published_or_final_version / Pathology / Master / Master of Philosophy

published_or_final_version / Psychiatry / Master / Master of Philosophy

published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

abstract / Medicine / Doctoral / Doctor of Philosophy

Vitamin A is known to have an important role in cellular differentiation and proliferation. In addition to regulating normal cellular processes vitamin A has also been shown to possess potent antineoplastic activity. The work in this dissertation characterizes the role of retinoic acid (RA) in cellular transformation and tumorigenesis with known oncogene involvement. These studies were initiated by examining the effects of RA on human carcinoma cell lines which express an activated c-ras gene. The bladder carcinoma, EJ/T24 (c-rasᴴ) and the two lung carcinoma cell lines, LXl (c-rasᴷ) and A2182 (c-rasᴷ), were not sensitive to RA. No inhibition of anchorage- or density-dependent growth was observed. Therefore, since these in vitro markers of transformation indicated a lack of effectiveness of RA on carcinomas containing a c-ras gene, retrovirally transformed cells were tested for RA sensitivity. Kirsten murine sarcoma, Balb/c murine sarcoma virus, and Simian sarcoma virus transformed NIH/3T3 and NRK cells were used in these studies. In contrast to the human carcinoma cell lines, anchorage-independent growth of some of the virally transformed cells was very sensitive to inhibition by RA. Anchorage-independent growth of KNRK and SSVNRK cells was sensitive to high concentrations (5 μM) of RA; whereas, Balb/cMSV3T3 and SSV3T3 were sensitive to 1-20 nM RA. BALB/cMSVNRK anchorage-independent growth was stimulated 3.5 fold by 1 μM RA. KNRK displayed a 60% reduction in anchorage-dependent growth at 10 μM RA while little inhibition was observed with the other retrovirally transformed cells. A high level of sensitivity to RA inhibition of anchorage-independent growth was correlated with the presence of cytoplasmic retinoic acid binding protein (CRABP). This indicated that CRABP may have some role in the inhibition of retrovirally induced cellular transformation. RA was shown to significantly reduce the incidence and size of Balb/cMSV3T3 cell tumors in nude mice. The inhibition of tumorigenesis in vivo therefore confirmed the results observed in vitro. To investigate the mechanism by which RA was acting to inhibit retroviral transformation, v-onc mRNA levels were examined. RA had no effect on v-onc mRNA levels in cell lines sensitive to the inhibition of transformation. The effect of RA on the relative rate of synthesis of p21, the transforming protein of KMSV and Balb/cMSV, was investigated. No effect of RA was observed in any of the cell lines. Also, GDP binding by p21 in KNRK cell was unchanged by RA treatment indicating that the functional activity of this transforming protein was not modified. RA does appear to be effective in inhibiting retrovirally induced cellular transformation and tumorigenesis. Evidence presented here indicates that this inhibition is not due to a direct effect of RA on the expression of the v-onc gene and/or gene product. Therefore, some other essential cooperating event(s) occurring within the cell are being acted upon by RA.

This research was an attempt to characterize previously constructed mutants with a specifically altered psbO gene which encodes a 33 kDa protein active in photosynthesis. This polypeptide was believed to function in stabilization of manganese ions during photolysis of water at the photosystem II. The initial phase of this work was concerned with determining the manganese content of the genetically manipulated PS II particles of the photosynthetically active cyanobacteria.We found however, that the results of the isolation procedure for PS II particles of photosynthetically active cyanobacteria as described by Burnap et al. was not reproducible in our research organism. This prevented the chemical characterization of function of these particles as had been planned.In the second phase of the research sequencing of the mutated gene was to be performed for several clones in order to determine the kinds of specific alterations that had been made. The mutated genes had been cloned into both pUC1 20 and pPGV5 vectors which were transformed into Escherichia OR (EQQJi) and the cyanobacterium Synechococcus PCC 7942, respectively.Several attempts were mad o isolate plasmid DNA from both the transformed E QQJI and cyanobacterium. Isolation of pUC120 DNA was not achieved due to the toxicity of the 33 kDa protein product of the psbO gene in sgJj. The pPGV5 plasmid isolation was successful and PCR-sequencing was performed. However, the sequencing did not result in a readable sequence. Instead, banding patterns showed more than one nucleotide per lane. Since pPGV5 contains a strong constitutive promoter, a large amount of mutant protein was being produced. Our findings suggested that transformed cyanobacteria may have been under pressure to revert the altered gene to wild-type. Thus, upon growth of a single colony to a larger volume, a heterogeneous population of cells with different sizes of plasmids may have resulted. Restriction analysis of isolated plasmid DNA confirmed the presence of multiple-sized plasmid molecules. Therefore, this research has shown that the previously constructed mutants are not stable enough to characterize for alterations in manganese binding. / Department of Biology

The 33 kDa manganese stabilizing protein (MSP) has been proposed to provide ligands to stabilize Mn ions in the water lysis reaction of photosystem II of photosynthesis. In previous research site-directed mutagenesis had been performed on regions of the psbO gene encoding two aspartic acid residues of MSP which were thought to have the potential to form carboxyl bridges with Mn ions. The purpose of this research was to analyze these mutants. Plasmids pUC120-33 (#1,3,5,7,9,11,15) containing mutant psbO genes could not be isolated from E.coli because the expressed MSP was toxic to the cells. However, a psbO mutant gene carried in pPGV5-33 (#7) was isolated from E.coli and transformed into cyanobacterium Svnechococcus PCC 7942. Cyanobacterial cells carrying the MSP mutant showed a susceptibility to intensive light (100 footcandles) with a decrease of 30% in the growth rate within the first 100 hours after inoculation. This result suggested a possible function of the MSP in protecting the oxygen evolving complex from intensive light exposure. However, the mutant appeared to revert after this time probably due to homologous gene recombination with the wild type gene. In order to further analyze the function of mutants without recombination occurring, the construction of an MSP deletion was attempted using insertion of a kanamycin cartridge into the middle of the psbO gene. The inactivated psbO gene was transformed into E.coli and transformants were selected by kanamycin resistance. However, plasmid DNA carrying the interrupted genes could not be isolated, probably due to toxicity of the expression product in E.coli cells. Thus, future studies should be directed to reconstruction of a deletion mutant by direct transformation into cyanobacterial cells. Once a deletion mutant has been constructed analyses of the site-directed mutations could be performed in cyanobacteria. / Department of Biology

Asthma is a common complex disease that affects millions of people around the world. Studies indicate the increase in prevalence of asthma worldwide during the past century and report asthma as an important cause of morbidity and mortality. Asthma can be considered as an important health condition in the UK that ranks amongst the countries with the highest rate of asthma prevalence, hospital admissions and mortality due to asthma. Asthma is caused by a combination of genetic and environmental factors. Genetics has an important role in development of asthma with the heritability of around 70% in most studies. To date, more than 100 asthma . associated genes have been identified but they account for only a small proportion of the heritability of asthma. The centerpiece of this thesis is the investigation of genetic association of cystatin and cathepsin genes with asthma and associated phenotypes including atopy and IgE levels. Cathepsinsl cystatins, as proteases and the related antiproteases have been suggested to have a role in airway remodeling. The investigation included three phases; initial association study, replication study in two independent samples sets and complementary analyses. Three sample panels were used in the studies; AUS1/UK1, MRC- AlMRC-E and DLM-4264. The results of this work identified CSTL 1 (cystatin like-1) associated with asthma and IgE levels.

Call number: LD2668 .T4 1986 K84 / Master of Science / Grain Science and Industry

Call number: LD2668 .T4 1957 E95 / Master of Science