All organisms must maintain an adequate level of thymidylate, which gets phosphorylated twice and then utilized by DNA polymerases for DNA replication that must precede cell division. Most organisms rely on classical thymidylate synthase (TSase) for this function. However, a subset of microorganisms – including a number of notable, widespread human pathogens – relies on an enzyme with a distinct structure and catalytic strategy. This enzyme is termed flavin-dependent thymidylate synthase (FDTS), as the flavin is required for thymidylate production. Because of this considerable orthogonality between FDTS and classical TSase, FDTS serves as a promising target for new therapeutics – one that could have only mild adverse effects on the host organism. FDTS catalyzes the reductive methylation of uridylate (2′-deoxyuridine-5′-monophosphate; dUMP) to yield thymidylate (2′-deoxythymidine-5′-monophosphate; dTMP). The methylene originally resides on CH2H4folate and is eventually transferred to the nucleotide. This methylene’s route to dUMP is unique in enzymology, and our experiments described herein strive to gain an understanding of the molecular details of its transfer. Compounds that mimic intermediates and transition states along this path are likely to bind FDTS tightly and could be leads for drugs, and our new insights could facilitate this. After methylene transfer is complete, a hydride transfer from flavin to the nucleotide occurs. We utilized rapid quench flow techniques in heavy water to follow the hydrogen transfers in FDTS; solvent isotope effects were measured and analyzed, furnishing evidence that the hydride transfer contributes to rate limitation. Reconstitution of the enzyme with unnatural flavins both reinforced these conclusions and suggested new hypotheses and experiments.

Electrocoagulation is an empirical (and largely heuristic) water treatment technology that has had many different applications over the last century. It has proven its viability by removing a wide range of pollutants. The approach to reactor design has been haphazard, however, with little or no reference to previous designs or underlying principles. This thesis reviewed these reactor designs, identifying key commonalities and synthesising a new design hierarchy, summarised by three main decisions: 1. Batch or continuous operation; 2. Coagulation only or coagulation plus flotation reactors, and; 3. Associated separation process if required. This design decision hierarchy thereby provides a consistent basis for future electrocoagulation reactor designs. Electrochemistry, coagulation, and flotation are identified as the key foundation sciences for electrocoagulation, and the relevant mechanisms (and their interactions) are extracted and applied in an electrocoagulation context. This innovative approach was applied to a 7 L batch electrocoagulation reactor treating clay-polluted water. Structured macroscopic experiments identified current (density), time, and mixing as the key operating parameters for electrocoagulation. A dynamic mass balance was conducted over the batch reactor, for the first time, thereby enabling the extraction of a concentration profile. For this batch system, three operating stages were then identifiable: lag, reactive, and stable stages. Each stage was systematically investigated (in contrast to the previous ad hoc approach) with reference to each of the foundation sciences and the key parameters of current and time. Electrochemical behaviour characterised both coagulant and bubble generation. Polarisation experiments were used to determine the rate-limiting step at each electrode�s surface. Consequently the appropriate Tafel parameters were extracted and hence the cell potential. At low currents both electrodes (anode and cathode) operated in the charge-transfer region. As the current increased, the mechanism shifted towards the diffusion-limited region, which increased the required potential. Polarisation experiments also define the operating potential at each electrode thereby enabling aluminium�s dissolution behaviour to be thermodynamically characterised on potential-pH (Pourbaix) diagrams. Active and passive regions were defined and hence the aluminium�s behaviour in an aqueous environment can now be predicted for electrocoagulation. Novel and detailed solution chemistry modelling of the metastable and stable aluminium species revealed the importance of oligomer formation and their rates in electrocoagulation. In particular, formation of the positively trimeric aluminium species increased solution pH (to pH 10.6), beyond the experimentally observed operable pH of 9. Thereby signifying the importance of the formation kinetics to the trimer as the active coagulant specie in electrocoagulation. Further leading insights to the changing coagulation mechanism in electrocoagulation were possible by comparison and contrast with the conventional coagulation method of alum dosing. Initially in the lag stage, little aggregation is observed until the coagulant concentration reaches a critical level. Simultaneously, the measured zeta potential increases with coagulant addition and the isoelectric point is attained in the reactive stage. Here a sorption coagulation mechanism is postulated; probably charge neutralisation, that quickly aggregates pollutant particles forming open structured aggregates as indicated by the low fractal dimension. As time progresses, pollutant concentration decreases and aluminium addition continues hence aluminium hydroxide/oxide precipitates. The bubbles gently sweep the precipitate through the solution, resulting in coagulation by an enmeshment mechanism (sweep coagulation). Consequently compact aggregates are formed, indicating by the high fractal dimension. Flotation is an inherent aspect of the batch electrocoagulation reactor via the production of electrolytic gases. In the reactor, pollutant separation occurs in situ, either by flotation or settling. From the concentration profiles extracted, original kinetic expressions were formulated to quantify these competing removal processes. As current increases, both settling and flotation rate constants increased due to the additional coagulant generation. This faster removal was offset by a decrease in the coagulant efficiency. Consequently a trade-off exists between removal time and coagulant efficiency that can be evaluated economically. A conceptual framework of electrocoagulation is developed from the synthesis of the systematic study to enable a priori prediction. This framework creates predictability for electrocoagulation, which is innovative and original for the technology. Predictability provides insights to knowledge transfer (between batch and continuous), efficient coagulant and separation path, to name just a few examples. This predictability demystifies electrocoagulation by providing a powerful design tool for the future development of scaleable, industrial electrocoagulation water treatment design and operation process.

