Spinal cord injury (SCI) results in extensive demyelination, leading to deleterious axon degeneration and inability of functional recovery. Remyelination has become a part of the fundamental strategy for SCI repair. Endogenous neural progenitor cells (NPCs) respond to SCI producing progenies and provide a possible source of regenerated oligodedrocytes for remyelination. During development of the central nervous system, glycogen synthase kinase-3 isoform beta (GSK-3β) is involved in multiple pathways that regulate oligodendrocyte differentiation and myelination, and thus may also play an important part in remyelination after SCI. This study aims to investigate (1) the role of GSK-3β in the differentiation of adult spinal cord derived-neural progenitor cells (ASC-NPCs); (2) whether AR-A014418 as a GSK-3β inhibitor, can promote oligodendroglial differentiation of ASC-NPCs; (3) the effect of LiCl, another GSK-3β inhibitor, on functional recovery after SCI; (4) the effects of LiCl on the myelin and axonal preservation after SCI. Neurosphere culture from adult mouse spinal cord was performed to test the effect of GSK-3β inhibitors, LiCl and AR-A014418, on differentiation of ASC-NPCs. Phenotyping of differentiated ASC-NPCs by immunocytochemistry (ICC) was performed to identify oligodendroglia progenitor cells (OPCs) at different stages. It was shown that LiCl (1 mM) and AR-A014418 (5 μM) promoted differentiation of OPCs as labeled by oligodendrocyte lineage-specific markers: PDGFR-α, NG2 and O4, while AR-A014418 was more potent in the OPC differentiation. Moreover, preliminary data from western blot confirmed that ARA014418 (5 μM) treatment increased the expression level of pGSK (inactive form of GSK-3) in differentiated ASC-NPCs. This suggests a possible strategy to modulate endogenous NPC response to SCI: to induce the preferential differentiation of NPCs into oligodendrocyte lineage by inhibiting GSK-3β activity and thus leading to enhanced remyelination by the differentiated oligodendrocytes. Basso Mouse Scale (BMS) open field test was used to evaluate the locomotive function of the spinal cord injured mice. The result showed that LiCl (4 mM, 200 μl) administration delivered locally at the lesion site by osmotic pump for 2 weeks improved functional recovery after SCI. Furthermore, immunohistochemistry (IHC) analyses revealed that LiCl treatment inhibited GSK-3β activity in the 〖Olig2〗^+ OPCs/oligodendrocytes, confirming LiCl as a GSK-3β inhibitor in vivo. Moreover, LiCl treatment better preserved myelin and axons detected by myelin basic protein (MBP) immunostaining and neurofilment-200 (NF-200) immunostaining respectively in the injured spinal cords. All together, the data from our in vitro and in vivo experiments suggested that LiCl treatment after spinal cord injury is beneficial for functional recovery by preventing the loss of myelin and axons after SCI and this effect is mediated via GSK-3β inhibition This study provided evidence for the involvement of GSK-3β in the regulation of OPC differentiation and the subsequent remyelination in the injured adult spinal cord. We propose GSK-3β as an important therapeutic target for SCI repair, LiCl as a potential candidate for SCI clinical treatment and the possibility to manipulate endogenous NPCs after SCI to enhance oligodendrocyte differentiation, remyelination, and ultimately better functional recovery.. / published_or_final_version / Anatomy / Master / Master of Philosophy

