published_or_final_version / Surgery / Master / Master of Philosophy

Bovine mammary glands respond to infection by foreign pathogens such as Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) through changes in gene expression. Monitoring the gene expression profiles will contribute to better understanding of the pathology of mastitis, and provide important selective markers for future animal breeding programs. Using cultured bovine mammary duct epithelial cells and somatic cells from infected bovine mammary glands, this study first examined the existence of Toll Like Receptors in these two systems. In cultured duct epithelial cells stimulated with E. coli LPS, both TLR 4 and 2 mRNA up regulation was detected at 2h-72h and 12h-48h respectively. For S. aureus LTA TLR 2 mRNA was up regulated at 48 and 72h whereas for TLR 4 mRNA expression up regulation was detected at 24, 48, and 72h in comparison to the Oh (p<0.05). In the case of PGN, an abundant structural component of S. aureus, the expression of TLR 2 mRNA was significant (p<0.05) at 72h whereas TLR 4 mRNA expression increased at 24, 48, and 72h. The expression of these receptors was also monitored in milk cells from cows infected with either E. coli or S. aureus. However, results obtained from the milk cells were inconclusive due to the high individual variability. Afterwards, differential gene expression profiles were monitored by the Differential Display Polymerase Chain Reaction technique in the cultured duct epithelial cells in response to E. coli and S. aureus structural components. A total of 6 candidate fragments were identified for E. coli LPS induction, whereas only one fragment was identified for S. aureus LTA induction. After LTA induction, a specific band was found to be up regulated and confirmed to be GCP-2, a chemokine involved in neutrophil recruitment. In contrast, PGN induction resulted in no change in GCP-2 levels. In different preparations of cultured duct epithelial cells both GCP-2 and IL-8 were confirmed by real time PCR to be up regulated by LTA with a significance of (p<0.01) when compared to the control cells. In the case of the E. coli identified bands, a different approach is necessary to potentially confirm the origin of these fragments. Further large scale screening of the GCP-2 and IL-8 genes in dairy cattle is necessary to test for their potential use as targets to differentiate the mastitis resistant from the mastitis prone cows.

Resistance (R) genes, a key factor in determining the resistance of plants, have been shown often to be highly allelic entities existing in duplicated regions of the genome. This characteristic suggests that R-gene acquisition may have arisen through frequent genetic rearrangements as a result of transient, reduced genome stability. Tabacco plants transgenic for a recombination construct exhibited reduced genome stability upon infection with a virulent pathogen (tobacco mosaic virus). The reduced genome stability manifested as an increase in recombination events in the transgene. Such increases were observed following a virulent pathogen attack. This increase in recombination was shown to be systemic and was observed prior to systemic viral movement suggesting the presence of a systemic recombination signal. Further molecular analyses revealed that specific R-gene loci experience a large frequency of rearrangements following a virulent pathogen encounter. The possible targeting of instability to R-gene regions may be controlled through epigenetic processes, in particular, DNA methylation. / xiii, 119 leaves ; 29 cm.

Ischemic stroke and ischemic heart disease (IHD) are complex disorders which are influenced by both environmental and genetic factors. To identify genetic risk factors to these disease, we conducted two association studies in a population where a significant founder effect has occurred: the French-Canadians. We studied 10 polymorphisms, in the following 9 genes, which have previously been implicated in the development of IHD and/or ischemic stroke: angiotensinogen (M235T), angiotensin-converting enzyme (287 by I/D), angiotensin II type I receptor (A1166C), coagulating factor V (Leiden mutation), platelet glycoprotein IIIa (PLA1/PLA2 alleles), plasminogen activator inhibitor-1 (4G/5G), apolipoprotein E (E2, E3, and E4 isoforms), methylenetetrahydrofolate reductase (C677T), and stromelysin-1 (5A/6A). / In the first study, we investigated the relationship between the gene variants listed above and 150 IHD patients and 113 controls, who matched on age, gender, and ethnic background. In the second study, we investigated the relationship between these same genetic variants and 97 French-Canadians with cerebrovascular disease (59 ischemic strokes, 38 TIAs) and 134 age and gender matched French-Canadian controls. (Abstract shortened by UMI.)

