Kinetic studies on the fidelity of DNA replication involving DNA templates containing O6-methylguanine

Production by N-nitroso compounds of O6-alkylguanine (O6-alkylG) in DNA directs the misincorporation of thymine during DNA replication, leading to G:C to A:T transition mutations, despite the fact that DNA containing O6>alkylG:T base-pairs is less stable than that containing O6-alkylG:C pairs. In the work presented in this thesis, the kinetics of incorporation by Klenow fragment of Escherichia coli DNA polymerase I of thymine (T), and of cytosine (C), opposite O6-meG in the template DNA strand were examined. Both T and C were incorporated opposite O6-meG much slower than nucleotides forming regular A:T or G:C base pairs. Using an excess of Klenow over DNA and various concentrations of dXTF and dCTP, the progress of incorporation of a single nucleotide in a single catalytic cycle of a preformed Klenow-DNA complex was measured (pre-steady state kinetics). The results were consistent with the kinetic scheme: 1. polymerase-DNA binds dNTP; 2. conformational change in polymerase; 3. formation of phosphodiester between the dNTP and the 3'-OH of primer; 4. conformational change of polymerase; 5. release of pyrophsphate. The results were analysed mathematically to identify the steps at which the rate constants differ significantly between the incorporation of T and C. The only significant difference was the 5-fold difference in the rates of formation of the phosphodiester bond (for dTTP, kforward = 3.9 s-1 and kback = 1.9 s-1 for dCTP, kforward = 0.7 s-1 and kback = 0.9 s-l). The equilibrium constants for each step suggest that the greatest change in the Gibbs' free energy occurs at the conformational change after polymerisation, and that while the formation of the phosphodiester bond to T is slightly exothermic, that to C is slightly endothermic. The Kms calculated from the rate constants (Km = 33.5 μM (24.0-46.7)* for both dTTP and dCTP [* 5% and 95% confidence limits]) were close to the approximate Kms obtained from Michaelis-Menten analysis of the initial rates of pre-steady state polymerisation (Km, = 30-35 μM for T and C). The measured progress of independently determined steady state experiments (i.e. polymerisation under conditions of excess DNA over Klenow) was close to that predicted from these calculated rate constants. The incorporation of the nucleotide following C in an O6-meG:C pair was much slower than that following T in an O^-meGiT pair. Taken with the available structural data (Kalnik et al., 1988a, b), this suggests that the discrimination in favour of the incorporation of T opposite O6-meG arises mainly because the T:O6-meG base-pair retains the Watson-Crick configuration (with the N1 of the purine juxtaposed to N3 of the pyrimidine), whereas the C:O6-meG base-pair is a wobble base pair with a distorted phosphodiester link 3' to the C. The slow incorporation of C opposite O6-meG, and of the next correct nucleotide following the incorporation of C, can be ascribed to the stereochemical problems encountered when forming the distorted phosphodiester links. The recent X-ray crystallography data (Beese et al., 1993) of a Klenow complexed with duplex DNA provided evidence that Klenow fragment interacts with the primer-template through the phosphodiester backbone, thus an incorporation event that produces a distortion in the phosphodiester backbone, such as the incorporation of C opposite O6-meG, could very well reduce the rate of its incorporation

Dust beneath the mist: state and frontier formation in the Central Highlands of Vietnam, the 1955-61 period

As an undergraduate student, I read about the distinctiveness of the Central Highlands of Vietnam and its inhabitants in comparison to the Kinh people, the dominant majority from the plains. I was often left with the feeling that these people and their cultures were teetering on the edge of endangerment with the arrival of Kinh settlers and the Vietnamese state. This is the typical storyline of state formation on the frontier, telling how a sophisticatedly organized and technologically powerful people arrived to reign over the margins (of its heartland domain) and the inhabitants there...Another facet to this story of state formation on the frontier, however, is missing from the prevailing literature. I am talking about a facet of the story that paints a less bleak picture of life on the frontier. It tells of how actors on the frontier try to make everyday relations bearable and operational, born out of attempts to understanding one another, albeit sometimes involving misunderstandings. This facet tells of mutability in the state formation process. This thesis explores this facet of the story and uses more nuanced conceptualizations of state, frontier, power, government and social orderings. I call this approach a 'recombinant model' of state formation. This thesis is about one particular period of Vietnamese state formation on the Central Highlands frontier, 1955-61 under the government of the First Republic. I argue that the prevailing approach to understanding Vietnamese state formation on the Central Highlands frontier conceptualizes the state as an a priori formed entity engaged in a process of immutable diffusion on the frontier. I call this the 'diffusion model' of state formation. Using recently available archival documents and the optic provided by the 'recombinant model', I argue that Vietnamese state formation between 1955 and 1961 on the Central Highlands frontier was a mutable process that produced a state form that was hybrid and recombinant in nature. I support this argument with a thick description of policy making and modification of policies by the government on four key issues: administrative standardization, frontier formation program of Dinh Dien, state formation via agrarian change and management of the land question. The story reconstructed through these archival documents does not fit nicely with the storyline advanced by the diffusion model. The recombinant model provides a better framework for understanding this story.The Australian Government via the International Post-Graduate Research Scholarship Award

