The cryopreservation of cells and tissues requires the addition of a cryoprotectant in order to prevent cellular damage caused by ice. Unfortunately, common cryoprotectants such as DMSO and glycerol exhibit significant toxicity which makes their use unfeasible for many clinical procedures. Our laboratory is interested in the development of alternative, non-toxic cryoprotectants which possess ice recrystallization inhibition (IRI) activity. Potent IRI activity has recently been discovered in certain small molecules, but the structural features required for this process are unclear. Herein we report the development of a library of O-aryl glycosides in order to probe the importance of electron density and hydrophobic moieties for IRI activity. It was found that the degree of electron density at the anomeric oxygen does not correlate with IRI ability in para-substituted aryl glycosides, nor does changing the position of the aryl substituent impart a predictable effect on activity. However, the addition of hydrophobic alkyl or acyl chains was beneficial for IRI activity; generally, increasing chain length was found to correlate with increasing activity. In some instances, an optimal alkyl chain length was identified, after which continued lengthening results in a loss of potency. We conclude from this study that a certain extent of hydrophobic character is beneficial for the IRI activity of aryl glycosides, and that a balance between hydrophobicity and hydrophilicity is required for optimum IRI ability. It is hoped that these findings will aid future efforts towards the rational design of novel cryoprotectants.

The impact of fatigue on the intermittent high intensity exercise undertaken during participation in team sports has not been extensively studied. Team sports are characterised not only by intennittent exercise, but also by the contribution of a wide range of skills. This thesis describes a series of studies conducted in a controlled environment to assess the influence of fluid ingestion and fatigue on selected soccer skills. The aim of the first study was to examine the effect of 90-min of high intensity shuttle running with and without water ingestion on a socc er-dribb ling test. The subjects were allocated to two randomly assigned trials either ingesting or abstaining from fluid intake during a 90 min intennittent exercise protocol (Loughborough Intermittent Shuttle Test: LIST). In the absence of water ingestion soccer skill deteriorated (p < 0.05) by 5% but was maintained when fluid was ingested. The principal aim of the second study was to understand further the mechanisms contributing to the deterioration observed during the LIST. Subjects completed the LIST ingesting a 6.4% carbohydrate electrolyte solution (CHO), placebo (CON) or no fluid (NON). Free fatty acids, cortisol and aldosterone responses were lower (P < 0.01) at the end of exercise during both CHO and CON in comparison to NON. There was no difference in respiratory exchange ratio between trials. Fluid ingestion did not appear to cause a shift in substrate metabolism even though there were differences in plasma FFA concentrations. The consumption of carbohydrate during exercise has been shown to increase physical performance, capacity and cognitive function. The aim of the third study was to assess the influence of a 6.4 % carbohydrate-electrolyte (CHO) placebo (CON) or no fluid (NON) on passing and dribbling soccer skills following the LIST. During the NON trial performance of the dribbling test followed a similar pattern to that in the first study and performance of the passing test decreased (p < 0.05). This reduction in performance was prevented during the CHO and CON trials. The purpose of the final study was to identify whether a rehydration strategy following the LIST would result in a recovery of skill performance. Subjects were allocated to two randomly assigned trials either ingesting a volume of fluid equivalent to 150% (L) or 9% (S) of body mass loss during the LIST, over a2h recovery period. During the recovery period serum sodium and osmolality returned to resting concentrations in the L trial but remained elevated in the S trial (P < 0.05). Despite body mass returning to resting values following the rehydration period, performance of the skills tests remained impaired. Deterioration in skill test performance may have been related to a reduction in neuromuscular control either by a reduction in muscle glycogen or by an increase in muscle damage during the no fluid trials. The mechanism responsible for the deterioration in skill performance remains to be elucidated.

To test the hypothesis that maternal dietary glucose restriction throughout pregnancy and lactation would perturb glucose transporter (GLUT) protein levels in offspring, isoenergetic diets containing graded levels of glucose (0, 12, 24 and 60%) were fed to pregnant rats and their offspring from gestation day (gd) 0 through postnatal day (pd) 49. Diets were defined as deficient (0%), restricted (12, 24%) or adequate (60%) in glucose. Plasma, small intestine, liver and kidney tissues were collected perinatally (gd20, birth, 12--24hrs postnatal), during lactation (pd7, 15, 21), post-weaning (pd28, 35, 49) and in adult controls. The proximal and distal regions of the small intestine responded differently to the dietary glucose restriction. Proximal small intestine GLUT2 protein levels did not change throughout postnatal development and remained unaltered with dietary glucose restriction, while distal small intestine GLUT2 protein expression changed throughout postnatal development and with dietary glucose restriction. These findings, together with information from the literature, indicate a dissociation between small intestine GLUT2 mRNA expression, GLUT2 protein levels and small intestine glycogen reserves.

