Thesis (Ph. D.)--University of Hong Kong, 2009. / Includes bibliographical references (leaves 149-163). Also available in print.

Thesis (Ph.D.)--University of Delaware, 2009. / Principal faculty advisor: Brian J. Bahnson, Dept. of Chemistry & Biochemistry. Includes bibliographical references.

Thesis (M.S.)--West Virginia University, 2001. / Title from document title page. Document formatted into pages; contains vi, 57 p. : ill. Includes abstract. Includes bibliographical references.

Thesis (Ph.D)--Biology, Georgia Institute of Technology, 2009. / Committee Chair: Weissburg, Marc; Committee Member: Hay, Mark; Committee Member: Kubanek, Julia; Committee Member: Webster, Donald; Committee Member: Yen, Jeannette. Part of the SMARTech Electronic Thesis and Dissertation Collection.

published_or_final_version / Biological Sciences / Master / Master of Philosophy

No description available.

After-cooking darkening (ACD), a gray-black discoloration, is one of the key quality defects of the potato. Previous work using a diploid population 13610 identified 14 single nucleotide polymorphism (SNP) markers, located on eight chromosomes, which have the strongest association with ACD. Seven of these markers are located in four major quantitative trait loci (QTL) on chromosomes 2, 4, 6, and 10. It is hypothesized that these 14 markers would also have significant relationship to ACD in other breeding populations. Therefore, their effects in another diploid population 14946 were analyzed by amplicon genotyping using high-resolution DNA melting (HRM). Eleven of the 14 SNP markers were confirmed to be significantly associated with ACD. Six of the 11 markers were located in the four major QTL regions. In addition, unlabelled probe assays using HRM were performed on a group of clones in the tetraploid population 14945. Five from the 14 markers were analyzed, and three showed significant relationship to ACD. The methodology established and the markers identified in this study could benefit breeding programs for developing lower ACD varieties.

Recent improvements in sequencing technologies have caused various interesting problems to arouse. Having millions of read sequences as the final product of sequencing genome at a lower cost compared to micro array era, has encouraged scientists to enhance previous methods in various areas of bioinformatics. Genotyping and generating genetic maps to study inherited genotypes in order to analyze specific traits in a population is one of the fields of bioinformatics that involves generating different genetic markers and identify polymorphisms in different individuals of a population. Presence/absence markers are the main focus of this thesis. This is one type of Restriction site Associate DNA (RAD) markers which is present in some samples and absent in others and is the sign of variation in the cut site of a restriction enzyme. However, the counts of markers in an experiment are highly correlated and calling true absence and presence is not a straightforward task which means any marker with zero count is not necessarily absent in the sample under study. This is also the case for non-zero count markers which are not necessarily present. A good model that can fit the data is able to make true calls. We propose two different contexts for designing such models as a solution to this problem and investigate their performance. On the other hand, utilizing features of next generation sequencing technology in an even more efficient way, requires the ability to multiplex high number of samples in a single experiment run. In that case, appropriate barcoding, that is robust to various sources of noise in the machine, becomes paramount. Designing such barcodes in an efficient way is a challenging task which is addressed in detail as another problem of this thesis. We make two contributions. One, we propose an algorithm for barcoding multiplexed RADSeq samples. Two, we propose an algorithm for the statistical selection of presence/absence markers on the basis of RADSeq data on two related individuals. Operating characteristics of our methods are explored using both simulated and real data.

Diagnosis of marginal zinc deficiency using current biochemical markers is unreliable due the lack of sensitivity or specificity. Metallothionein (MT) levels in various fluids and tissues have been shown to reflect zinc status, but measurement of the protein has proved difficult in plasma and white blood cells. An alternative to measuring MT protein is the assay of MT mRNA in blood cells. Northern blotting was not sensitive enough to detect basal levels of MT from human T cells. A sensitive RTPCR assay was developed which detected and measured human MT-2A mRNA in T cells. A competitive MT-2A DNA standard with an 80 base-pair deletion was constructed that co-amplified with the MT-2A mRNA during RT-PCR. After analysis of the PCR products by gel electrophoresis, the ratio of the added standard to MT-2A was used to quantify the MT-2A mRNA expression. Competitive RT-PCR was also carried out for β-actin to correct for RNA degradation and loading. Capillary electrophoresis combined with laser-induced fluorescence detection can also allow sensitive and rapid analysis of RT-PCR reactions. The RT-PCR assay was used to measure MT-2A gene expression in T cells from human volunteers in zinc deficiency studies. In a severe zinc deprivation study (>0.5 mg Zn per day) most of the samples were too degraded for accurate analysis, showing the requirement of a degradation control for quantitative RT-PCR. In a marginal zinc deprivation study, human volunteers were given a diet that was changed from 15mg Zn/day to 5mg Zn/day. After 50 days on the low zinc diet, each individual showed a decrease in T lymphocyte MT-2A mRNA levels (30-80% of baseline value). The T lymphocyte MT-2A mRNA levels increase upon zinc repletion (15mg Zn/day) after about two weeks. There were no apparent changes in plasma zinc concentration during the study. This suggests that T-lymphocyte mRNA measurements may be a sensitive index of zinc status in humans.

Osteocalcin, which is produced by the osteoblast, and the urinary pyridinium compounds (pyridinoline and deoxypyridinoline), which are derived from collagen, are widely used as markers of bone turnover. Osteocalcin was extracted from bone in 20% formic acid and separated using a linear 4-60% acetonitrile gradient containing 0.1% TFA at a flow rate of 1ml/min. The standard curve was linear up to 15 <I>μ</I>g of osteocalcin injected, with inter- and intra-assay coefficients of variation of 6.9 and 8.8% respectively while recovery of osteocalcin added to bone extracts averaged 102.7 ± 6.16 %. Having developed this assay it was then used in a series of experiments designed to study the biological function of osteocalcin in bone. Plasma osteocalcin levels decreased with age and showed large between-animal variations; the variability over 24 h was also large but there was no evidence of consistent circadian rhythm. In bone, changes in osteocalcin levels tended to parallel those for calcium whereas pyridinium crosslink levels tended to increase with age. Neither were sufficiently sensitive to detect differences in bone turnover in lambs of the same age but growing at different rates though osteocalcin levels in blood and in bone were sensitive to P-deficiency in sheep but not in pigs and there was little evidence indicating that osteocalcin plays any direct role in the mineralisation process. In separate studies adult sheep were treated with a bone antiresorptive agent, Ibandronate, and its effects on the metabolism and excretion of the pyridinium crosslinks was examined. At rates which have been shown to be effective in reducing bone resorption in humans this compound had little effect on the overall rate of excretion of these crosslinks in these sheep but did alter the proportions excreted in free or in peptide bound form.