Thesis (Ph. D.)--Georgia State University, 2007. / Naveen Donthu, committee chair; Detmar Straub, Corliss Thornton, Sevo Eroglu, committee members. Electronic text (149 p. : ill.) : digital, PDF file. Title from file title page. Description based on contents viewed Dec. 10, 2007. Includes bibliographical references (p. 142-149).

Thesis (M.S.)--University of Nevada, Reno, 2005. / "December, 2005." Includes bibliographical references (leaves 73-77). Online version available on the World Wide Web.

Among the genetic mutations involved in carcinogenesis, TP53 mutation is a frequent event in many types of cancer. P53 is a transcription factor that regulates activities such as cell cycle arrest, apoptosis, DNA repair and angiogenesis. The majority of TP53 mutations are missense mutations that accumulate in cancer and are often retained in distant metastases. The effects of the mutant p53 proteins include loss of function, dominant-negative effects over wild-type (WT) p53 and possible acquisition of new properties (gain-of-function). However, some of these properties may differ from one mutant p53 protein to another. These differences could have implications for the in vivo behaviour of tumours carrying particular mutations and hence patient prognosis. The aim of this project was to investigate the phenotypic variation between cells transformed with different p53 mutants. This was achieved by constructing a range of TP53 mutants (R175H, G245S, R248W, R248Q, R273H, R282W) using PCR-based mega-primer site directed mutagenesis. These mutants were cloned into a mammalian bi-cistronic expression vector (designed for the co-expression of WT and mutant TP53 from a single plasmid) to allow transient expression in NCI-H358 cells (p53 null). Regard to the method for PCR site directed mutagenesis, the main technical difficulty with conventional methods was the insufficiency of the mutant TP53 product yield (75%). This thesis has modified these methods by carrying over the start template to a second round of PCR and increasing the MgCl2 concentration. This modified PCR-based site directed mutagenesis method has demonstrated an increased mutant TP53 product yield (100%). The tetracycline expression system is the most widely used for conditional inducible systems in mammalian cells, although high background expression has been a main problem. The ecdysone inducible system potentially allows for the study of the conditional expression of the exogenous reporter gene even though it may be cell lethal or alter the phenotype during the selection of transfectants. This system relies on two independent transfections of two plasmids namely pVgRXR and pIND. However, disruption of the regulatory element within the plasmid during stable integration can result in silence or high background expression of the exogenous reporter gene. A previous study reported a transient luciferase reporter assay to screen the cell line stably transfected with pVgRXR plasmid. However, there is no suitable method to screen the subsequent pIND transfection. This thesis has demonstrated a real time RT-PCR strategy to screen for the background expression problem associated with the ecdysone expression system. However, due to the project’s time limitations, a transient expression system rather than a stable expression system was used. The metastasis related cellular activity of WT/mutant TP53 transfected NCI-H358 cells was examined using a range of in vitro functional assays including a proliferation assay, a p21 promoter binding activity assay, a colony formation assay, and a migration assay. To extend the study, this thesis also employed real-time RT-PCR to examine the mRNA expression level of three metastatic related genes, VEGF, HER-2, and E-cadherin, in the WT/mutant TP53 transfected NCI-H358 cells. The results showed that different WT/mutant TP53 transfected cell linse could contribute to markedly different cellular activity. Among these mutants, R175H produced the highest cellular proliferation activity, the strongest dominant-negative activity over the WT on the p21 promoter binding activity and apoptosis activity, and the greatest effect on cellular migration. Furthermore, the real-time PCR results showed that the WT p53 inhibited transcription of key metastasis-related genes such as VEGF and HER-2. Considered with recent literature, this led me to postulate a feedback amplification cycle involving defective p53 and HER-2 mRNA expression. In conclusion, cancer cells with the R175H mutant could contribute to aggressive tumours. This conclusion, based on the in vitro data, is consistent with some clinical observations and animal model experiments. In the past few years it has become apparent that epigenetic changes also play a vitally important role in the cancer developmental process. Recent studies have reported the p53 protein can contribute in methylation which is one of the processes involved in epigenetic modification. This thesis employed a very new PCR-based AMP technique to examine the change of the global genome methylation pattern as a result of knocked-out p53 protein. The results showed defective p53 protein expression may associate with the global genome methylation pattern changes. However, it is important to note that antibiotic reagents, which were used for stable transfectant selection, could also contribute to the global genome methylation changes. In conclusion, this thesis has successfully developed two new methods. One allows the generation of a genetic mutant construct using PCR-based site directed mutagenesis while the other screens the tightly regulated ecdysone reporter system. In terms of effect of p53 in in vitro cell activity, this thesis has postulated that the R175H mutation is associated with much more aggressive metastatic cellular activity. Finally, this thesis also reported that loss of p53 expression could also result in changes in the global genome methylation pattern.

