Nerve cells are organized into complex networks that comprise the building blocks of our nervous system. Neurons communicate by transmitting messenger molecules released from synaptic vesicles. Alterations in neuronal circuitry and synaptic signaling contribute to a wide range of neurological conditions, often with consequences for movement. Intrinsic neuronal networks in the spinal cord serve to coordinate vital rhythmic motor functions. In spite of extensive efforts to address the organization of these neural circuits, much remains to be revealed regarding the identity and function of specific interneuron cell types and how neuromodulation tune network activity. In this thesis, two novel genes initially identified as markers for spinal neuronal populations were investigated: Slc10a4 and Dmrt3. The orphan transporter SLC10A4 was found to be expressed on synaptic vesicles of the cholinergic system, including motor neurons, as well as in the monoaminergic system, including dopaminergic, serotonergic and noradrenergic nuclei. Thus, it constitutes a novel molecular denominator shared by these classic neuromodulatory systems. SLC10A4 was found to influence vesicular transport of dopamine and affect neuronal release and reuptake efficiency in the striatum. Mice lacking Slc10a4 displayed impaired monoamine homeostasis and were hypersensitive to the drugs amphetamine and tranylcypromine. These findings demonstrate that SLC10A4 is capable of modulating the modulatory systems of the brain with potential clinical relevance for neurological and mental disorders. The transcription factor encoded by Dmrt3 was found to be expressed in a population of inhibitory commissural interneurons originating from the dorsal interneuron 6 (dI6) domain in the spinal cord. In parallel, a genome-wide association study revealed that a non-sense mutation in horse DMRT3 is permissive for the ability to perform pace among other alternate gaits. Further analysis of Dmrt3 null mutant mice showed that Dmrt3 has a central role for spinal neuronal network development with consequences for locomotor behavior. The dI6 class has been suggested to take part in motor circuits but remains one of the least studied classes due to lack of molecular markers. To further investigate the Dmrt3-derived neurons, and the dI6 population in general, a Dmrt3Cre mouse line was generated which allowed for characterization on the molecular, cellular and  behavioral level. It was found that Dmrt3 neurons synapse onto motor neurons, receive extensive synaptic inputs from various neuronal sources and are rhythmically active during fictive locomotion. Furthermore, silencing of Dmrt3 neurons in Dmrt3Cre;Viaatlx/lx mice led to impaired motor coordination and alterations in gait, together demonstrating the importance of this neuronal population in the control of movement.

Mutations in the SLC4A1 gene result in misfolding and trafficking defects of the human erythroid (AE1) and kidney (kAE1) forms of the anion exchanger 1 glycoprotein. This affects the amount of functional protein at the cell surface, resulting in hematological and renal diseases. In this thesis, the role of the quality control system of molecular chaperones (cytosolic and ER) was examined during the biosynthesis of wild type and mutant AE1 in different cellular models. The hypothesis to be tested is that molecular chaperones are responsible for the intracellular retention of AE1 mutants. Chaperones were found to interact with AE1 and kAE1 in vitro and in vivo (HEK-293, K562, MDCK cells). Disruption of the calnexin-AE1 interaction in K562 cells did not affect the cell surface levels of wild type or mutant erythroid AE1. AE1 also trafficked to the cell surface in mouse embryonic fibroblasts completely deficient in calnexin or calreticulin. In contrast, in MDCK cells, disruption of the calnexin-kAE1 interaction allowed functional dominant (R589H, R901stop), but not misfolded kAE1 mutants (kSAO, G701D), to escape the ER and traffic to the cell surface. Calnexin is therefore not required for the cell surface expression of erythroid AE1, but can be responsible for the intracellular retention of certain kAE1 mutants in cells with the complete complement of molecular chaperones. Components involved in membrane glycoprotein folding and quality control (calnexin, ERp57, Hsc70, Hsp70), were lost at later stages during the differentiation of CD34+ erythroid progenitor cells. This suggests that the loss of molecular chaperones may facilitate the massive production of red cell glycoproteins, allowing erythroid AE1 mutants to escape quality control, traffic to the plasma membrane, and be present in mature red blood cells. These studies demonstrate that the role chaperones play varies, depending on cellular context. By understanding the cellular context and factors involved, therapeutic strategies may be tailored to deal with protein misfolding diseases, and in the case of kAE1, rescue the cell surface trafficking of misfolded, but functional, transport protein using pharmacological modulators.

