tolerance during DNA replication using an error-prone DNA polymerase complex. The complex is comprised of the ImuA, ImuB, and ImuC proteins that are found in approximately one-third of bacteria, including high priority antimicrobial resistant pathogens, such as Pseudomonas aeruginosa. Previous in vivo studies have shown that TLS increases beneficial bacterial mutations as the error-prone DNA polymerase, ImuC, lacks proof-reading activity. However, how ImuA and ImuB proteins contribute to the polymerase mechanism is unknown. Thus, the goal of this study is to characterize the TLS proteins in vitro to determine how ImuA and ImuB associate with ImuC to promote error- prone replication. ImuA and ImuBNΔ34 were successfully purified for biochemical characterization from the homolog Myxococcus xanthus. Using size-exclusion chromatography coupled to multi-angle light scattering, both ImuA and ImuBNΔ34 are trimers in solution. Each protein also binds DNA independently as assessed by fluorescence polarization. Interestingly, both proteins bind ssDNA and a 3’ overhang substrate mimicking the DNA replication intermediate with the highest affinity. DNA binding assays further confirm these proteins can form a DNA-ImuA-ImuBNΔ34 complex. Using bacterial two-hybrid assays, the ImuA- ImuB interaction occurs in the C-terminal region of both proteins. Overall, these results suggest that ImuA and ImuB may recruit and stabilize ImuC on DNA for replication past damaged DNA, providing the first insights into the ImuA and ImuB molecular mechanism. / Thesis / Master of Science (MSc)

Inhibiting inflammatory processes or eliminating inflammation represents a logical role in the suppression and treatment strategy of cancer. Several studies have shown that anti-inflammatory drugs (NSAIDs) have promise as anticancer agents while reducing metastases and mortality. NSAIDs are seriously limited by side effects and their toxicity, which can become cumulative with their long-term administration for chemoprevention. The huge development in nanotechnology allows the drugs to exhibit novel and significantly improved properties compared to the large particles of the respective bulk compound, leading to more targeted therapy and reduced dosage. The overall aim of this thesis is to add to our understanding of cancer prevention and treatment through studying the genotoxicity mechanisms of NSAIDs agents in lymphocytes. In this study, the genotoxicity mechanisms of NSAID in bulk and nanoparticles forms a strategy to prevent and minimise the damage in human lymphocytes. Aspirin nano (ASP N) caused a significant decrease in deoxyribonucleic acid (DNA) damage compared to aspirin bulk (ASP B). Also, ibuprofen nano (IBU N) showed a significant reduction in DNA damage compared to ibuprofen bulk (IBU B). Micronuclei (MNi) decreased after ASP N, ASP B and IBU N in prostate cancer patients and healthy individuals, and the ibuprofen bulk showed a significant increase of MNi formation in lymphocytes from healthy and prostate cancer patients when compared to untreated lymphocytes from prostate cancer patients. In order to study the geno-protective properties of these drugs, the protective effect of NSAIDs and the quantification of the DNA repair capacity in lymphocytes was studied. ASP N was found to increase the DNA repair capacity and reduced the reactive oxygen species (ROS) formation significantly more than ASP B. Finally, the role of NSAIDs on some key regulatory signal transduction pathways in isolated lymphocyte cells was investigated by studying their effect on ataxia-telangiectasia-mutated kinase (ATM) and ataxia-telangiectasia and Rad3-related kinase (ATR) mRNA. ATM mRNA significantly increased after treatment with ASP B, ASP N and IBU N. ATR expression also increased after treatment with IBU B and IBU N, but was only significant with IBU N. These findings indicate that a reduction in particle size had an impact on the reactivity of the drug, further emphasising the potential of nanoparticles as improvement to current treatment options.

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