Thesis (Ph. D.)--University of Wisconsin--Madison, 1964. / Typescript. eContent provider-neutral record in process. Description based on print version record. Bibliography: leaves 146-147.

Thesis (M.S.)--Rochester Institute of Technology, 1994. / Typescript. Includes bibliographical references (leaves 218-221).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1969. / Typescript. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliography.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1959. / Typescript. Abstracted in Dissertation abstracts, v. 20 (1959) no. 2, p. 469-470. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [107]-114).

The purpose of this study was to examine the phenomenon of aging at the cellular level in vitro. Some of the biological mechanisms underlying the aging process, which may invoke a change at the level of the DNA were studied. Morphological changes were analysed with phase-contrast microscopy and functional changes by the use of tritiated thymidine in combination with autoradiography as well as by cell treatment with colchicine. A method is described for obtaining "aged" or "old" cells in vitro. In the human cells (human embryonic kidney) as well as the rat, mouse, and Syrian hamster cells, morphological changes in vitro are basically the same as aging progresses. These include transformation to a polygonal, epithelial -1ike shape; binucleation; an accumulation of "age pigments" around the nucleus; the appearance of ragged edges of the cell membrane; an increase in the overall cell size; and a loss of a regular (often parallel) orientation to adjacent cells. The mitotic rate and DNA-synthesizing capacity in "young" and "aged" cells were examined using autoradiography and cell treatment with colchicine. Evidence is presented that DNA-synthesizing aged cells are non-proliferating while DNA-synthesizing young cells are mitotically active. The significance of DNA-synthesis in non-dividing "aged" cells is discussed. The number of population doublings (generation times or cell divisions) that it takes hamster and mouse cells to age in vitro was also investigated. Thirteen and six cell generation times were found to cause hamster and mouse cells to age with a loss of proliferative capacity. The effect of various molarities of 4-nitroquinoline-1 oxide (4-NQO), on DNA of aged cells, which results in an unscheduled DNA-repair synthesis, was studied using autoradiography. It appears that an aged cell responds to these concentrations in much the same way as young cells; however, there does seem to be a slightly greater sensitivity to toxic doses of 4-NQO in aged cells. Autoradiographic studies also revealed that the duration of DNA-repair is the same in both aged and young cells, but the former appear to have a decreased capacity to repair damage to the DNA of the pretreated cells caused by the 4-NQO. The significance of this apparent decrease in the DNA-repair capacity of "aged" cells is discussed. Mouse cells, aged in vitro, were exposed to human adenovirus type 12 (strain Huie). Evidence is presented that this external agent stimulated these aged cells to increase DNA-synthesis and also pushed them into mitosis (for at least one cell division). The possibility that this might be an accountable mechanism for the observation of an accumulation of mutations in aged cells is evaluated. Aged cells were examined for frequencies and types of chromosome aberrations after exposure to adenovirus type 12. Among the most common are chromatid breaks and double fragments. As well, old cells exhibited a much higher frequency of chromosome aberrations than young cells after viral exposure. A comparison of this in vitro system of cell aging with an in vivo system is presented. The application of all of the aforementioned results and observations concerning the cellular aging process to the problem of carcinogenesis and neoplasia is emphasized. / Science, Faculty of / Zoology, Department of / Graduate