Mutations in the SLC4A1 gene result in misfolding and trafficking defects of the human erythroid (AE1) and kidney (kAE1) forms of the anion exchanger 1 glycoprotein. This affects the amount of functional protein at the cell surface, resulting in hematological and renal diseases. In this thesis, the role of the quality control system of molecular chaperones (cytosolic and ER) was examined during the biosynthesis of wild type and mutant AE1 in different cellular models. The hypothesis to be tested is that molecular chaperones are responsible for the intracellular retention of AE1 mutants. Chaperones were found to interact with AE1 and kAE1 in vitro and in vivo (HEK-293, K562, MDCK cells). Disruption of the calnexin-AE1 interaction in K562 cells did not affect the cell surface levels of wild type or mutant erythroid AE1. AE1 also trafficked to the cell surface in mouse embryonic fibroblasts completely deficient in calnexin or calreticulin. In contrast, in MDCK cells, disruption of the calnexin-kAE1 interaction allowed functional dominant (R589H, R901stop), but not misfolded kAE1 mutants (kSAO, G701D), to escape the ER and traffic to the cell surface. Calnexin is therefore not required for the cell surface expression of erythroid AE1, but can be responsible for the intracellular retention of certain kAE1 mutants in cells with the complete complement of molecular chaperones. Components involved in membrane glycoprotein folding and quality control (calnexin, ERp57, Hsc70, Hsp70), were lost at later stages during the differentiation of CD34+ erythroid progenitor cells. This suggests that the loss of molecular chaperones may facilitate the massive production of red cell glycoproteins, allowing erythroid AE1 mutants to escape quality control, traffic to the plasma membrane, and be present in mature red blood cells. These studies demonstrate that the role chaperones play varies, depending on cellular context. By understanding the cellular context and factors involved, therapeutic strategies may be tailored to deal with protein misfolding diseases, and in the case of kAE1, rescue the cell surface trafficking of misfolded, but functional, transport protein using pharmacological modulators.

In this study, we address job sequencing and tool switching problem arising in Flexible Manufacturing Systems. We consider a single machine with limited tool slots on its tool magazine. The available tool slots cannot accommodate all the tools required by all jobs, therefore tool switches between jobs are required. A single tool transporter with limited capacity is used in transporting the tools from the storage area to the machine. Our aim is to minimize the number of tool transporter movements. We provide two mixed integer linear programming formulations of the problem, one of which is based on the traveling salesman problem. We develop a Branch-and-Bound algorithm powered with various lower and upper bounding techniques for optimal results. In order to obtain good solutions in reasonable times, we propose Beam Search algorithms. Our computational results reveal the satisfactory performance of the B&amp / B algorithm for moderate sized problems. Moreover, Beam Search techniques perform well for large-sized problems.