Patients undergoing elective surgery typically fast for 8-12 hours before surgery. However, oral preoperative carbohydrate ingestion may increase postoperative insulin sensitivity and reduce complications. To determine the effects of carbohydrate supplementation prior to CABG or spinal surgery, 38 patients were randomized to receive a carbohydrate supplement or to fast for 12 hours preoperatively. Baseline and postoperative measurements of insulin sensitivity were completed using the short insulin tolerance test and homeostasis model assessment (HOMA). Patient discomfort was measured immediately before surgery. Insulin sensitivity was not significantly different between groups. However, the supplemented group experienced a significantly smaller rise in glucose levels following surgery (p=0.03) and had higher postoperative HOMA-β scores (p=0.02). Fasted patients were significantly more thirsty (p=0.01), hungry (p=0.04) and anxious (p=0.01) before surgery and experienced a significantly longer hospital stay (p=0.008). Carbohydrate supplementation improved outcomes, warranting re-evaluation of fasting practices prior to major surgery.

The relationship between substrate oxidation (RER) and food intake (FI) is undefined. This study examined the effects of RER modified by a glucose pre-load (GL), exercise (EX) and GL with EX on, FI and energy balance (NEB) in normal-weight boys (9-12 y) and men (20-30 y). Subjects (15 boys, 15 men) were randomized with treatments of either water or GL followed by either EX or rest. Measures included RER, energy expenditure (EE)(kcal/kg), subjective appetite, FI(kcal/kg) measured at a pizza lunch and NEB (kcal/kg). FI(kcal/kg) was reduced by GL(p < 0.0001), and further decreased with GL ingested prior to EX(p = 0.0254). RER was increased with GL(p < 0.0001) and EX(p = 0.0043), and was higher in men compared to boys (p = 0.007). There was no association between RER and FI(kcal/kg). In conclusion, there was no relationship between RER and FI, suggesting that FI is not affected by substrate oxidation.

Patients undergoing elective surgery typically fast for 8-12 hours before surgery. However, oral preoperative carbohydrate ingestion may increase postoperative insulin sensitivity and reduce complications. To determine the effects of carbohydrate supplementation prior to CABG or spinal surgery, 38 patients were randomized to receive a carbohydrate supplement or to fast for 12 hours preoperatively. Baseline and postoperative measurements of insulin sensitivity were completed using the short insulin tolerance test and homeostasis model assessment (HOMA). Patient discomfort was measured immediately before surgery. Insulin sensitivity was not significantly different between groups. However, the supplemented group experienced a significantly smaller rise in glucose levels following surgery (p=0.03) and had higher postoperative HOMA-β scores (p=0.02). Fasted patients were significantly more thirsty (p=0.01), hungry (p=0.04) and anxious (p=0.01) before surgery and experienced a significantly longer hospital stay (p=0.008). Carbohydrate supplementation improved outcomes, warranting re-evaluation of fasting practices prior to major surgery.

The relationship between substrate oxidation (RER) and food intake (FI) is undefined. This study examined the effects of RER modified by a glucose pre-load (GL), exercise (EX) and GL with EX on, FI and energy balance (NEB) in normal-weight boys (9-12 y) and men (20-30 y). Subjects (15 boys, 15 men) were randomized with treatments of either water or GL followed by either EX or rest. Measures included RER, energy expenditure (EE)(kcal/kg), subjective appetite, FI(kcal/kg) measured at a pizza lunch and NEB (kcal/kg). FI(kcal/kg) was reduced by GL(p < 0.0001), and further decreased with GL ingested prior to EX(p = 0.0254). RER was increased with GL(p < 0.0001) and EX(p = 0.0043), and was higher in men compared to boys (p = 0.007). There was no association between RER and FI(kcal/kg). In conclusion, there was no relationship between RER and FI, suggesting that FI is not affected by substrate oxidation.

The effect of maternal dietary glucose on perinatal phosphoenolpyruvate carboxykinase (PEPCK) gene expression was investigated in this study. Pregnant rats were fed isoclaoric diets containing graded levels of glucose (0%, 12%, 24% and 60%) from gestation day 2 to lactation day 15. The developmental profiles of PEPCK gene expression in fetal and neonatal liver and kidney were analyzed by northern blot. In the liver, feeding glucose free and glucose restriction (12% and 24%) diets precociously induced PEPCK gene expression at day 21 of gestation. In the kidney, PEPCK mRNA (2.8 kb) was detected at birth in the glucose free group, 12-16 hours postnatally in control group; it was not visualized until day 3 in the 12% and 24% glucose restriction groups. In our study, two species of RNA (1.8 kb and 2.8 kb) were hybridized with PEPCK cDNA probes, and there was a relationship between maternal dietary glucose levels and the 1.8 kb RNA fragment in the kidney.

