Thesis (Ph. D.)--Ohio State University, 2007. / Title from first page of PDF file. Includes bibliographical references (p. 132-151).

Thesis (M. Sc.)(Biochemistry)--University of Pretoria, 2006. / Includes bibliographical references. Available on the Internet via the World Wide Web.

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The oral intake of green tea, black tea and some of the tea constituents has been demonstrated to protect against various diseases and show protective effects on the cell. Tea is able to regulate gene expression, regulate enzymes, regulate the cell cycle, cause cell cycle arrest, induce apoptosis, and inhibit the proteasome. The aim of this study was to find proteins by a proteomic approach that may be modulated in mice drinking tea in contrast to control animals that receive water. Three groups; control, low dose, and a high dose tea group were chosen to determine the effect of tea on protein regulation of C57BL/6 male mice. Daily liquid consumption was measured, and even though the high dose group consumed less liquid they still ingested more tea than the low dose group at the end of the study. Weight gain was measured for all the groups but no significant differences were found. Some differences were found in organ weights of the low and high dose groups. There was no dose dependent effect for the liver and small intestine, while the colon showed a positive and the pancreas a negative dose dependent effect. Small intestine and liver proteins were separated by one and two dimensional gel electrophoresis. No significant differences were found for the small intestine and liver when the proteins were separated by tricine SDS PAGE. However some significant differences were found on the glycine SDS PAGE gels of both the small intestine and the liver. The small intestine had three significant bands at 66kDa, 45kDa and 10kDa. The three significant liver bands were at 110kDa, 66kDa and 14kDa. HPLC analysis of the liver 66kDa band showed that the band consisted of only one protein while the 14kDa band consisted of possibly two proteins. MS analysis of the 14kDa band identified the proteins as hypothetical protein XP_358319 (15 190Da) and immunoglobulin Ą chain (13 140Da). Although the identified proteins match the molecular weight of the 14kDa band these results will need to be confirmed by MudPIT. Thirty 2DE spots of the small intestine were regulated by tea. Ten of these spots were analyzed by MALDI TOF MS, but only seven of these proteins were identified. These proteins were S-phase kinase associated protein p19, hypothetical protein XP_903753, unnamed protein product, adenylyl cyclase-associated protein 2, developmental control protein, lysosomal acid phosphatase, and cytochrome P450 (CYP2D13). All seven the small intestinal proteins will need to be confirmed by de novo sequencing, to ensure the positive identification of the proteins. Currently there is no 2DE map in literature of the small intestine. This study will provide the first 2DE map of the murine small intestine proteins. Thirty three 2DE spots of the liver were regulated by tea. Twenty of these were analyzed by MALDI TOF MS, but only fifteen of these proteins were idenitifed. These regulated proteins are: superoxide dismutase, and glutathione peroxidase that are antioxidant enzymes to counteract oxidative stress, detoxification enzymes like glutathione S-transferase mu-1, glutathione peroxidase theta-1 and cytochrome b5. Annexin A4 is able to help stabilize plasma proteins and the cytoskeleton and may induce apoptosis, keratin 8 may help with network formation and reinforcement of cellular membranes, malate dehydrogenase for energy expenditure and ketohexokinase in carbohydrate metabolism, while ubiquitin conjugating enzyme E2 plays a role in protein turn over. Other identified proteins include inosine-triphosphate-pyrophosphatase, triosephosphate-isomerase, and myoglobin. This study provides a novel 2DE map for liver protein regulation by tea. This was the first study that has taken a proteomic approach to the identification of the overall regulation of proteins by tea. The aim of this study was met by identifying the tea regulated proteins and elaborating on the protective effects and possible cancer chemo preventative effects of tea. / Dissertation (MSc (Biochemistry))--University of Pretoria, 2007. / Biochemistry / unrestricted

Microbiota are present in large numbers and as a diverse population within the gastrointestinal tract. There are approximately 400 different species of microbiota which may be beneficial, harmful or both, but each play an important role in the regulation and modulation of the hosts’ bowel processes (McOrist et al. 2008; Dethlefsen et al. 2008). Many of these colon microbiota allow for saccharolytic fermentation of non-digestible dietary fibres and carbohydrates into by-products and intermediates, followed by a subsequent conversion into short chain fatty acids (SCFAs) (mainly n-butyric acid, propionic acid and acetic acid) each of which play an important role in maintaining colon homeostasis (Topping & Clifton 2001). A balance of ‘good’ microbiota (e.g., Bacteroides spp./ Bifidobacteria spp.) and ‘bad’ microbiota (e.g., Veilonellae) and the optimal production of various SCFAs within the gut could possibly allow for proper functioning of the large intestine and assist in decreasing the onset of various colonic disorders such as Irritable Bowel Syndrome (IBS). The sample group for the study consists of male and female patients, with an average age of 40 to 50 years old, whom of which have been diagnosed with either constipation IBS (C-IBS) or diarrhoea IBS (D-IBS) via the Rome III criteria system for IBS diagnosis. DNA and SCFA extractions were optimised for human stool, colonic fluid and tissue biopsy sample obtained from the aforementioned patients. Optimization steps allowed for starting material with high analysis integrity. Different methods of microbiota analysis, such as ARISA, were investigated; however, real-time qPCR was selected as the best method to identify and quantify specific microbiota. Extracted SCFAs were separated via gas chromatography and identified and quantified via Mass Spectrometry. Significant changes in microbial content and SCFA profiles were found to be associated with healthy and IBS patients. Results obtained would however be influenced by external factors typical of clinical studies of this nature. This study allows for opportunities for future research into understanding IBS.