Human cells possess multiple nucleoside transporters (NTs) that belong to either the human equilibrative or concentrative NT (hENT: hENT1/2/3/4; hCNT: CNT1/2/3) families. In the kidney, coupling of apical hCNT3 activities to basolateral hENT1/2 activities is hypothesized to mediate renal nucleoside proximal tubular absorption while apical ENT1 may have a role in secretion. The overall aim of this research was to increase understanding of the roles of hENTs and hCNTs in renal handling of physiological nucleosides and anti-cancer nucleoside analog drugs. This was achieved by investigating the distribution of hENTs and hCNTs in human kidney tissue and the function of hENTs and hCNTs in cellular uptake and transepithelial fluxes of nucleosides in cultured human renal proximal tubule cells (hRPTCs). Immunolocalization of hCNT3 and hENT1 in human kidney tissue revealed that hENT and hCNT3 were present in apical membranes of proximal tubules. Production and characterization of adherent hRPTC cultures demonstrated endogenous hCNT3, hENT1, and hENT2 activities. These results provided evidence for the involvement of hCNT3, hENT1, and hENT2 in renal handling of nucleosides. Comparison of adherent hRPTC cultures derived from kidneys from different individuals demonstrated that hCNT3 activities varied between cultures. Also, the extent of cellular uptake of fludarabine, an anti-cancer nucleoside drug, and degree of cytotoxicity was reflected in the different hCNT3 activities observed between cultures. These results suggested that hCNT3 plays an important role in fludarabine renal handling and is a determinant of potential renal toxicities. Production of polarized monolayer cultures of hRPTCs on transwell permeable inserts enabled the functional localization of hCNT3 and hENT1 to apical membranes and hENT2 to basolateral membranes. Transepithelial flux studies demonstrated that (i) apical-to-basolateral fluxes of adenosine were mediated by apical hCNT3 and basolateral hENT2, (ii) basolateral-to-apical fluxes of 2′-deoxyadenosine were mediated, in part, by apical hENT1 and basolateral hOATs, and (iii) apical-to-basolateral fluxes of fludarabine, cladribine, and clofarabine were mediated by apical hCNT3. These studies showed that coupling of apical hCNT3 to basolateral hENT2 mediates proximal tubular nucleoside reabsorption, that coupling of basolateral human organic anion transporters (hOATs) to apical hENT1 mediates proximal tubular nucleoside secretion, and that hCNT3 is a key determinant of fludarabine proximal tubular reabsorption and cytoxicity.

Glucocorticoids are widely used in clinical therapy. However, they cause adverse effects including insulin resistance and Type 2 diabetes, which are characterised by decreased glucose transport into the muscles and fat. How glucocorticoids inhibit glucose transport remains unclear. Insulin stimulates glucose uptake via the insulin receptor substrate (IRS)-1 / phosphoinositide-3-kinase (PI3K) / protein kinase B (AKT) pathway and promotes the redistribution of GLUT4 from intracellular storage compartments to the plasma membrane (PM). Insulin-stimulated phosphorylation of AKT substrate of 160 kDa (AS160), a Rab-GTPase activating protein is downstream of AKT and appears to be essential for exposure of GLUT4 at the PM and glucose uptake. This is mediated through the association of phosphorylated AS160 (at the key residue T642) with 14-3-3 in the cytosol. The mildly insulin-responsive GLUT1 mediates basal glucose uptake in adipocytes. It is also subject to regulated trafficking like GLUT4. This study aimed to determine the level at which glucocorticoids inhibit glucose uptake in adipocytes. Effects of the synthetic glucocorticoid dexamethasone (Dex) and the natural glucocorticoid cortisol, on GLUT1 and GLUT4 function were examined. Candidates for the glucocorticoid-mediated inhibition of GLUT1- and GLUT4-mediated glucose uptake were investigated. These were glycogen synthase kinase (GSK) 3β (an AKT substrate) for GLUT1-mediated glucose transport; and adaptor protein containing PH domain, PTB domain, and leucine zipper motif (APPL)-1 (an AKT-interacting protein) and AS160 for GLUT4-mediated glucose transport. Dex and cortisol significantly decreased basal glucose uptake by 50% (p<0.05) in SGBS and 3T3-L1 adipocytes. Similarly, insulin-stimulated