There were two purposes to the current set of studies. The first was to test the validity of the Adaptiveness of Defenses Scale (ADS), a new measure developed to assess variability within ego defense mechanisms. Study 1 addressed the validity of the ADS with a sample of 104 male undergraduates. The ADS was hypothesized to account for significant unique variance on dependent measures (the PAI, ECR, and final grade in introductory psychology) beyond that accounted for by the Defense-Q. Regression analyses showed that a different pattern emerged. The Defense-Q and ADS accounted for different aspects of the dependent variables, with the Defense-Q predicting PAI Somatization scale scores and the ADS predicting ECR avoidance scale scores. Study 2 addressed the validity of the ADS with a sample of 64 male federal inmates. The ADS was hypothesized to account for significant unique variance on dependent measures (the BPI, PCL-R, and PAS) beyond that accounted for by the Defense-Q. As with Study 1, the two measures tended to predict different aspects of the dependent measures. The Defense-Q predicted immature defense factor scores on the BPI as well as BPI total score, and it predicted affective instability factor scores on the PAS, as well as PAS total score. Conversely, the ADS predicted PCL-R total and Factor 1 scores. The relation between the Defense-Q ADP similarity score and the PCL-R interpersonal facet was significantly positive, while the relation between the ADS total score and this facet was significantly negative. The results of Studies 1 and 2 suggest that the ADS captures an aspect of defensive functioning different from that measured by the Defense-Q, likely related to interpersonal functioning. Study 3 examined the relation between defenses (measured by the Defense-Q and ADS) and correctional variables (offense history, institutional incidents, institutional charges, urinalysis outcomes, and correctional program outcomes), as well as examined Aboriginal and non-Aboriginal differences. The differences between Aboriginal and non-Aboriginal inmates tended to show that non-Aboriginal inmates were older and had more aggressive criminal histories as well as higher PCL-R total and Factor 1 scores. They also tended to have more problematic institutional adjustments, likely because of their higher levels of psychopathy. The Defense-Q was significantly related to general offence history for Aboriginal inmates and the ADS was significantly related to violence history for non-Aboriginal inmates, but other significant relations were sparse. The results of the current series of studies provides preliminary support for the idea that individual defenses have their own ranges of adaptiveness, as the ADS scores male undergraduates and male inmates were significantly different for the same defenses. Given the pattern of the relations of the ADS to the dependent variables, it appears that the ADS is sensitive to interpersonal functioning.

There were two purposes to the current set of studies. The first was to test the validity of the Adaptiveness of Defenses Scale (ADS), a new measure developed to assess variability within ego defense mechanisms. Study 1 addressed the validity of the ADS with a sample of 104 male undergraduates. The ADS was hypothesized to account for significant unique variance on dependent measures (the PAI, ECR, and final grade in introductory psychology) beyond that accounted for by the Defense-Q. Regression analyses showed that a different pattern emerged. The Defense-Q and ADS accounted for different aspects of the dependent variables, with the Defense-Q predicting PAI Somatization scale scores and the ADS predicting ECR avoidance scale scores. Study 2 addressed the validity of the ADS with a sample of 64 male federal inmates. The ADS was hypothesized to account for significant unique variance on dependent measures (the BPI, PCL-R, and PAS) beyond that accounted for by the Defense-Q. As with Study 1, the two measures tended to predict different aspects of the dependent measures. The Defense-Q predicted immature defense factor scores on the BPI as well as BPI total score, and it predicted affective instability factor scores on the PAS, as well as PAS total score. Conversely, the ADS predicted PCL-R total and Factor 1 scores. The relation between the Defense-Q ADP similarity score and the PCL-R interpersonal facet was significantly positive, while the relation between the ADS total score and this facet was significantly negative. The results of Studies 1 and 2 suggest that the ADS captures an aspect of defensive functioning different from that measured by the Defense-Q, likely related to interpersonal functioning. Study 3 examined the relation between defenses (measured by the Defense-Q and ADS) and correctional variables (offense history, institutional incidents, institutional charges, urinalysis outcomes, and correctional program outcomes), as well as examined Aboriginal and non-Aboriginal differences. The differences between Aboriginal and non-Aboriginal inmates tended to show that non-Aboriginal inmates were older and had more aggressive criminal histories as well as higher PCL-R total and Factor 1 scores. They also tended to have more problematic institutional adjustments, likely because of their higher levels of psychopathy. The Defense-Q was significantly related to general offence history for Aboriginal inmates and the ADS was significantly related to violence history for non-Aboriginal inmates, but other significant relations were sparse. The results of the current series of studies provides preliminary support for the idea that individual defenses have their own ranges of adaptiveness, as the ADS scores male undergraduates and male inmates were significantly different for the same defenses. Given the pattern of the relations of the ADS to the dependent variables, it appears that the ADS is sensitive to interpersonal functioning.