Mice homozygous for the disrupted renal type 11a sodium/phosphate (Na/Pi) cotransporter gene, Npt2, (Npt2 KO) exhibit renal Pi wasting and hypercalciuria, predisposing factors for renal stone formation. We observed that Npt2 KO mice, but not wild-type littermates form renal stones. The renal stones were evident in newborn, weanling and adult mice and composed of calcium (Ca) and Pi. The presence of renal calcification correlated with the absence of Npt2 gene expression and the presence of genes responsible for the synthesis (1alpha-hydroxylase) and catabolism (24-hydroxylase) of 1,25-dihydroxyvitamin D, whose elevated levels contribute to the hypercalciuria and renal calcification in Npt2 KO mice. The renal calcification was associated with increased osteopontin (OPN) mRNA expression and colocalized with OPN, the latter associates with renal stones in vivo and inhibits Ca mineralization in vitro). These data demonstrate that hyperphosphaturia and hypercalciuria, secondary to Npt2 gene disruption, are sufficient for the development of renal calcification.

The work reported here was done at the Wheeler Orchid Collection and Species Bank and the Department of Biology at Ball State University. We have developed a research teaching program with two applied research goals: genetically transforming and DNA fingerprinting orchid tissue. As part of their molecular biology education, students have investigated the genetic transformation of orchids for mitigating viral symptoms and the identification of unknown orchids by DNA fingerprinting. In a second application of the technology, DNA fingerprinting has been used to determine evolutionary relationships and to quantify genetic diversity among orchids.This dissertation details the background and need for this project and the research that was done to start it. As the early work has, developed and students have added their contributions, the data have developed into two papers formatted for submission to scientific journals. They are included as results.The first is a project designed to insert exognenous DNA into orchid tissue. The soil microbe Agrobacterium tumefaciens causes crown-gall tumors to develop in its plant hosts by inserting DNA into their cells which then controls the biosynthesis of development-controlling hormones. A. tumefaciens which has been disarmed has been routinely used to bioengineer dicotyledonous plants but its use has been rare on monocotyledons. In this paper, we report that A. tumefaciens transformed embryonic orchid tissue and caused alteration in its normal developmental course.The second paper details the DNA fingerprinting of tissue from Aplectrum hymale, a terrestrial orchid native to this climate. Three populations of A. hymale have been sampled and DNA extracted from the tissue samples. RAPD primers were used to prime PCR amplifications of random sequences of the DNA and the amplified DNA was visualized by gel electrophoresis. Loci of the resulting bands were treated as potentially multiallelic gene loci and heterozygosity between and within subpopulations was calculated. We report that the three populations could be partially differentiated by this procedure and that the two populations located nearest to each other yielded the least between -ubpopulation heterozygosity. We report very high levels of genetic diversity between individuals within small subpopulations in spite of the fact that these subpopulations are considered to be primarily clonal in reproductive nature. / Department of Biology

T-cell acute lymphoblastic leukemia (T-ALL) is a childhood cancer created through genetic alterations; most commonly upregulation of TALI and LMOI oncoproteins. T-ALL is treated with radiation and chemotherapy, but malignant T-cells are resistant to apoptotic stimulation. To study this disorder, AKR-DP-603 cells were transduced to express both oncoproteins. Western blots verified protein expression and each population was treated with etoposide. Caspase-3 and Annexin-V/FITC apoptosis assays were performed following treatment. When the response of control cells was compared to engineered cells, no difference was observed from the Annexin-V/FITC assay, and only LM01 cells showed a difference in the caspase-3 assay. Furthermore, cells were transfected with siRNA to TALI and LM01 and the apoptotic response was re-tested. Complete silencing was verified by Western and apoptotic activity varied in the TALI population for both assays. These differences might indicate that cells resisted etoposide induction and following silencing were sensitized apoptotic induction. / Department of Biology