glucose uptake was decreased by 50% (p<0.001 for SGBS; p<0.05 for 3T3-L1) and 30% (p<0.05 for both) at 1 nM and 100 nM insulin respectively. Similar results were observed with differentiated primary human preadipocytes and human adipose explants. Dex-mediated inhibition of basal glucose uptake was limited to insulin-sensitive cell types implying that glucocorticoids may regulate GLUTs at steps common to GLUT1 and GLUT4 trafficking. Dex-mediated reduction in glucose uptake correlated with the reduction in basal and insulin-stimulated expression of GLUT1 and GLUT4 to the PM without changes in total GLUT1/4 expression. Dex did not alter total expression or phosphorylation of proximal insulin-signalling molecules up to and including AKT but increased FOXO1 expression, and modified GSK3β-S9 phosphorylation. Dex did not alter total APPL1 expression or subcellular distribution. Dex significantly decreased 1nM-insulin stimulated AS160-T642 phosphorylation by 50% (p<0.05) in SGBS and 3T3-L1 adipocytes via the glucocorticoid repector (GR). This correlated with reduced AS160:14-3-3 interaction. Similar results were obtained for AS160-T642 basal phosphorylation. At 1nM insulin, AS160-T642 phosphorylation is maximal at sub-maximal glucose uptake, i.e. AS160 phosphorylation significantly contributes to glucose uptake. RU486 significantly prevented but did not fully abrogate the Dex-mediated reduction in glucose uptake suggesting additional Dex-induced defects. In conclusion, glucocorticoids inhibit glucose uptake at a level distal to AKT by GR-dependent mechanisms. A role for GSK3β or APPL1 in glucocorticoid-mediated inhibition of glucose uptake requires further investigations. FOXO1 represents a suitable candidate for mediating the Dex-induced defects. Of significance, perturbation in AS160-T642 phosphorylation contributes to Dex-mediated inhibition of glucose uptake. Thus, AS160 presents a novel therapeutic target in the improvement of glucocorticoid-mediated inhibition of glucose uptake.

AM plants acquire Pi via two pathways; the direct uptake pathway via plant roots and the AM pathway via external fungal hyphae and colonised cortical cells. It has been assumed that these two pathways are additive and therefore in non-responsive plants the AM pathway is often considered to be non-functional. However, data from ³²P uptake studies indicates that the AM pathway is functional in many non-responsive symbioses and in some instances supplies the majority of plant P. In recent years the high-affinity Pi transporters involved in both direct and AM Pi uptake pathways have been identified. They are expressed at the root epidermis and the symbiotic interface of colonised cortical cells and respond to the P and AM status of the plant. The overall objective of the work described in this thesis was to characterise Pi uptake via the AM pathway in barley, a non-responsive AM host, using an approach which integrated physiological measurements of plant responsiveness and AM contribution with investigations of gene expression and functional characterisation of the plant Pi transporters. A preliminary survey of field-grown barley demonstrated the persistence of AM colonisation under commercial cropping regimes in southern Australia and highlighted the relevance of AM studies to commercial agriculture. Under glasshouse conditions AM colonisation of barley induced depressions in growth and P uptake compared to NM controls. Growth depressions were unrelated to percent colonisation by two AM fungal species and could not readily be explained by fungal C demand; the strong correlation between growth and P content suggested that P was the limiting factor in these experiments. However, a compartmented pot system incorporating ³²P-labelling demonstrated that the AM pathway is functional in colonised barley and, in the interaction with G. intraradices, contributed 48% of total P. This suggested that P flux via the direct uptake pathway is decreased in AM barley. The expression of three Pi transporters, HvPT1, HvPT2 and HvPT8 was investigated in colonised roots. HvPT1 and HvPT2 have previously been localised to the root epidermis and root hairs and are involved in Pi uptake via the direct pathway whilst HvPT8 is an AM-inducible Pi transporter which was localised by in-situ hybridisation to colonised