α-Isopropylmalate synthase (α-IPMS) is responsible for catalysing the first committed step in leucine biosynthesis. This pathway is found in plants and microorganisms, including pathogenic bacteria such as Mycobacterium tuberculosis and Neisseria meningitidis. α-IPMS catalyses a Claisen condensation reaction between α-ketoisovalerate (KIV) and acetyl coenzyme A (AcCoA) to form the product α-isopropylmalate (IPM). This enzyme undergoes feedback inhibition by the end product of the pathway, leucine. This regulation allows the control of the rate leucine biosynthesis. This project focuses on the α-IPMS enzymes from M. tuberculosis and N. meningitidis (MtuIPMS and NmeIPMS). These α-IPMS enzymes are homodimeric in structure. Each monomer consists of a catalytic domain which comprises of a (β/α)8 barrel fold, two subdomains and a regulatory domain, to which the allosteric binding of the natural inhibitor leucine occurs. The mechanism by which the allosteric binding of leucine leads to a decrease in enzymatic activity is not yet fully understood. Citramalate synthase (CMS) is responsible for catalysing the first committed step of threonine-independent isoleucine biosynthesis. This enzyme is extremely similar to α-IPMS in both the reaction which it catalyses and the catalytic and regulatory domain structure. CMS catalyses a Claisen condensation reaction between pyruvate and AcCoA to produce citramalate (CM). CMS is also feedback inhibited by the end product of its pathway, isoleucine. The similarity between α-IPMS and CMS enzymes resulted in and examination of the inhibitor selectivity of MtuIPMS. Amino acids in the leucine binding site were altered to their counterparts in the isoleucine binding site of the CMS enzyme to see if the selectivity of the leucine binding site could be interchanged. Results from this study show that it is possible to change inhibitor selectivity with a single amino acid substitution. However, changing the selectivity from leucine to isoleucine was unsuccessful. Instead, one of the MtuIPMS variants displayed significantly increased sensitivity to an alternative amino acid, norvaline. The MtuIPMS variants were expressed and purified using immobilised metal affinity chromatography and size-exclusion chromatography. These variants were then kinetically characterised and displayed similar binding affinities and turnover rates for the natural substrates to the wild-type enzyme. As expected changes to the leucine binding pocket had drastic effects on the sensitivity of the enzyme to its natural inhibitor. This work is described in Chapter 2 of this thesis. The mechanism by which the regulatory signal is transferred from the allosteric leucine binding site to the catalytic site in α-IPMS is not fully understood. NmeIPMS variants were created based on preliminary molecular dynamic simulations which indicated that significant changes in residue contacts were associated with leucine binding. Chapter 3 describes studies that explore the effect of single amino acid substitutions of NmeIPMS. The NmeIPMS variants were expressed and purified similarly to MtuIPMS, using immobilised metal affinity chromatography and size-exclusion chromatography. Variants were subsequently characterised via mass spectrometry, differential scanning fluorimetry and kinetic assays. It was found that each variant generated retained sensitivity to leucine but displayed significant differences in the catalytic efficiencies with AcCoA. One of the generated variants also displayed a significant increase in thermal stability. Results are drawn together in Chapter 4 along with future directions of this research. This chapter details knowledge gained into protein structure and allosteric mechanisms in this thesis.

Background: Anthracyclines, in particular Doxorubicin (DOX), are highly effective chemotherapeutic agents in the breast cancer setting, which are limited by their cardiotoxic side effects. Recently, the introduction of Trastuzumab (TRZ), a novel monoclonal antibody against the HER2 receptor, in the breast cancer setting compounds the issue of DOX mediated cardiac dysfunction. Amongst the potential mechanisms for the deleterious effects of this drug-induced cardiomyopathy, the relationship between nitric oxide synthase 3 (NOS3) and oxidative stress has gained recent attention. Objective: To determine the role of NOS3 in a clinically relevant female murine model of DOX+TRZ induced heart failure. Methods: A total of 120 C57Bl/6 female mice [60 wild type (WT) and 60 NOS3 knockout (NOS3-/-)] were treated with either 0.9% saline, DOX (20 mg/kg), TRZ (10 mg/kg), or DOX+TRZ. Serial echocardiography was performed daily for a total of 10 days, after which the mice were euthanized for histological and biochemical analyses. Results: As compared to WT, NOS3-/- mice demonstrated increased cardiotoxicity following treatment with DOX. This effect was potentiated with DOX+TRZ combination therapy. In WT female mice receiving DOX+TRZ, left ventricular ejection fraction (LVEF) decreased from 75±3% at baseline to 46±2% at day 10 (p<0.05). In the NOS3-/- group, LVEF decreased from 72±3% at baseline to 35±2% at day 10 (p<0.05). LVEF was significantly lower in NOS3-/- mice than WT at day 10 in those receiving DOX+TRZ (p<0.05). As compared to WT, NOS3-/- mice also demonstrated increased mortality following treatment with DOX+TRZ, corroborating the echocardiographic findings. Histological analysis using light and electron microscopy demonstrated increased loss of cell integrity including myofibrillar degradation, cytoplasmic vacuolization, and enlargement of the smooth endoplasmic reticulum in both the WT and NOS3-/- mice treated with DOX+TRZ. There was no significant difference, however, in the degree of cardiac remodeling between the WT and NOS3-/- groups. There was an increasing trend in the degree of cardiac apoptosis in both WT and NOS3-/- mice treated with DOX+TRZ therapy. Conclusion: Congenital absence of NOS3 potentiates the cardiotoxic effects of DOX+TRZ in an acute female murine model of chemotherapy-induced cardiomyopathy.

No description available.