In the study, we choose the companys that had conducted IPOs or SEOs between 2005 and February 2006 as studying samples. The purpose of the study is to investigate how the issuing firms choose among different underwriting mechanisms and the factors influencing the choice of underwriting mechanisms. Empirical result shows that issuing factors are the important factors that influence the choice of underwriting mechanisms. The firms that conduct IPOs usually trend to choose bookbuilding method, but the firms that conduct SEOs trend to choose fixed price method instead. On the other hand, we also find that issuing scale is another significant factor influencing the choice of underwriting mechanisms and the effect is more apparent on the firms that conduct SEOs. The firms with bigger issuing scale trend to choose bookbuilding method but the firms with smaller issuing scale trend to choose fixed price rather than bookbuilding method.

Myotonic dystrophy type 2 (DM2) is caused by the extreme expansion (from < 30 repeats in normal individuals to ~ 11,000 for the full mutation in certain patients) of the repeating tetranucleotide CCTG•CAGG sequence in the intron of the zinc finger protein 9 (ZNF9) gene. The genetic instabilities of the CCTG•CAGG repeats were investigated to evaluate the molecular mechanisms responsible for these massive expansions. The effects of replication, recombination, repair and transcription on the genetic instabilities have been investigated in COS-7 cells and E. coli model systems. A replication assay was established in COS-7 cells wherein the CCTG•CAGG repeats cloned proximal to the SV40 origin of replication resulted in expansions and deletions in a length and orientation-specific manner, whereas the repeats cloned distal to the same origin were comparatively stable. These results fit with our data obtained from biochemical studies on synthetic oligonucleotides since these biochemical studies revealed that the d(CAGG)26 oligomer had a marked propensity to adopt a hairpin structure as opposed to its complementary d(CCTG)26 that lacked this capacity. Furthermore, a genetic assay in E. coli was used to monitor the intramolecular frequency of recombination. This assay revealed that the tetranucleotide repeats were indeed hot spots for recombination. Moreover, studies conducted in SOS-repair mutants showed that recombination frequencies were much lower in a SOS¯ strain as compared to a SOS+ strain. However, experiments conducted to ascertain the level of induction of the SOS response revealed that the SOS pathway was not stimulated in our studies. These results revealed that although breaks may occur within the repeats, the damage is most likely repaired without induction of the SOS response contrary to previous beliefs. Thus, a complex interplay of replication, recombination, and repair is likely responsible for the expansions observed in DM2.

Vascular endothelial growth factor receptor-2 (VEGFR2) is a key angiogenic factor, and angiogenesis is an important physiological process associated with neovascularization, growth, and metastasis of many different tumors. The mechanism of VEGFR2 gene expression was investigated in MiaPaCa-2, Panc-1, and AsPC-1 pancreatic cancer cells transfected with a series of VEGFR2 promoter deletion/mutated constructs, and the results indicated that the GC-rich –60 to –37 region of the promoter was essential for VEGFR2 expression in these cell lines. EMSA and ChIP assays showed that Sp proteins are expressed and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies on Sp proteins demonstrated that Sp1, Sp3, and Sp4 all contributed to VEGFR2 gene/protein expression in pancreatic cancer cells. VEGFR2 gene expression was also investigated in ZR-75 and MCF-7 breast cancer cells. ZR-75 cells treated with 10 nM 17b-estradiol (E2) increased VEGFR2 mRNA levels/protein expression. The VEGFR2 promoter was induced by E2 in ZR-75 cells, and analysis of the VEGFR2 promoter identified the GC rich -60 to -37 region that was required for E2-mediated transactivation. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in ZR-75 cells and bind the proximal GC-rich region of the VEGFR2 promoter. RNA interference was used to determine the relative contributions of Sp proteins on hormonal regulation of VEGFR2 through ER/Sp complexes, and interestingly, in ZR-75 cells, hormone-induced activation of VEGFR2 involves ERa/Sp3 and ERa/Sp4 but not ERa/Sp1. In MCF-7 cells treated with 10 nM E2, VEGFR2 mRNA levels were decreased. Analysis of the VEGFR2 promoter revealed that the same GC-rich region important for E2-mediated upregulation in ZR-75 cells was responsible for E2-dependent downregulation of VEGFR2 gene expression in MCF-7 cells. EMSA and ChIP assays confirmed that Sp1, Sp3, and Sp4 proteins are expressed in MCF-7 cells and bind to the proximal GC-rich region of the VEGFR2 promoter. RNA interference studies showed that Sp1, Sp3, and Sp4 are involved in the E2-mediated downregulation of VEGFR2 in MCF-7 cells, and ERa/Sp protein-promoter interactions are accompanied by recruitment of the corepressor SMRT using the ChIP assay.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains x, 225 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 113-119).

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 204-231). Available also in a digital version from Dissertation Abstracts.

Thesis (Ph. D.)--University of Texas at Austin, 2003. / Vita. Includes bibliographical references. Available also from UMI Company.