Riverine sustainment 2012

Student Integrated ProjectIncludes supplementary materialThis technical report analyzed the Navy's proposed Riverine Force (RF) structure and capabilities for 2012. The Riverine Sustainment 2012 Team (RST) examined the cost and performance of systems of systems which increased RF sustainment in logistically barren environments. RF sustainment was decomposed into its functional areas of supply, repair, and force protection. The functional and physical architectures were developed in parallel and were used to construct an operational architecture for the RF. The RST used mathematical, agent-based and queuing models to analyze various supply, repair and force protection system alternatives. Extraction of modeling data revealed several key insights. Waterborne heavy lift connectors such as the LCU-2000 are vital in the re-supply of the RF when it is operating up river in a non-permissive environment. Airborne heavy lift connectors such as the MV-22 were ineffective and dominated by the waterborne variants in the same environment. Increase in manpower and facilities did appreciable add to the operational availability of the RF. Mean supply response time was the biggest factor effecting operational availability and should be kept below 24 hours to maintain operational availability rates above 80%. Current mortar defenses proposed by the RF are insufficient.N

Nitrate Respiration Protects Hypoxic Mycobacterium tuberculosis Against Acid- and Reactive Nitrogen Species Stresses

There are strong evidences that Mycobacterium tuberculosis survives in a non-replicating state in the absence of oxygen in closed lesions and granuloma in vivo. In addition, M. tuberculosis is acid-resistant, allowing mycobacteria to survive in acidic, inflamed lesions. The ability of M. tuberculosis to resist to acid was recently shown to contribute to the bacillus virulence although the mechanisms involved have yet to be deciphered. In this study, we report that M. tuberculosis resistance to acid is oxygen-dependent; whereas aerobic mycobacteria were resistant to a mild acid challenge (pH 5.5) as previously reported, we found microaerophilic and hypoxic mycobacteria to be more sensitive to acid. In hypoxic conditions, mild-acidity promoted the dissipation of the protonmotive force, rapid ATP depletion and cell death. Exogenous nitrate, the most effective alternate terminal electron acceptor after molecular oxygen, protected hypoxic mycobacteria from acid stress. Nitrate-mediated resistance to acidity was not observed for a respiratory nitrate reductase NarGH knock-out mutant strain. Furthermore, we found that nitrate respiration was equally important in protecting hypoxic non-replicating mycobacteria from radical nitrogen species toxicity. Overall, these data shed light on a new role for nitrate respiration in protecting M. tuberculosis from acidity and reactive nitrogen species, two environmental stresses likely encountered by the pathogen during the course of infection

3D printing optimization using recycled HDPE filaments made from used milk jugs

3D Printing has gained popularity over the years for its improved affordability and great versatility. There is a wide variety of 3D printing technologies and a wide spectrum of materials that could be 3D printed, ranging from metallic to nonmetallic. Fused deposition modelling (FDM) has emerged as the most common technology used in desktop/home 3D printers and it is most typically used for printing thermoplastics such as acrylonitrile butadiene styrene (ABS) and polylactic acid (PLA). High density polyethylene (HDPE) is a thermoplastic widely used for packaging. The sheer availability of recycled HDPE (rHDPE) from used packaging encourages efforts to apply it in 3D printing, in particular FDM printing. However, rHDPE is a challenging filament material to work with, with critical problems such as part detachment from print bed and high warpage. The focus of this project would be to determine the optimal conditions that would mitigate, if not eliminate, the print bed adhesion problem associated to printing rHDPE filaments. This was done by carrying out a series of tests, one leading to the next. Finally, a comparison between human perception on part removability and shear strength of print bed adhesion was done to identify the necessary range of print bed adhesion and corresponding parameters.Bachelor of Engineering (Mechanical Engineering

Impact of mothers' work-family boundary on family dinner

Recent research has reported promising multiple benefits of family eating together. However, the antecedents of family dinner have yet to be fully explored. The present study investigated the association of working mothers’ work-family boundary strength (flexibility ability, flexibility willingness, and permeability) with their family dinner outcomes (frequency of family dinners and quality of dinner interactions). In total, 131 Singaporean working mothers completed a survey questionnaire. We analysed the unique contributions of each boundary strength variables in predicting the family dinner outcomes. Results indicated that work flexibility ability which allows mothers to fit their eating time around their child’s schedule was positively correlated with higher family dinner frequency. However, work flexibility ability was insufficient to promote family dinner, but needed to be accompanied by the ability to keep work separate from family during afterhours. Low permeability of the family domain was necessary for mothers to reduce interference from work, allocate adequate eating time with family, and to direct their personal resources to maintain family dinner. Family flexibility willingness was negatively associated with dinner satisfaction while time spent on eating with family was positively associated with dinner satisfaction. Contrary to our predictions, permeability of the family did not predict the quality of dinner interactions. Quality of interactions was positively associated with dinner satisfaction. Findings from this study provide an in-depth look at mothers’ boundary management choices and constraints that influence their achievement of work-life reconciliation. Keywords: working mothers, family dinner frequency, quality of dinner interactions, boundary flexibility, boundary permeabilityBachelor of Arts in Psycholog

Determinants of audit fees in the banking industry.

This project explores the determinants of audit fees in the banking industry

Are NTU Bachelor of Accountancy undergraduates equipped for assurance services?

This research evaluates if the NTU BAcc curriculum structure helps students to develop the relevant competencies for providing assurance services. It also identifies areas for curriculum improvement and suggests recommendations to enhance the curriculum, aiming to propel NTU towards world-class standards

Comparison of EVA adopters and non adopters.

We seek to determine whether poor performance is the primary factor that motivates firms to adopt EVA initially. We also investigated whether the adoption of EVA improves the performance of firms with respect to 14 accounting variables