Interactions between carbohydrates and proteins are increasingly being recognized as crucial in many biological processes, such as cellular adhesion and communication. In order to investigate the interactions of carbohydrates and proteins, the development of efficient analytic technologies, as well as novel strategies for the synthesis of carbohydrates, have to be explored. To date, several methods have been exploited to analyze interactions of carbohydrates and proteins, for example, biosensors, nuclear magnetic resonance (NMR); enzyme-linked immunosorbent assays (ELISA), X-ray crystallography and array technologies. This thesis describes the development of novel strategies for the synthesis of carbohydrates, as well as new efficient strategies to Quartz Crystal Microbalance- (QCM-) biosensors and carbohydrate microarrays technologies. These methodologies have been used to probe carbohydrate-lectin-interactions for a range of plant and animal lectins. / QC 20100915

Subtilase cytotoxin (SubAB) is the prototype of a novel class of AB₅ cytotoxins produced by Shiga-toxigenic Escherichia coli (STEC). The A subunit (SubA) is a serine protease that cleaves the ER chaperone BiP causing cell death by a previouslyundetermined mechanism. The B subunits of AB₅toxins typically recognise host cell glycan receptors and direct the subcellular transport of the A subunit. Although the function of SubA and its intracellular substrate have been elucidated, the B subunit (SubB) is relatively uncharacterised. The subcellular trafficking pathway of SubAB was initially examined. SubAB conjugated to Oregon Green 488 (SubAB-OG) was internalised by Vero cells by 5 min, and co-localised with its ER target BiP within 30 min. When Vero cells were incubated with SubAB-OG and either Alexa Fluor 594-conjugated Cholera toxin B subunit (CtxBAF594) or Texas Red-conjugated Shiga toxin B subunit (StxB-TR), individual cells exhibited differential toxin uptake. This was shown to be cell cycle-dependent, in which, SubAB-OG was preferentially internalised by cells migrating through G1 and early S phases. In contrast, CtxB-AF594 was taken up by cells in S through M phases and by a majority of cells in G1, while StxB-TR endocytosis occurred in cells traversing G1. Fluorescent SubAB co-localised with the clathrin marker transferrin, but not with Caveolin-1 (a marker for cholesterol-associated caveolae) and was subsequently trafficked via a retrograde pathway to the TGN, Golgi and ER. The clathrin inhibitor phenylarsine oxide prevented SubAB entry and BiP cleavage in SubAB-treated Vero, HeLa and N2A cells, while cholesterol depletion did not, demonstrating that, unlike either Stx or Ctx, SubAB internalisation is exclusively clathrin-dependent. Identification of the SubB receptor was initially approached using toxin overlay assays in which Vero cell glycolipid extracts were separated by thin-layer chromatography and overlaid with SubAB. SubAB exhibited a high affinity for particular acidic species in the ganglioside fraction. However, none co-migrated with commercial glycolipid standards. SubAB-OG also exhibited an affinity for the oligosaccharide structures of chimeric LPS from GM₂ and GM₃ bacterial receptor mimic constructs in an LPS toxin overlay assay. Glycan array analysis revealed that SubB possessed a unique affinity for carbohydrate receptors with a terminal Neu5Gcα(2→3)Galβ disaccharide. Monovalent receptor analogues with distal Neu5Gc or Neu5Gcα(2→3)Galβ and highly-sialylated α₁-AGP did not prevent endocytosis of SubAB-OG, BiP cleavage or cytotoxicity in Vero cells. This indicated that SubAB has a greater affinity for the host cell receptors than the receptor analogues and may engage multiple receptors displayed on a lipid bilayer. In addition to mediating toxin binding and subcellular trafficking, CtxB and StxB can also potentiate the immune response to co-administered antigen. Accordingly, the systemic immunomodulatory properties of SubB administered by the i.p. route were assessed in mice. Using SubAA₂₇₂ as a bystander antigen, SubB significantly increased mouse anti-SubAA₂₇₂ titres to levels that were comparable to those obtained using Alum adjuvant. However, when admixed with structurally-unrelated OVA, SubB did not significantly affect anti-OVA titres whereas Alum and CtxB did. This indicated that SubB may function as a systemic carrier protein (rather than an adjuvant) for particular antigens. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1363363 / Thesis (Ph.D.) - University of Adelaide, School of Molecular and Biomedical Science, 2009