cortical cells. Using promoter::GFP gene fusions the localisation of HvPT8 to arbuscule-containing cortical cells was confirmed in living roots from transgenic barley. Quantitative real-time PCR analysis of the expression of these three Pi transporters indicated that HvPT1 and HvPT2 were expressed constantly, under all conditions regardless of AM colonisation status and indicated that decreased P flux via the direct pathway is not related to expression of these transporters. HvPT8 was induced in AM colonised roots. However, the level of expression was not related to flux via the AM pathway or arbuscular colonisation. The HvPT8 transporter was further characterised by constitutive over-expression in transgenic barley. ³²P uptake assays in excised roots demonstrated increased Pi uptake from low P solution compared to wild-type roots and confirmed that HvPT8 is a functional Pi transporter with high-affinity transport properties. This is the first report of characterisation of an AM-inducible Pi transporter in planta. When these transgenic plants were grown in solution culture there was no increase in growth or P uptake relative to wild-type or transgenic controls and growth in soil and AM colonisation were also unaffected in these transgenic lines. The data presented in this thesis highlights the importance of combined physiological and molecular approaches to characterising plant AM interactions. The persistence of AM colonisation in barley in the field indicates the importance of improving our understanding of symbiotic function in non-responsive plants. Future efforts should be directed towards understanding the signals which regulate P flux via both the direct and AM pathways with the ultimate aim of enhancing AM responsiveness of non-responsive species. Making the direct and AM pathways additive in nonresponsive species should be a key aim of future research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313311 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

AM plants acquire Pi via two pathways; the direct uptake pathway via plant roots and the AM pathway via external fungal hyphae and colonised cortical cells. It has been assumed that these two pathways are additive and therefore in non-responsive plants the AM pathway is often considered to be non-functional. However, data from ³²P uptake studies indicates that the AM pathway is functional in many non-responsive symbioses and in some instances supplies the majority of plant P. In recent years the high-affinity Pi transporters involved in both direct and AM Pi uptake pathways have been identified. They are expressed at the root epidermis and the symbiotic interface of colonised cortical cells and respond to the P and AM status of the plant. The overall objective of the work described in this thesis was to characterise Pi uptake via the AM pathway in barley, a non-responsive AM host, using an approach which integrated physiological measurements of plant responsiveness and AM contribution with investigations of gene expression and functional characterisation of the plant Pi transporters. A preliminary survey of field-grown barley demonstrated the persistence of AM colonisation under commercial cropping regimes in southern Australia and highlighted the relevance of AM studies to commercial agriculture. Under glasshouse conditions AM colonisation of barley induced depressions in growth and P uptake compared to NM controls. Growth depressions were unrelated to percent colonisation by two AM fungal species and could not readily be explained by fungal C demand; the strong correlation between growth and P content suggested that P was the limiting factor in these experiments. However, a compartmented pot system incorporating ³²P-labelling demonstrated that the AM pathway is functional in colonised barley and, in the interaction with G. intraradices, contributed 48% of total P. This suggested that P flux via the direct uptake pathway is decreased in AM barley. The expression of three Pi transporters, HvPT1, HvPT2 and HvPT8 was investigated in colonised roots. HvPT1 and HvPT2 have previously been localised to the root epidermis and root hairs and are involved in Pi uptake via the direct pathway whilst HvPT8 is an AM-inducible Pi transporter which was localised by in-situ hybridisation to colonised cortical cells. Using