The cells of the licorice plant, Glycyrrhiza glabra, were cultured as a "single cell" suspension. Their growth behaviour, yield and metabolic products were studied. The suspension cultures of the licorice plant were established from the friable calluses obtained from the radicle, cotyledon and hypocotyl of the germinated seeds. The single cells, regardless of their origin showed little difference in cell size and morphology. After an apparent adjustment to the medium, the cells required 11-13 days of incubation to reach the maximum cell yield of 1.2 gm/100 ml medium, dry weight. During the growth period, the pH of the growth medium decreased from pH 5.6 to pH 4.7 in the first few days and then increased to about pH 6. A level of 10% coconut milk in PRL-4-CM medium was found to support good cell growth; the lower the coconut milk level, the longer the growth period required to reach the maximum cell yield. It was also found that 0.57% yeast extract could be used to replace the coconut milk in the PRL-4-CCM medium. The metabolites detected and examined in the licorice single cell suspension culture included a volatile apple aroma, a polysaccharide pectin-like material, steroids and triterpenoids. The analyses of the licorice cell volatile apple aroma found under anaerobic conditions indicated the presence of ethanol and some related esters. The monosaccharides found in the pectin-like polysaccharide hydrolysate were glucose, fructose, galactose, arabinose, xylose, galacturonic acid and glucuronic acid. The pectin-like material in the cell preparations reached a maximum yield of 1.1 mg/ml after one month of growth. Glycyrrhizinic acid, the common licorice constituent found in the root, could not be detected in the suspension cultures. However, several other related compounds which gave typical steroid and triterpenoid reactions were found. Sorbitol and fructose were found to be the two major sugars which accumulated in free form in the licorice cell medium. / Land and Food Systems, Faculty of / Graduate

Castor (Ricinus communis L.) is a high-yielding oilseed crop native to tropical Africa. The seed contains ~60% oil by weight, yielding approximately 1,200 kg of oil per hectare. The oil is composed of ~90% ricinoleic acid, a unique hydroxylatty acid. Its unique composition provides castor oil with distinctive characteristics important for industrial use. Unfortunately, this valuable oilseed has not been widely cultivated in the United States since 1972, due in part to the presence of ricin in the seed. Ricin is a highly toxic lectin found in the endosperm of mature castor seed. This project sought to silence ricin production through the introduction of an RNAi element into the castor genome. The RNAi vector (pC1-RKO) containing a segment of ricin mRNA and its inverted repeat separated by a chalcone synthase A intron from pFGC5941 enclosed in a pCambia1301 backbone was created, verified via sequencing, and transformed into Agrobacterium tumefaciens for castor transformation. Fungal contamination was a serious concern; successful disinfestation used a 10-minute wash with 0.1% mercuric chloride (w/v). Media supplemented with 6-benzylaminopurine generated healthier shoots from embryo axes dissected from mature seed compared to thidiazuron-treated mesocotyls dissected from mature seed. Short treatments of thidiazuron on 6-benzylaminopurine initiated shoot cultures showed greater shoot proliferation on embryo axes dissected from mature seed. Rooting occurred with incubation on half-strength medium containing naphthaleneacetic acid or indole-3-butyric acid; however, naphthaleneacetic acid produced hardier roots which better survived acclimatization. Inoculation of embryo axis explants after 2 days pre-culture improved survivability. Likewise, transformations using A. tumefaciens cultures of 0.5 O.D.600 and lower did not lead to downstream bacterial contamination. The pCambia1304 vector was used as a test plasmid for refinement of the transformation protocol. Of the 870 pCambia1304 inoculation explants, 2 survived hygromycin screening and showed gusA activity. Of the 2,500 pC1-RKO inoculated explants, 6 survived hygromycin selection and rooted. Further analysis via PCR, end-point RT-PCR, and Western and dot-blotting showed these to be non-transformed and ricin content unaffected.

The broad objective of this research was to develop transgenic poinsettia that express tryptophan decarboxylase (TDC) capable of protecting poinsettia against whitefly. An effective and efficient in vitro micro propagation and proliferation technique of poinsettia ‘Prestige Red’ was successfully developed in this study and this protocol can be used for potential development of transgenic poinsettia. Poinsettia ‘Prestige Red’ was successfully infected by Agrobacterium rhizogenes producing hairy roots at the site of infection. Investigations of more effective PGR concentrations are necessary in order to develop transgenic poinsettia through hairy roots. Stem disks of poinsettia ‘Eckespoint Pollys Pink’ developed into somatic embryos when they were transformed by A. tumefaciens harboring TDC. A. tumefaciens-mediated transformation of poinsettia through somatic embryogenesis is cultivar dependent. Additional research into more effective PGR combinations, antibiotic concentrations and antinecrosis chemicals is required in order to develop transgenic poinsettia harboring TDC through somatic embryogenesis using A. tumefaciens.

Thesis (Ph. D.)--University of Wisconsin, 1953. / Typescript (carbon copy). eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves [38]-40).

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