published_or_final_version / Physiology / Doctoral / Doctor of Philosophy

Prostaglandin E2, postulated to be immunosuppressive to the tumor bearing host, is produced and excreted in elevated quantities by many tumors. Arachidonic acid, the precursor molecule for PGE2, is released from membrane phospholipids by phospholipase A2. Phospholipase A2 has been proposed as the rate limiting enzyme in the production of prostaglandin E2.Phospholipase A2 from different sources varies in substrate specificities, pH optima, and Ca ++ concentration requirements. Therefore, the determination of its specific activity depends on the development of appropriate incubation, extraction, and identification methodologies.This study attempted to develop methodologies for determination of PLA2 activity using enzymes from snake venom, mouse liver, and normal and tumored mouse mammarytissue. The method of substrate preparation, kind of substrate, amount of protein, length of incubation, and addition of KC1 and deoxycholate were varied. Reaction products were extracted and isolated with hexame, and methylated with diazomethane. The methyl esters were identified by gas liquid chromatography. Quantitative analyses were based on proportionality of experimental peak areas to internal standard peak area.Activity could not be demonstrated with snake venom or liver PLA2 preparations. Low specific activity was obtained in some tumor and normal mammary tissue extracts. These studies will be used as a basis for developing an optimal assay system for PLA2 from normal and tumored mouse mammary tissue.

Diss. (sammanfattning) Uppsala : Univ., 2004. / Härtill 4 uppsatser.

Univ., Diss.--Münster, 2004.

Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2004. / Includes bibliographical references (leaves 94-103). Also available in electronic version. Access restricted to campus users.

Thesis (Ph. D.)--University of Texas at Austin, 1998. / Vita. Includes bibliographical references (leaves 195-227). Available also in a digital version from Dissertation Abstracts.

Thèse (M.Sc.)--Université Laval, 2003. / Bibliogr.: f. 89-104. Publié aussi en version électronique.

Thesis (Ph. D.)--West Virginia University, 2006. / Title from document title page. Document formatted into pages; contains viii, 156 p. : ill. (some col.). Includes abstract. Includes bibliographical references.

No description available.

Doctor of Philosophy / Department of Agronomy / William T. Schapaugh Jr / Harold N. Trick / Demands on value-added crops have been raised to improve agricultural, industrial, and economical value. Currently, transgene application is one of most effective methods to satisfy these demands. Success in herbicide-resistant soybean (Glycine max (L.) Merr.) has boosted genetic engineering to be used for biochemical, nutritional, cultural, and physiological improvements. The objectives of this study were to establish transgenic soybean lines with attenuated phospholipase D[Alpha] (PLD[Alpha]) activity in the seed, test the alteration of fatty acid profiles affected by transgene and somaclonal variation, and evaluate the physiological alteration of transgenic lines by both transgene and somaclonal variation. To change fatty acid profile in soybean seed, we attenuated PLD[Alpha] enzyme activity by an RNA interference construct using the PLD[Alpha] gene sequence. Two transgenic soybean lines were established by particle inflow gun bombardment of co-bombarding pSPLDi and pHG1 transgenes, and evaluated for the presence and expression of transgenes thoroughly through the T[subscript]5 generation. PLDα-suppressed soybean lines were characterized by decreased PLD[Alpha] enzyme activity and PLD[Alpha] protein both during seed development and in mature seeds. The PLD[Alpha]-attenuated transgenic lines, SW1-7-1-1 and SW1-7-1-2, contain 36% and 49% oleic acid in the filed and greenhouse evaluations, respectively, which are equivalent to the mid-oleic acid soybean lines improved by conventional breeding and mutagenesis. Phenotypic and genetic analysis of the transgenic lines suggested the possibility that the multi-copy transgene integration formed direct or indirect repeats by random ligation during integration and organization of transgenes in the soybean genome, and the transgene cluster with tandem repeats may consequently increase the probability of transgene silencing. Various factors, such as high humidity and temperature, result in the loss of seed viability. Fayette seed stored for two months since harvest exhibited about 95% viability; however Fayette seeds stored for 33 months at room temperature and uncontrolled relative humidity become non-viable. PLDα-attenuated transgenic soybean seeds have been produced by transformation. PLD-suppressed transgenic soybean seeds have maintained viability when stored for 33 months at room temperature. Germination of transgenic seed stored for 33 months ranged from 30 to 50%. Increased leakage of electrolytes associated with the loss of viability was observed in null-transgenic and background seeds versus transgenic seed. The increase in electrolyte leakage may have been induced by lipid peroxidation and free radical formation which can generate oxidative damage in the cell and subsequently decrease seed viability. Differences in the ultrastructure of cotyledon tissue were observed between PLD[Alpha]-suppressed soybean and the background cultivar. The loss of viability in the background cultivar was consistent with observations of the plasma membrane being detached from the cell wall complex and disorganization of oil bodies. Stresses caused by temperatures higher or lower than ambient are one of agricultural problems that reduce crop productivity in many areas and diverse species. To overcome the uncertainty of environmental fluctuations, efforts continue to improve high and low temperature tolerance in crops. PLD[Alpha]-suppressed transgenic events were produced by antisense suppression driven by constitutive and seed-specific promoters using the particle inflow gun (PIG) bombardment method. Nine fertile transgenic events suppressed the expression of PLD[Alpha] protein. PLD{Alpha] enzyme activity in T[subscript]1 seed was observed to be reduced by 25 percent compared to the non-transgenic control. When soybean seedlings were exposed to lethal freezing temperature, increased electrolyte leakage associated with oxidative damage and biophysical changes were observed in non-transgenic soybean, whereas membrane stability and integrity were maintained in transgenic soybean seedlings. The early growth of PLD[Alpha]-attenuated soybean seedlings was recovered from extreme heat-shock (45 °C) and freezing treatments (-8 °C). The disruption of the plasma membrane and organelles was observed in freeze-stressed non-transgenic control seedlings. On the other hand, the structures of the plasma membrane, oil bodies, and cell organelles in transgenic seedlings were partially sustained after enduring freezing and thawing stresses.