The aim of this study was to develop an <I>in vitro</I> mammary model, based on rat mammary explants, which could be used to examine the effects of amino acid profile and concentration in the media on protein synthesis. Secondly, to ascertain whether these responses to amino acids, in particular leucine and α ketoisocaproic acid, were transmitted through the mTOR/p70 S6 kinase signalling pathway. Mammary explant protein synthesis was found to be stimulated up to twofold in response to graded levels of a complete mixture of amino acids (2 x and 4 x, normal rat plasma concentrations). The acute (1 h) stimulation of protein synthesis was at the level of translation. Inhibition of mTOR by rapamycin did not block the stimulation of protein synthesis by amino acids. In fact, when total amino acid concentrations were increased 0.5 to 4-fold, p70 S6 kinase activity decreased, despite the fact that protein synthesis was elevated up to 2.5 fold. When explants were incubated with either leucine or its transamination product α ketoisocaproic acid at 4 x normal levels in the presence of other amino acids (1 x), p70 S6 kinase activity was increased. There was a tendency for p70 S6 kinase activity to be blocked when transamination was inhibited. The failure to decrease protein synthesis by inhibition of transamination, despite the fact that p70 S6 kinase activity was inhibited, suggests that other translation factors may be more important in regulating mammary protein synthesis. This Phd thesis demonstrates a novel role for amino acids in mammary protein synthesis, whereby amino acids modulate the activity of the translation regulator p70 S6 kinase. In particular leucine and its transamination are important in the regulation of p70 S6 kinase activity. This provides the starting point for future studies exploring the role of translation factors in the regulation of mammary protein synthesis.

Pyruvate kinase M2 (PKM2) has been studied for decades on its role in cancer metabolism. Recently, PKM2 is highlighted again for its new function: promoting gene transcription by acting as a protein kinase. Moreover, the PKM2 levels in patient circulation have been used as a diagnostic marker for many types of cancers. However, it remains unclear whether PKM2 in blood circulation has any physiological or pathological function. In my dissertation, I demonstrate that PKM2 released from cancer cells facilitates tumor growth by promoting tumor angiogenesis. Our experiments show that PKM2 promotes endothelial cell proliferation, migration and survival. Only the dimeric PKM2, not the tetrameric PKM2 possesses the activity in angiogenesis promotion. Our results further indicate that PKM2 regulates angiogenesis by integrin αvβ3 activation and integrin redistribution. I also found that PKM2 enhances drug resistance of cancer cells expressing integrin αvβ3.

Le cœur est un organe central au fonctionnement du système cardiovasculaire. Il est physiologiquement compartimenté et est constitué de cellules spécialisées qui régulent les impulsions électriques ainsi que la contraction du myocarde. Le cœur adapte le flux sanguin en fonction des besoins du corps. En condition pathologique, le cœur recourt toutefois à des mécanismes compensatoires. Au niveau physiologique, la compensation s’observe par l’hypertrophie des cardiomyocytes qui, bien que bénéfique à court terme, exacerbe à long terme la fonction cardiaque. L’activation des « mitogen activated protein kinases » (MAPK) contribue autant au maintien de la fonction physiologique qu’à la détérioration pathologique du myocarde et serait également une cause de l’hypertrophie observée. Parmi les 5 groupes de MAPK connues, la MAPK p38 est formée de 4 isoformes dont les sérine/thréonine kinases p38α et p38γ sont exprimées de façon prédominante dans le cœur. Les p38 partagent les mêmes activateurs, mais leurs effecteurs diffèrent. Bien que le rôle de p38α semble impliqué dans l’aggravement des troubles cardiaques, celui de p38γ ne semble pas redondant à p38α et demeure incompris. Cette isoforme possède un motif de liaison aux domaines PDZ, unique chez les MAP kinases. Également, chez les cellules cardiaques, elle transloque au noyau en condition de stress. Le but de l’étude ici est de comprendre le rôle de p38γ et de ses motifs uniques sur la structure et la taille des cardiomyocytes. Afin de répondre au but de l’étude, plusieurs mutants adénoviraux de p38 ont été conçus. Un des mutants ne possède pas le motif de liaison aux domaines PDZ, deux autres contrôlent la localisation cellulaire soit au noyau, soit au cytoplasme, et un autre mutant est muté au site de phosphorylation. Des cardiomyocytes en culture ont été infectés par les différents mutants en présence de leur activateur en amont ou de la β-galactosidase. Les réseaux d’α-actinine, ainsi que la taille des cardiomyocytes, ont été observés par microscopie. Les observations effectuées montrent que p38γ entraîne une désorganisation des réseaux d’α-actinine lorsqu’il est phosphorylé. Également, il facilite l’hypertrophie des cardiomyocytes en présence de son activateur s’il est forcé hors du noyau ou en l’absence de son motif de liaison aux domaines PDZ. En conclusion, les résultats obtenus suggèrent que p38γ exerce bel et bien un rôle dans le maintien structural des cardiomyocytes par l’intermédiaire de l’α-actinine.

