The E2F1 pathway plays an important role in coordinating early cell cycle progression. Deregulation of the E2F1 pathway, which may be brought about by somatic mutations in genes such as INK4A, RB1 and CDK4, is found in a majority of cancers. The transcription factor E2F1 is able to activate genes involved in proliferation as well as apoptosis, but the mechanisms that govern these opposing biological effects remain poorly understood. In this study, I would describe how E2F1 activity can be regulated by two novel post-translational modifications which result in different functional consequences. It was found that PRMT1 asymmetrically methylates E2F1 at R109, while PRMT5 symmetrically methylates R111 and R113. The symmetric dimethyl marks on E2F1 promoted the recruitment of Skp2, a component of the E3 ubiquitin ligase complex, which coincided with decreased protein stability and transcriptional activity, as well as enhanced cell proliferation and reduced apoptosis. The asymmetric dimethyl marks on E2F1 was found to hinder symmetric dimethylation, leading to reduced cell proliferation and enhanced apoptosis. The competition between PRMT1 and PRMT5 at the E2F1 RG-rich motif was found to be regulated by the adjacent cyclin A binding site. Induction of DNA damage, which resulted in decreased cyclin A level, corresponded to an increase in PRMT1 and decrease in PRMT5 binding. This study uncovers a new mechanism in E2F1 regulation and establishes the importance of arginine methylation in cell proliferation and apoptosis.

Circadian clocks are biochemical mechanisms that allow eukaryotic and some prokaryotic organisms to coordinate their physiology with daily environmental changes. It enables organisms to increase their fitness by taking advantage of beneficial environmental conditions while also avoiding or restricting certain sensitive processes during harsh conditions. Similarly, post-translational histone modifications allow eukaryotic organisms to regulate gene expression in response to environmental or developmental factors. Some post-translational modifications of histones are associated with active transcription while others are associated with repressed transcription depending upon the location, type and degree of modification. Trimethylation of lysine 4 on the N-terminal tail of histone H3 (H3K4me3) near a gene's promoter has been linked to active transcription of that gene in several organisms. The purpose of the current study was to investigate whether the amount of H3K4me3 at promoters of three specific genes shows a circadian rhythm in Chlamydomonas reinhardtii, a unicellular green alga. Two of the genes had previously been shown to display a circadian rhythm of expression with opposite phase (LHCBM6 and JMJD6-like2), while the third gene is constitutively expressed (RACK1). Quantitative PCR was used to determine the amount of immunoprecipitated H3K4me3 over a circadian cycle. It was hypothesized that H3K4me3 amount at the JMJD6-like2 and LHCBM6 promoter would show a circadian rhythm with a phase correlating directly with the phase of each gene’s rhythm of expression. Conversely, the H3K4me3 amount at the RACK1 promoter was predicted to not show a circadian rhythm, as the gene is constitutively expressed. Instead, results showed that H3K4me3 amount exhibits a circadian rhythm with identical phase for all three genes. ANOVA confirmed that the rhythms were not significantly different between the three genes. General histone H3 amount at promoters did not show a circadian rhythm across any of the three genes. Since recent genome-wide studies in mouse liver revealed a circadian rhythm of H3K4me3 amount with identical phase at the promoter of many genes with diverse expression, the findings presented here suggest that C. reinhardtii might show a similar global regulation of rhythmic H3K4me3 as in mice and that, therefore, this feature has been preserved during eukaryotic evolution.

