No description available.

No description available.

Thesis (Ph. D.)--Lehigh University, 2000. / Includes vita. Includes bibliographical references (leaf 70).

No description available.

No description available.

Copper-containing nitrite reductase (NiR) is a homotrimer of two cupredoxin domains and catalyzes the single electron reduction of NO2- to NO during dissimilatory denitrification. To investigate the evolution of NiR, methods of mutagenic library generation and high-throughput variant screening from E. coli colonies were developed. These methods allow for facile screening of 105 mutants for folding efficiency or substrate specificity. Initial proof of principle studies yielded several variants that oxidized the artificial substrate ο-dianisidine up to 8 times faster than wild type NiR, suggesting that this methodology has the potential to engineer NiR to acquire other reductase functions. A crystal structure was solved for a putative multicopper oxidase (MCO) and NiR homologue from Arthrobacter sp. (AMMCO) to 1.8 Å resolution. The overall folds of AMMCO and NiR are very similar (r.m.s.d. of 2.0 Å over 250 Cα atoms); Like NiR, AMMCO is a trimer with type-1 Cu sites in the N-terminal domain of each monomer; however, the active site of AMMCO contains trinuclear Cu site characteristic of MCOs instead of a the mononuclear type-2 Cu site found in NiR. Detailed structural analysis supports the theory that two-domain MCOs similar to AMMCO were intermediaries in the evolution of NiR and the more common three-domain MCOs. The physiological function of AMMCO remains uncertain, but genomic, crystallographic and functional analysis suggests that the enzyme is involved in metal regulation. Considering the extensive similarity between AMMCO and NiR, particularly at the active site, engineering a trinuclear cluster into NiR appears feasible with a modest number of alterations to the polypeptide chain. With the aid of my newly developed high-throughput screening technique and site-directed mutagenesis, the mononuclear NiR active site was remodelled into a trinuclear Cu site similar to that of MCO. A crystal structure of this variant was solved to 2.0 Å and the presence of three copper atoms at the engineered cluster was confirmed by Cu-edge anomalous diffraction data. Although the trinuclear copper cluster is present and catalyzes the reduction of oxygen, achieving rates of catalysis seen in native MCOs has proven more difficult. With the framework provided, further engineering NiR into a robust MCO is likely to provide further insights into the structural basis of oxygen reduction by trinuclear copper sites.

As workplace demands change, a need has developed for alternatives to traditional education. With advancements in electronic telecommunication technologies, distance education has become a viable alternative to traditional classrooms for working professionals. Efficiency and cost effectiveness are driving many programs to place oncampus students and distance students in the same courses at the same time. This phenomenon has resulted in the placement of students with vastly different backgrounds, levels of expertise, and levels of motivation in the same classrooms. Often a professor will teach to one learning style, leaving some students in the dust, never to get on track. Without face-to-face contact with an instructor, this can leave distance education students feeling isolated and alone. There is a continuing need for the development of alternative instruments to assess self-directed learning (Brockett & Himestra, 1991). We must develop a means of determining an individual??s readiness for self-directed learning, as well as a device for measuring the efficiency of programs designed to foster the attitudes and skills which are involved in increased self-directedness in learning (Guglielmino, 1977). Self-directed learning readiness is important to a learner??s success in distance education programs. Inorder for an educator to tailor instruction to the unique attributes of each student, there is a need for an instrument that will identify the learner??s stage of self-directedness or degree of dependency and for an instrument that will determine the educator??s default teaching style at the beginning of a course. Such an instrument will help instructors increase their learners?? level of self-direction and will improve the overall quality, student satisfaction, and student retention in distance learning courses. The purpose of this study is to develop and pilot test two instruments based upon the Staged Self Directed Learning Model (Grow, 1991) and the key assumptions of andragogy: one measuring the self-directed learning readiness of a student in the context of an individual course and the other measuring the teaching style of the instructor in the context of the same course. The data will be analyzed and given to the instructor to give him/her an idea of the self-directed learning readiness level of students enrolled in the course. A report will be generated to show matches and mismatches between the instructor??s teaching style and the self-directed learning readiness level of the students. A decision model will be developed to suggest teaching strategies that minimize mismatches and facilitate the growth of students from dependent to self-directed through the course.