Docosahexaenoic acid (22:6n-3) and arachidonic acid (20:4n-6) are deposited in large amounts in membrane phospholipids of the developing central nervous system (CNS). High levels of 22:6n-3 are found in synaptic terminals and retina, and are important for normal visual development and function. 20:4n-6 and22:6n-3 are supplied in human milk. In contrast, infants fed formula rely completely on endogenous synthesis of 20:4n-6 and 22:6n-3 from linoleic (18:2n-6) and a-linolenic (18:3n-3) acid, respectively. Levels of 22:6n-3 in the blood lipids of infants fed formula are lower than in infants fed human milk. Concern over the supply of 22:6n-3 led to clinical trials in which premature infants were fed formulas containing fish oils as a source of 22:6n-3. Piglets, which have a similar lipid metabolism and perinatal timing of the brain growth spurt to humans, have a lower percentage of 22:6n-3 in blood, liver and CNS tissues when fed formula with 30% of fatty acids as18:2n-6 and 0.8% 18:3n-3, compared to sow milk. It was hypothesized that the low blood and tissue 22:6n-3 in formula-fed piglets was due to inappropriate quantities and/or ratios of dietary 18:2n-6 and 18:3n-3 limiting the synthesis of 22:6n-3. Thus, the main objectives of this thesis were to determine. (1) if 22:6n-3 is an essential dietary nutrient for the term gestation piglet, (2) if appropriate quantities and ratios of 18:2n-6 and 18:3n-3 in formula will support CNS membrane accretion of 20:4n-6 and 22:6n-3, comparable to piglets fed varying amounts of 22:6n-3 in natural milk, and (3) if lower blood phospholipid 22:6n-3 consistently reflects reduced 22:6n-3 in the CNS. Initial studies (Experiment I) showed that formula with 4% 18:3n-3 supported a similar percentage of22:6n-3 in piglet liver and CNS membrane lipids to sow milk, but was associated with lower brain weight. Deposition of 22:6n-3 in brain was influenced by the formula 18:3n-3 content. The 18:2n-6:18:3n-3 ratio (22:1and 37:1) seemed to be important, however, when formulas contained 1% 18:3n-3. Low levels of fish oil in formula, similar to those used in clinical trials, were effective in supplying 22:6n-3 to the developing piglet brain (Experiment II). The efficacy of 18:3n-3 in supporting the deposition of 22:6n-3 in the brain was estimated to be at least 20% that of dietary 20:5n-3 plus 22:6n-3. With increasing dietary fish oil, however, levels of eicosapentaenoic acid (20:5n-3) increased and 20:4n-6decreased in plasma, liver and retina, but not brain (Experiment III). This suggests regulatory mechanisms may exist to maintain relatively constant levels of 20:4n-6 and 20:5n-3 in brain. Milk 22:6n-3 varies with maternal intake of 22:6n-3. The effect of milk 22:6n-3 content was studied in piglets fed milk with 0.1% or 1.5% 22:6n-3 obtained from sows fed usual pig diets containing vegetable fats without or with fish oil, respectively (Experiment IV). Consumption of 1.5 vs 0.1% 22:6n-3 from sow milk resulted in 300% higher 22:6n-3 in liver and blood phospholipids and 11% higher 22:6n-3 in cerebrum of nursing piglets. Despite similar milk 20:4n-6, the % 20:4n-6 in tissues other than the brain was lower in piglets fed high22:6n-3 sow milk. Thus, high intakes of n-3 fatty acids decrease 20:4n-6 in piglet liver and blood lipids. The blood phospholipid % 22:6n-3 in piglets fed formulas containing 18:2n-6 and 18:3n-3 but not their long-chain derivatives, was lower than in piglets fed 22:6n-3 in natural milk, consistent with published findings in formula-fed infants. However, in contrast to circulating lipids, formulas with 4% 18:3n-3 maintained similar levels of 22:6n-3in the piglet CNS compared to milk. These studies show that blood phospholipid 22:6n-3 and 20:4n-6 are not specific indices of effects in CNS lipids. This thesis has shown (1) 22:6n-3 is not essential in the diet of the term piglet, if adequate 18:3n-3 is given, (2) fish oils are an effective source of 22:6n-3 for deposition in the developing brain, (3) high dietary n-3fatty acids interfere with 20:4n-6 metabolism, and (4) blood lipid 20:4n-6 and 22:6n-3 do not accurately reflect CNS fatty acids. / Land and Food Systems, Faculty of / Graduate

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