<p>Probably no group of organisms has been as important as bacteriophages when it comes to the understanding of fundamental biological processes like transcriptional control, DNA replication, site-specific recombination, e.t.c.</p><p>The work presented in this thesis is a contribution towards the complete understanding of these organisms. Two proteins, integrase, and Cox, which are important for the choice of the life mode of bacteriophage P2, are investigated. P2 is a temperate phage, i.e. it can either insert its DNA into the host chromosome (by site-specific recombination) and wait (lysogeny), or it can produce new progeny with the help of the host protein machinery and thereafter lyse the cell (lytic cycle). The integrase protein is necessary for the integration and excision of the phage genome. The Cox protein is involved as a directional factor in the site-specific recombination, where it stimulates excision and inhibits integration. It has been shown that the Cox protein also is important for the choice of the lytic cycle. The choice of life mode is regulated on a transcriptional level, where two mutually exclusive promoters direct whether the lytic cycle (Pe) or lysogeny (Pc) is chosen. The Cox pro-tein has been shown to repress the Pc promoter and thereby making tran-scription from the Pe promoter possible, leading to the lytic cycle. Further, the Cox protein can function as a transcriptional activator on the parasite phage, P4. P4 has gained the ability to adopt the P2 protein machinery to its own purposes.</p><p>In this work the importance of the native size for biologically active integrase and Cox proteins has been determined. Further, structure-function analyses of the two proteins have been performed with focus on the protein-protein interfaces. In addition it is shown that P2 Cox and the P2 relative Wphi Cox changes the DNA topology upon specific binding. From the obtained results a mechanism for P2 Cox-DNA interaction is discussed.</p><p>The results from this thesis can be used in the development of a gene delivery system based on the P2 site-specific recombination system.</p>

The scientific endeavors that took place at Hanford Engineer Works, beginning in World War II and continuing thereafter, are often overlooked in the literature on the Manhattan Project, the Atomic Energy Commission, and in regional histories. To historians of science, Hanford is described as an industrial facility that illustrates the perceived differences between academic scientists on the one hand and industrial scientists and engineers on the other. To historians of the West such as Gerald Nash, Richard White, and Patricia Limerick, Hanford has functioned as an example of the West's transformation during in World War II, the role of science in this transformation, and the recurring impacts of industrialization on the western landscape. This thesis describes the establishment and gradual expansion of a multi-disciplinary research program at Hanford whose purpose was to assess and manage the biological and environmental effects of plutonium production. By drawing attention to biological research, an area in which Hanford scientists gained distinction by the mid 1950s, this study explains the relative obscurity of Hanford's scientific research in relation to the prominent, physics-dominated national laboratories of the Atomic Energy Commission. By the mid 1960s, with growing public concern over radiation exposure and changes in the government's funding patterns for science, Hanford's ecologically relevant research provided a recognizable and valuable identity for the newly independent, regionally-based research laboratory. With funding shifts favoring the biological and environmental sciences in the latter half of the twentieth-century, Hanford scientists were well prepared to take advantage of expanding opportunities to carve out a permanent niche on the border of American science. / Graduation date: 2003

RNA metabolism includes all the processes required for RNA synthesis, maturation, and degradation in living cells. Ribonucleases (RNases) are involved in RNA maturation and degradation, two essential processes in gene expression and regulation in both prokaryotes and eukaryotes. Oligoribonuclease (Orn) has an important role in eliminating small oligonucleotides (nano-RNA), the last step in mRNA degradation. In E. coli, Orn is the only essential exoribonuclease. The enzyme has been shown to form a stable dimer, both in solution and in the crystalline form. Analysis of the three-dimensional structure of Orn allowed us to hypothesize that dimerization is essential for enzyme catalysis. In order to test the hypothesis, I analyzed a number of deletion and point mutants of Orn and determined that tryptophan 143 is essential for dimerization. A W143A mutant is unable to dimerize and has very little activity, similar to that of an active site mutant (D162A). The atomic structure of the W143A mutant, solved at a resolution of 1.9 Å, showed that although the overall three-dimensional fold is similar to that of the wild-type protein, minor differences exist that could account for the monomeric behavior in solution. A flexible Arg174 is repositioned into the cavity created by the missing Trp143. In this new orientation Arg174 protrudes into a hydrophobic pocket in the dimerization interface and is proposed to produce sufficient unfavorable interactions to keep the monomers apart in solution. All these data suggest that dimerization of Orn is essential for its activity. The human homolog of Orn, also known as small fragment nuclease (Sfn), has been shown to degrade short single-stranded RNA, the last step in mRNA decay. In order to determine the mechanism of action of Sfn and its role in the cell, we solved the crystal structure of a truncated form of Sfn at a resolution of 2.6 Å. This mutant form of Sfn lacks the C-terminal 21 amino acids (Sfn-∆C21) yet is as efficient as full length Sfn on model substrates. Interestingly, Sfn is not as active as E. coli Orn in in vitro assays. Analysis of the atomic structure revealed that the active site cleft in Sfn is narrower than the corresponding active site in E. coli. We propose a model for how this narrower cleft may explain the lower in vitro activity. Bacillus subtilis does not have an Orn homolog and until recently, the enzyme responsible for nano-RNA degradation in this organism was unknown. YtqI (also termed nrnA or nanoRNase), a protein unrelated to E. coli Orn, was recently shown to be responsible for the digestion of oligonucleotides in B. subtilis. In order to better understand the mechanism of action of YtqI, I solved its crystal structure at a resolution of 2.0 Å. The nuclease has a RecJ-like fold with two globular domains connected via a flexible linker that forms a central groove. On one side of the groove, the larger N-terminal domain harbors the putative active site, while on the opposite side, the C-terminal domain includes a putative RNA binding domain. The structure of YtqI provides insights into how this enzyme binds and digests oligoribonucleotides. The studies described here provide a better understanding of the mechanism of action for several exoribonucleases that act on nano-RNA oligonucleotides - Oligoribonuclease from E. coli, its close homolog in humans (Small fragment nuclease), as well as a functional homolog in Bacillus (YtqI). This work is relevant to understanding RNA metabolism, which is an essential process for survival of both eukaryotic and prokaryotic organisms.