The purpose of this study was to determine and understand the variables affecting the revival of the draft horse industry in the United States. A qualitative study was conducted using 31 purposively (Lincoln and Guba, 1985) selected draft horse industry participants who were drawn from three case studies, conducted in East Texas, Russia (Siberia), and Northern Indiana. Structured and semi-structured interviews were utilized. Major findings emerged with this research showed that the culture of practicality, inventiveness, and risk taking has allowed communities of individuals to achieve financial success where others have been forced to abandon their businesses and seek other sources of livelihood. Some of the findings stood alone with no seeming connection to the other findings. Other findings appeared to be intertwined with one another. All of the emerging findings contributed to the renewal and engagement of individuals in the draft horse industry revival. The findings are deliberately not listed in any particular order. The scope of the study did not include a method to determine whichfinding or series of findings preceded the others. The major findings to emerge from this study are as follows. Most of the participants in the revival were or are middle aged men and women. This finding melds well with another finding that determined the draft horse business was the second, third, or fourth career of the participants in this study. Many of the participants operate their businesses in rural non-farm locations. Owners and publishers of trade magazines played a pivotal role in this industry revival. Geography and international connections played a role. The Amish communities across the U.S. played a pivotal role in keeping relic technologies alive and maintaining seed stock for the revival of the draft breeds. Specific markets for horses and equipment have been carved out by many of the participants. Self-directed learning and andragogy were exhibited by almost all of the participants. The role of university and extension personnel in this revival was essentially non-existent.

Phosphotriesterase (PTE) isolated from the soil bacterium Flavobacterium sp. is a member of the amidohydrolase superfamily. PTE catalyzes the hydrolysis of a broad spectrum of organophosphate triesters including the insecticide paraoxon, and the chemical warfare agents; GF, sarin, and soman. In addition, PTE has been shown to catalytically hydrolyze the lethal nerve agent, VX. However, the rate of VX hydrolysis is significantly slower. PTE was subjected to directed evolution studies to identify variants with enhanced activity towards VX hydrolysis. First generation libraries targeted amino acid residues in the substrate binding site. The H254A mutation displayed a 4-fold enhancement in kcat and a 2-fold enhancement in kcat/Km over wild type PTE. The double mutant H254Q/H257F was isolated from the second generation libraries and displayed a 10-fold enhancement in kcat and a 3-fold enhancement in kcat/Km. In addition, H254Q/H257F displayed a 9-fold enhancement in kcat/Km for the hydrolysis of the VX analog, demeton-S. An in vivo selection approach utilizing organophosphate triesters as the sole phosphorus source is discussed. The selection is based on co-expressing PTE with the phosphodiesterase (GpdQ) from E. aerogenes. Substrate specificity of GpdQ was investigated using a small library of structurally diverse organophosphate diesters and phosphonate monoesters. Results obtained from the in vivo growth assays showed that GpdQ enabled E. coli to utilize various organophosphate diesters and phosphonate monoesters as the sole phosphorus source. Cells co-expressing PTE and GpdQ were tested for their ability to utilize two different organophosphate triesters as the sole phosphorus source. The results from this experiment indicate that the growth rate is limited by the phosphotriesterase activity. Protein translocation to the periplasm was proven advantageous for in vivo selection since it overcomes the limitation of intercellular delivery of the substrate of interest. Translocation of PTE to the periplasmic space of E. coli was examined. Two signal peptides were tested; the native leader peptide from Flavobacterium sp. and the signal sequence of alkaline phosphatase. The results obtained from cellular fractionation indicated that neither signal peptides were able to translocate PTE to the periplasm and that the protein remained in the cytoplasm.

Thesis--PlanB (M.S.)--University of Wisconsin--Stout, 2002. / Field problem. SUPPLEMENTARY BINDER STORED IN ARCHIVES. Includes bibliographical references.