The enzyme 3-deoxy D-arabino-heptulosonate 7-phosphate (DAH7P) synthase catalyses the first step of the shikimate pathway. This pathway lies at the heart of bacterial metabolism, and is responsible for the synthesis of a variety of compounds essential to the chemistry of life; from the aromatic amino acids phenylalanine, tyrosine and tryptophan, to a number of aromatic and non-aromatic natural products. This thesis describes the design, synthesis and evaluation of inhibitors of DAH7P synthase. These inhibitors exploit a variety of strategies to interrupt the activity of DAH7P synthase, ranging from simple substrate mimicry to inhibitors that mimic unstable reaction intermediates; inhibitors that exploit metal coordination and entropic effects, and inhibitors that gain improved potency by interacting with multiple sites. In Chapter Two, the synthesis of a mimic for a proposed unstable reaction intermediate is described, and its interaction with DAH7P synthase characterised. The compound was prepared in twelve steps from D-arabinose, and was found to be a slow-tight binding inhibitor of Escherichia coli DAH7P synthase. In Chapter Three, a number of compounds are prepared that were designed to bind to the phosphoenolpyruvate subsite of the DAH7P synthase active site. The binding of these compounds to the enzyme is investigated in order to gain an understanding of the factors involved in DAH7P synthase inhibition. The enantiomeric phospholactates were prepared, and the extent of inhibition of E. coli DAH7P synthase was shown to be dependent on compound chirality. Several other phosphoenolpyruvate-like molecules were prepared, and were also shown to be effective DAH7P synthase inhibitors. In Chapter Four extended compounds are designed that will bind the enzyme by multiple interactions at both substrate binding sites. Four compounds were prepared, and an increase in inhibitory potency was observed. In Chapter Five computational techniques are explored to aid the interpretation of the inhibition of DAH7P synthase by the compounds prepared in these studies. Several approaches for more potent inhibition of this enzyme are outlined and discussed.

Sialic acids are prevalent in many organisms and facilitate a range of cellular processes in both bacteria and mammals. Whilst a variety of sialic acids are present in nature, N-Acetylneuraminic acid (NANA) is the most common and plays a key role in the pathogenesis of a select number of neuroinvasive bacteria such as Neisseria meningitidis. These pathogens coat themselves with polysialic acids, mimicking the exterior surface of mammalian cells and consequentially concealing the bacteria from the host’s immune system. NANA is synthesised in prokaryotes via a condensation reaction between phosphoenolpyruvate and N-acetylmannosamine. This reaction is catalysed by the domain swapped, homodimeric enzyme, N-acetylneuraminic acid synthase (NANAS). Each NANAS monomer is comprised of two distinct domains; a catalytic domain linked to an antifreeze protein-like (AFPL) domain. This thesis outlines research into the role of the AFPL domain using a range of structural and kinetic analyses to compare variant enzymes to the natural, NmeNANAS enzyme. An investigation was also made into the evolutionary relationships between NANAS and other bacterial sialic acid synthases such as Legionaminic acid synthase and Pseudaminic acid synthase.

Elevated circulating Ang II activates neurons in the forebrain cardiovascular regulatory areas to cause sympatho-excitation and hypertension. We hypothesized that circulating Ang II causes neuronal activation in the SFO and thereby activates efferent pathways to the PVN, and chronically causes activation of aldosterone production in magnocellular neurons in PVN and SON, which amplifies neuronal activation in the PVN and central sympatho-excitatory pathways. The aim of the present study was to determine the pattern of neuronal activation in forebrain nuclei by circulating Ang II and to elucidate where in the hypothalamus Ang II may stimulate aldosterone biosynthesis. Dose related effects of circulating Ang II on BP were first assessed. Wistar rats instrumented with telemetry probes were infused subcutaneously with Ang II 150 and 500 ng/kg/min for 14 days. The subcutaneous infusion of Ang II at 150 ng/kg/min increased blood pressure gradually up to 20 mmHg and at 500 ng/kg/min up to 60 mmHg. Ang II at 500 ng/kg/min increased plasma Ang II by 4-fold. To assess effects of circulating Ang II on CNS pathways, Wistar rats were implanted subcutaneously with minipumps infusing 150 and 500 ng/kg/min Ang II for 1, 4 and 14 days. Three patterns of neuronal activation were observed by sc infusion of Ang II. The SFO was activated during the first day and remained activated for 4 days, but at 14 days showed diminished activation. MnPO did not show significant activation during the first day but, after several days the activation was high and then less by 14 days. Parvocellular PVN (pPVN), magnocellular PVN (mPVN) and SON showed an initial activation that increased over time. Chronic intracerebroventricular infusion of an aldosterone synthase inhibitor or a mineralocorticoid receptor (MR) blocker attenuated the increase in Fra expression in PVN but not SON, and prevented the decrease in SFO after 14 days infusion of Ang II. A significant increase in mRNA expression of steroidogenic acute regulatory protein (StAR), a rate limiting enzyme in aldosterone production was found in glia cells of PVN and SFO assessed by rt-PCR after 3 days subcutaneous infusion of Ang II at 500 ng/kg/min. Total expression of aldosterone synthase (CYP11B2) mRNA was increased in SFO, MnPO, SON and PVN after 3 days of infusion of Ang II. After 14 days no significant changes were observed in the expression of StAR or CYP11B2 mRNA. In comparison, in adrenal StAR mRNA expression increased after 3 days but no longer after 14 days. In contrast, CYP11B2 mRNA expression in adrenal increased after both 3 and 14 days of infusion. These findings may support our hypothesis that chronic elevation of circulating Ang II increases neuronal activity in CVOs, presumably leading to activation of the PVN and SON to induce an increase in aldosterone production in magnocelular PVN and SON. In the second phase activation of CVOs appears to diminish, but an aldosterone-dependent amplifying mechanisms, causes sustained activation of the PVN and thereby hypertension.