Extensive emphasis on selection for milk yield with minimal attention to the animals' functional performance has increased the yield of North American dairy cattle. The high intensity of selection for production traits such as milk yield, protein yield and fat yield has also brought about a rapid increase in genetic relationships among animals. In dairy cattle, correlated response to selection for milk yield includes fertility and susceptibility to diseases. Although the high producing cows have greater net profit, they also have higher mammary and discarded milk costs associated with high production. Long-term genetic selection against clinically diagnosed diseases might be useful to diminish their incidence. The Scandinavian countries have incorporated the health traits into their selection indices. Canadian breeding programs realize the need to consider traits other than the yield in selection decisions. The objective of this study was to determine the genetic variability of various clinically diagnosed health traits. Data from 171 herds of the Ontario milk-recording program were used. These herds are collaborating with the University of Guelph (Dr. David Kelton) to record health traits. A major impediment to estimating heritabilities for the majority of the disorders was that the progeny group size per sire was not large enough to enable detecting a significant difference among sires. Hence, heritability estimates were not obtained for all the health disorders included in the study. The progeny group size per sire has to be increased such that there are at least 5 cases per progeny group to enable detecting a difference among sires. Heritability estimates for retained placenta and displaced abomasum in the first lactation were 0.067 and 0.212 respectively. The heritability estimate of cystic ovaries in the second lactation was 0.092. In the third lactation, the heritability estimate of mastitis was 0.10 whereas retained placenta had a heritability of 0.25.

The aim of this thesis was to investigate the possibility of using buccal cells derived from a multi layered epithelial tissue from the oral mucosa as a model to identify potential biomarkers of genomic instability in relation to normal ageing and premature ageing syndromes such as AD and DS. A buccal micronucleus cytome assay was developed and used to investigate biomarkers for DNA damage, cell proliferation and cell death in healthy young, healthy old and young Down’s syndrome cohorts. Cells with micronuclei, karyorrhectic cells, condensed chromatin cells and basal cells increased significantly with normal ageing (P<0.0001). Cells with micronuclei and binucleated cells increased (P<0.0001) and condensed chromatin, karyorrhectic, karyolytic and pyknotic cells decreased (P<0.002) significantly in Down’s syndrome relative to young controls. The buccal micronucleus cytome assay was used to measure ratios of buccal cell populations and micronuclei in clinically diagnosed Alzheimer’s patients compared to age and gender matched controls. Frequencies of basal cells (P<0.0001), condensed chromatin cells (P<0.0001) and karyorrhectic cells (P<0.0001) were found to be significantly lower in Alzheimer’s patients, possibly reflecting changes in the cellular kinetics or structural profile of the buccal mucosa. Changes in telomere length were investigated using a quantitative RTm-PCR method to measure absolute telomere length (in Kb per diploid genome) and show agerelated changes in white blood cells and buccal cell telomere length (in kb per diploid genome) in normal healthy individuals and Alzheimer’s patients. We observed a significantly lower telomere length in white blood cells (P<0.0001) and buccal cells (P<0.01) in Alzheimer’s patients relative to healthy age-matched controls (31.4% and 32.3% respectively). However, there was a significantly greater telomere length in hippocampus cells of Alzheimer’s brains (P=0.01) compared to control samples (49.0). Buccal cells were also used to investigate chromosome 17 and 21 aneuploidy. A 1.5 fold increase in trisomy 21 (P<0.001) and a 1.2 fold increase in trisomy 17 (P<0.001) was observed in buccal cells of Alzheimer’s patients compared to age and gender matched controls. Chromosome 17 and chromosome 21 monosomy and trisomy increase significantly with age (P<0.001). Down’s syndrome, which exhibits similar neuropathological features to those observed in Alzheimer’s disease also showed a strong increase in chromosome 17 monosomy and trisomy compared to matched controls (P<0.001). However, aneuploidy rate for chromosome 17 and 21 in the nuclei of hippocampus cells of brains from Alzheimer’s patients and controls were not significantly different. Observations that AD individuals have altered plasma folate, B12 and Hcy levels compared to age-matched controls who have not been clinically diagnosed with AD were investigated. Genotyping studies were undertaken to determine whether polymorphisms within particular genes of the folate methionine pathway contributed to AD pathogenesis. Correlations between folate, B12 and Hcy status with previously determined buccal micronucleus assay cytome biomarkers for DNA damage, cell proliferation and cell death markers was investigated. Lastly, the potential protective effects of phytonutrient polyphenols on genomic instability events in a transgenic mouse model for AD were investigated. We determined the effects of curcumin and GSE polyphenols on DNA damage by testing the mice over a 9 month period utilizing a buccal micronucleus cytome assay, an erythrocyte micronucleus assay and measuring telomere length in both buccal cells and olfactory lobe brain tissue. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313395 / Thesis (Ph.D.) -- The University of Adelaide, School of Molecular and Biomedical Science, 2008

v, 107 leaves, [22] leaves of plates : ill ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Biochemistry, 1988