Thesis (Ph. D.)--University of Helsinki. / Includes bibliographical references (p. 86-93).

Thesis (M.A.)--Oregon State University, 2007. / Printout. Includes bibliographical references (leaves 113-120). Also available on the World Wide Web.

Two deeply buried, well-stratified, and well-dated components dating to the Early Holocene period were excavated at the Richard Beene site (41BX831) in Bexar County, Texas. This thesis utilizes both qualitative (interpretation of maps) and quantitative (unconstrained clustering) spatial analysis techniques to identify site structure and assess post-depositional disturbance by analyzing patterns among artifact categories, selected artifacts, and features from these components. Results of spatial analysis are compared to expectations of the archaeological record based on previous research. Each component revealed a distinct pattern. The Lower Medina component (ca. 6900 B.P.) is well preserved and spatial analysis showed clear distinctions between domestic and peripheral zones. The Upper Perez component (8800 B.P.) is a fluvial lag deposit of displaced artifacts and fire-cracked rock features. Results of spatial analysis confirmed that most, if not all, of this component is disturbed, revealing no site structure.

The objective of this longitudinal study was to study the relationship between changes in prevalence of resistant enteric bacteria associated with mean monthly doses (MMD) of various antibiotics used in each of two host species. From January 2004 – January 2007, monthly composite swine fecal samples and human wastewater samples representing various production and occupational cohorts, respectively, were collected from 19 geographically unique locations in east- and south-central Texas. Bacterial isolates cultivated on CHROMagar-E.coliTM and DifcoTM mEnterococcus (ME) were tested for susceptibility to multiple antibiotics by microbroth dilution using the SensititreTM system. The relationship between the prevalence of resistant bacteria, sampling period, and antibiotic use within each host species was assessed in a generalized linear model adjusted for the dependence of responses within location using a binomial distribution and logit link function in STATA® ver. 9.2. For the swine E. coli isolates, the relationship between tetracycline resistance and level of chlortetracycline (CTC) use in swine illustrated a dose-response relationship, with odds ratios (OR) of 1.20 and 1.81 (P < 0.05) for second- and third-level categories of MMD relative to baseline (zero-use) respectively. When considered by swine production groups, intake boar isolates had an elevated relative odds of resistance to tetracycline (OR = 1.51, P < 0.05), and the nursery units had an elevated odds (OR = 2.61, P < 0.05) of exhibiting resistance to ceftiofur, relative to pigs housed in the farrowing barns. Regarding swine Enterococci isolates, those swine from locations that utilized tylosin had an elevated OR of 3.54 (P < 0.05) of exhibiting resistance to tylosin, relative to those locations that used no tylosin. At this juncture, an apparent occupational risk of harboring tetracycline-resistant E. coli, and the apparent sparing effect (Enterococcus spp.) associated with exposure to swine production, remain unexplained. This study demonstrated that the prevalences of tetracycline- and tylosin-resistant enteric bacteria swine were dependent on CTC and tylosin use in feed, respectively. Swine production group-effects on the prevalence of tetracycline, ceftiofur, and erythromycin resistance were also important. This study provides a better understanding of the relationships between antibiotic prescribing practices at the ecologic level and the relative odds of carriage of resistant bacteria within two host species in a vertically integrated agri-food system.