Background: Anthracyclines, in particular Doxorubicin (DOX), are highly effective chemotherapeutic agents in the breast cancer setting, which are limited by their cardiotoxic side effects. Recently, the introduction of Trastuzumab (TRZ), a novel monoclonal antibody against the HER2 receptor, in the breast cancer setting compounds the issue of DOX mediated cardiac dysfunction. Amongst the potential mechanisms for the deleterious effects of this drug-induced cardiomyopathy, the relationship between nitric oxide synthase 3 (NOS3) and oxidative stress has gained recent attention. Objective: To determine the role of NOS3 in a clinically relevant female murine model of DOX+TRZ induced heart failure. Methods: A total of 120 C57Bl/6 female mice [60 wild type (WT) and 60 NOS3 knockout (NOS3-/-)] were treated with either 0.9% saline, DOX (20 mg/kg), TRZ (10 mg/kg), or DOX+TRZ. Serial echocardiography was performed daily for a total of 10 days, after which the mice were euthanized for histological and biochemical analyses. Results: As compared to WT, NOS3-/- mice demonstrated increased cardiotoxicity following treatment with DOX. This effect was potentiated with DOX+TRZ combination therapy. In WT female mice receiving DOX+TRZ, left ventricular ejection fraction (LVEF) decreased from 75±3% at baseline to 46±2% at day 10 (p<0.05). In the NOS3-/- group, LVEF decreased from 72±3% at baseline to 35±2% at day 10 (p<0.05). LVEF was significantly lower in NOS3-/- mice than WT at day 10 in those receiving DOX+TRZ (p<0.05). As compared to WT, NOS3-/- mice also demonstrated increased mortality following treatment with DOX+TRZ, corroborating the echocardiographic findings. Histological analysis using light and electron microscopy demonstrated increased loss of cell integrity including myofibrillar degradation, cytoplasmic vacuolization, and enlargement of the smooth endoplasmic reticulum in both the WT and NOS3-/- mice treated with DOX+TRZ. There was no significant difference, however, in the degree of cardiac remodeling between the WT and NOS3-/- groups. There was an increasing trend in the degree of cardiac apoptosis in both WT and NOS3-/- mice treated with DOX+TRZ therapy. Conclusion: Congenital absence of NOS3 potentiates the cardiotoxic effects of DOX+TRZ in an acute female murine model of chemotherapy-induced cardiomyopathy.

The blood brain barrier (BBB), formed by endothelial cells lining the lumen of the brain capillaries, is a restrictively permeable interface that only allows transport of specific compounds into the brain. Cardiolipin (CL) is a mitochondrial- specific phospholipid known to be required for the activity and integrity of the respiratory chain. The current study examined the role of cardiolipin in maintaining an optimal mitochondrial function that may be necessary to support the barrier properties of the brain microvessel endothelial cells (BMECs). Endothelial cells have been suggested to obtain most of their energy through an-aerobic glycolysis based on studies of cells that were obtained from the peripheral vasculatures. However, here, we showed that the adult human brain capillary endothelial cell line (hCMEC/D3) appeared to produce ~60% of their basal ATP requirement through mitochondrial oxidative phosphorylation. In addition, RNAi mediated knockdown of the CL biosynthetic enzyme cardiolipin synthase (CLS), although did not grossly affect the mitochondrial coupling efficiency of the hCMEC/D3 cells, did seem to reduce their ability to increase their mitochondrial function under conditions of increased demand. Furthermore, the knockdown appeared to have acted as a metabolic switch causing the hCMEC/D3 cells to become more dependent on glycolysis. These cells also showed increase in [3H]-2-deoxyglucose uptake under a low glucose availability condition, which might have served as a mechanism to compensate for their reduced energy production efficiency. Interestingly, the increase in glucose uptake appeared correlated to an increase in [3H]-2-deoxyglucose glucose transport across the knockdown confluent hCMEC/D3 monolayers grown on Transwell® plates, which was used in our study as an in vitro model for the human BBB. This suggests that changes in the brain endothelial energy status may play a role in regulating glucose transport across the BBB. These observations, perhaps, also explain why the brain capillary endothelial cells were previously observed to possess higher mitochondrial content than those coming from non-BBB regions (Oldendorf et al. 1977).

Includes bibliography / 108 leaves, [24] 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, 1988