promoter::GFP gene fusions the localisation of HvPT8 to arbuscule-containing cortical cells was confirmed in living roots from transgenic barley. Quantitative real-time PCR analysis of the expression of these three Pi transporters indicated that HvPT1 and HvPT2 were expressed constantly, under all conditions regardless of AM colonisation status and indicated that decreased P flux via the direct pathway is not related to expression of these transporters. HvPT8 was induced in AM colonised roots. However, the level of expression was not related to flux via the AM pathway or arbuscular colonisation. The HvPT8 transporter was further characterised by constitutive over-expression in transgenic barley. ³²P uptake assays in excised roots demonstrated increased Pi uptake from low P solution compared to wild-type roots and confirmed that HvPT8 is a functional Pi transporter with high-affinity transport properties. This is the first report of characterisation of an AM-inducible Pi transporter in planta. When these transgenic plants were grown in solution culture there was no increase in growth or P uptake relative to wild-type or transgenic controls and growth in soil and AM colonisation were also unaffected in these transgenic lines. The data presented in this thesis highlights the importance of combined physiological and molecular approaches to characterising plant AM interactions. The persistence of AM colonisation in barley in the field indicates the importance of improving our understanding of symbiotic function in non-responsive plants. Future efforts should be directed towards understanding the signals which regulate P flux via both the direct and AM pathways with the ultimate aim of enhancing AM responsiveness of non-responsive species. Making the direct and AM pathways additive in nonresponsive species should be a key aim of future research. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1313311 / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2008

Doctor of Philosophy(PhD) / The aims of this project were to contribute to the elucidation of the role of the cholinergic system in attention and memory, two cognitive processes severely compromised in Alzheimer’s disease (AD), and to evaluate and develop tools for the functional molecular imaging of this system with a view to improving knowledge of AD and other neurological disorders. Towards the first aim, the specific anti-cholinergic toxin 192 IgG-saporin (SAP) was administered to female Sprague-Dawley rats via either an intracerebroventricular (icv) or an intracortical route and animals were tested with a vibrissal-stimulation reaction-time task and an object recognition task to evaluate their attentional and mnemonic function, respectively. The second aim was approached in two ways. Firstly, relative neuronal densities from animals with icv lesions were assessed with both ex vivo and in vitro autoradiography with the specific cholinergic radiopharmaceuticals [123I]iodobenzovesamicol (123IBVM) and 125I-A-85380, ligands for the vesicular acetylcholine transporter and the nicotinic acetylcholine receptor, respectively. Secondly, a number of in vivo and in vitro studies were performed on a novel and unique molecular imaging system (TOHR), with which it had been hoped initially to image eventually SAP-lesioned animals, with a view to measuring and ameliorating its performance characteristics and assessing its in-principle suitability for small-animal molecular imaging. The behavioural studies support a critical role for the cholinergic system in normal attentional function. Additionally, in accord with literature evidence, no significant impairment was observed in mnemonic function. It is postulated however that the results observed in the intracortically-lesioned animals support the published hypothesis that cholinergic projections to the perirhinal cortex are critical for object-recognition memory. In autoradiographic studies, SAP-lesioned animals demonstrated reduced uptake of 123IBVM in multiple regions. A reduction of nicotinic receptors was also seen in SAP-lesioned animals, a novel finding supportive of the excellent characteristics of radioiodinated I-A-85380. Examination of the performance characteristics of the TOHR support in principle its utility for targeted small-animal molecular imaging studies.