published_or_final_version / Obstetrics and Gynaecology / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / toc / Surgery / Doctoral / Doctor of Philosophy

published_or_final_version / abstract / Medicine / Doctoral / Doctor of Philosophy

published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

published_or_final_version / Anatomy / Master / Master of Philosophy

The production of methyl mercury, an acute neurotoxin which readily accumulates in the tissue of organisms, is a biologically mediated process facilitated by sulfate reducing bacteria in aquatic sediments. In-situ capping is a frequently considered risk management strategy for contaminated sediments. Since placement of an in-situ cap will induce anaerobic conditions that are known to be favorable for the growth of sulfate reducing bacteria, there is justifiable concern that capping could increase mercury methylation in underlying sediments. This research builds an understanding of the effects of in-situ capping on underlying biogeochemical processes and elucidates their importance in controlling methyl mercury production. Laboratory experiments and mathematical models were implemented to simulate mercury methylation in redox conditions likely to be induced by capping using sediment from different environments. Mathematical descriptions of processes known to be involved in methylation were incorporated into the model to quantify the effects of these processes. Observations in both well-mixed slurry conditions and intact sediment columns showed that methyl mercury concentrations are strongly dependent upon biogeochemical conditions. Results from experiments with sediment spanning a range of redox conditions and organic contents suggested that sulfate reduction rates, aqueous speciation, and solid phase partitioning are involved in limiting methylation depending on bulk geochemical characteristics. A model with a mechanistic basis that incorporates the effects of these processes provides a useful means of qualitatively and quantitatively considering their cumulative impact in limiting methyl mercury production. High methyl mercury concentrations observed in some lab experiments suggest that there is reason to be concerned about anoxic conditions induced by capping; however, not all anoxic conditions led to equivalent increases in methyl mercury. Experimental and modeling results suggest that in a high organic environment, in-situ capping may produce conditions which accelerate methylation in (formerly) surficial sediment while in a low organic environment, with an overall lower potential for methylation, capping can be expected to have a less dramatic effect. Over time, two processes will temper capinduced increases in methyl mercury. Increases will only last until sulfide builds up to inhibitory levels in underlying sediment or until organic carbon is depleted and overall bacterial activity slows. By providing a more fundamental understanding of the effects of capping on mercury methylation, the results of this research will aid in identifying situations and conditions in which cap-induced increases in methyl mercury have the potential to limit the effectiveness of the management strategy. / text

Uncoupling proteins (UCPs) are localized on the inner mitochondrial membrane (IMM) and “uncouple” the electrochemical proton gradient formed by the electron transport chain (ETC) from ATP production. Though the prototypical uncoupling protein 1 (UCP1) is known to mediate the cold-induced thermogenesis in rodents and human neonates, the physiological and biochemical functions of the homologs UCP2-5 are still under debate. Our research focuses on UCP3, the homolog prevalently expressed in skeletal muscle (SKM), the most important metabolic organs. UCP3 has long been speculated to have a pivotal role in maintaining the mitochondrial metabolism. Several biochemical roles have been attributed to UCP3, including the regulation of fatty-acid transport and oxidation, reactive oxygen species (ROS) scavenging and calcium uptake. And several proteins have been identified to directly bind with UCP3 and facilitate its function. But to further understand how UCP3 relates to different aspects of mitochondrial functions, a more comprehensive profile of the UCP3 interaction partners is needed. We performed a mass spectrometry-based experiment and successfully identified a list of over 170 potential proteins that may directly or indirectly interact with UCP3, and several novel functions of UCP3 are implied by these protein-protein interactions. Additionally, researches have shown that the metabolic defects are important contributing factors to the epigenetic changes. Considering the roles of UCP3 in sustaining the normal mitochondrial metabolism, we hypothesized that UCP3 has a novel function in regulating the genomic DNA methylation processes. The data we obtained from the pilot study confirms that loss of UCP3 will lead to aberrant DNA methylation changes. But further experiment is still needed to investigate the regulatory pathway between UCP3 and DNA methylation. The physiological role of UCP3 in defending against cancer, diabetes and obesity has been investigated, but the mechanisms how UCP3 protect the organism from these diseases have not been elucidated. Our research sheds light on the understanding of UCP3 functions and may be of significant therapeutic benefit in the prevention and treatment of these diseases. / text

abstract / Medicine / Doctoral / Doctor of Philosophy