Doctor of Philosophy (Medicine) / Glutamate transporters play a role in removing extracellular excitatory neurotransmitter, L-glutamate into the cells. The rate of the uptake depends on the density of the transporters at the membrane. Some studies claimed that glutamate transporters could transit between the cytoplasm and the membrane on a time-scale of minutes. The present study examined the distribution of glutamate transporter GLAST predominantly expressed in rat cortical cultured astrocytes between the membrane and the cytoplasm by using deconvolution microscopy and then analyzing the images. The regulation of the distribution of GLAST was studied in the presence of glutamate transporter substrate (D-aspartate), purinergic receptor activators (α,β-methylene ATP, adenosine), neuroleptic drugs (clozapine, haloperidol), ammonia (hyperammonia) and Na+/K+-ATPase inhibitors (ouabain, digoxin and FCCP). It was demonstrated that the translocation of GLAST towards the plasma membrane was induced by D-aspartate, α,β-methylene ATP, adenosine, clozapine and ammonia (at 100 μM and very high concentrations of 10 mM). However, the inhibition of Na+/K+-ATPase activity had an opposite effect, resulting in redistribution of GLAST away from the membrane. It has previously been claimed that the membrane-cytoplasm trafficking of GLAST was regulated by phosphorylation catalysed by protein kinase C delta (PKC-delta). Involvement of this mechanism has, however, been put to doubt when rottlerin, a PKC-delta inhibitor, used to test the hypothesis showed to inhibit Na+/K+-ATPase-mediated uptake of Rb+, suggesting that rottlerin influenced the activity of Na+/K+-ATPase. As Na+/K+-ATPase converts ATP to energy and pumps Na+, K+ ions, thus helping to maintain normal electrochemical and ionic gradients across the cell membrane. Its inhibition also reduced D-aspartate transport and could impact on the cytoplasm-to-membrane traffic of GLAST molecules. Furthermore, rottlerin decreased the activity of Na+/K+-ATPase by acting as a mitochondrial inhibitor. The present study has focused on the inhibition of Na+/K+-ATPase activity by rottlerin, ouabain and digoxin in homogenates prepared from rat kidney and cultured astrocytes. The activity of Na+/K+-ATPase was measured by the absorption of inorganic phosphate product generated from the hydrolysis of ATP and the fluorescent transition of the dye RH421 induced by the movement of Na+/K+-ATPase. This approach has a potential to test whether the rottlerin effect on Na+/K+-ATPase is a direct inhibition of the enzyme activity. Rottlerin has been found to block the activity of Na+/K+-ATPase in a dose-dependent manner in both rat kidney and astrocyte homogenates. Therefore, rottlerin inhibited the activity of Na+/K+-ATPase directly in a cell-free preparation, thus strongly indicating that the effect was direct on the enzyme. In parallel experiments, ouabain and digoxin produced similar inhibitions of Na+/K+-ATPase activity in rat kidney while digoxin blocked the activity of Na+/K+-ATPase to a greater extent than ouabain in rat cortical cultured astrocytes. In a separate set of experiments, Na+/K+-ATPase in the astrocytic membrane was found to be unsaturated in E1(Na+)3 conformation in the presence of Na+ ions and this could explain the differences between the effects of digoxin and ouabain on the activity of Na+/K+-ATPase in rat astrocytes. In addition, it was found that at low concentrations of rottlerin, the activity of Na+/K+-ATPase was increased rather than inhibited. This effect was further investigated by studying rottlerin interactions with membrane lipids. The activity of Na+/K+-ATPase has been reported to be regulated by membrane lipids. The enzyme activity can be enhanced by increasing fluidity of the lipid membrane. I have, therefore, proposed that rottlerin binds to the membrane lipids and the effects of rottlerin on Na+/K+-ATPase are mediated by changes in the properties (fluidity) of the membrane. The hypothesis was tested by comparing rottlerin and a detergent, DOC (sodium deoxycholate), for their binding to the lipids by using a DMPC (1,2-Dimyristoyl-sn-Glycero-3-Phosphocholine) monolayer technique. DOC has been shown to both increase and inhibit activity of Na+/K+-ATPase in a manner similar to that displayed by rottlerin. The effects of rottlerin and DOC on the DMPC monolayers were studied by measuring the surface pressure of DMPC monolayers and surface area per DMPC molecule. I established that both rottlerin and DOC decreased the surface pressure of DMPC monolayers and increased the surface area per DMPC molecule. This indicates that both rottlerin and DOC penetrated into the DMPC monolayers. If rottlerin can interact with the lipids, changes in fluidity of the lipid membrane cannot be ruled out and should be considered as a possible factor contributing to the effects of rottlerin on the activity of Na+/K+-ATPase. Overall, the study demonstrates that rottlerin is not only a PKC-delta inhibitor but can have additional effects, both on the enzyme activities (Na+/K+-ATPase) and/or on lipid-containing biological structures such as membranes. The findings have implication not only for studies where rottlerin was used as a supposedly specific PKC-delta inhibitor but also for mechanisms of its toxicity.