No description available.

Thesis (Ph. D.)--University of Hawaii at Manoa, 1995. / Includes bibliographical references (leaves 115-129). / Microfiche. / ix, 129 leaves, bound ill. 29 cm

Plants possess integrated signalling networks that mediate the responses to various environmental conditions. Mitogen-activated protein kinases (MAPKs) constitute a highly conserved family of enzymes in eukaryotes, and in plants MAPK-based signal transduction modules regulate a large number of physiological processes, including responses to environmental stresses and phytohormones. Regulated dephosphorylation of active MAPKs is a key component of the control of MAPK signalling cascades, and in mammals, members of the MAPK phosphatase (MKP) sub-class of dual-specificity tyrosine phosphatases have been recognized as key players for inactivating MAPKs. Five MKP homologues are found in Arabidopsis thaliana, but only limited information is available concerning their properties and biological roles. Based on initial data derived from my reverse genetics and protein interaction studies of these five potential MKPs, as well as gene function information in the literature, I chose to focus on two putative Arabidopsis MKPs, AtMKP2 and Indole-3-Butyric Acid-response 5 (IBR5). By using a combination of genetic and biochemical studies, I established that the previously uncharacterized MKP designated AtMKP2, participates in the regulation of cellular homeostasis in ozone-challenged tissue, and can influence the activation state of two MAPKs, MPK3 and MPK6. AtMKP2-suppressed plants displayed significantly prolonged MPK3 and MPK6 activation during ozone treatment, and recombinant AtMKP2 was able to dephosphorylate both phospho-MPK3 and phospho-MPK6 in vitro, providing direct evidence that AtMKP2 may target these oxidant-activated MAPKs. A mutation in IBR5, one of the five potential AtMKPs, was previously reported to confer reduced sensitivity to auxin and ABA in Arabidopsis. My protein interaction studies demonstrated that IBR5 and MPK12 are physically coupled and that the C-terminus of MPK12 is essential for its interaction with IBR5. In vitro dephosphorylation assays indicated that recombinant phosphoMPK12 is efficiently dephosphorylated by IBR5. In transgenic plants with reduced expression of the MPK12 gene, root growth is hypersensitive to exogenous auxins, consistent with the lower auxin sensitivity reported for ibr5 mutants. Taken together, my data demonstrate for the first time that both AtMKP2 and IBR5 are bona fide Arabidopsis MAPK phosphatases and that they serve as important regulators of oxidative stress and auxin signalling, respectively, in Arabidopsis.

Cell survival in conditions of severe oxygen deprivation depends on a wide variety of biochemical modifications, which result in a large-scale suppression of metabolism, preventing [ATP] from falling to fatally low levels. We investigated whether AMP-activated protein kinase (AMPK) has a role in the coordination of cellular modification during hypoxia, which leads to a regulated state of metabolic suppression in the goldfish (Carassius auratus). Energy charge, AMPK activity, protein and gene expression, as well as the translational capacity and phosphorylation state of a downstream target were measured in goldfish tissues during exposure to hypoxia (-0.3 mg 02/L) for up to 12 h. AMPK activity in the goldfish liver increased by 4-fold at 0.5 h hypoxia and was temporally associated with a —11-fold increase in calculated AMPfree/ATP. No change was observed in total AMPK protein or relative gene expression of identified AMPK isoforms. Changes in AMPK activity were also associated with a decreased rate of protein synthesis and an increase in the phosphorylated form of eukaryotic elongation factor-2 (eEF2; relative to total eEF2). Increases in AMPK activity were not seen in hypoxic goldfish muscle, brain, heart or gill, nor was a significant alteration in cellular energy charge seen in muscle. Still, the present study is the first to show that AMPK activity increases in liver in response to short-term severe hypoxia exposure in a hypoxia-tolerant fish. The decreased rates of protein synthesis, a well known component of metabolic suppression, combined with increased phosphorylation of eEF2, a downstream target of AMPK, potentially implicate the kinase in the cellular effort to suppress metabolism in hypoxia-tolerant species during oxygen deprivation.

The natural history of CML has been transformed in recent years by the introduction of Glivec[superscript TM] (imatinib mesylate), an ABL kinase inhibitor, which provides the new treatment paradigm for chronic phase CML. While the majority of patients with CP-CML respond very well to imatinib, there are approximately 15% of patients who fail to respond, or respond suboptimally. While the major cause of secondary imatinib resistance can be attributable to kinase domain mutations, the underlying cause of primary resistance is yet to be elucidated. Utilizing the phosphorylation of the adaptor protein Crkl, an immediate downstream partner of BCRABL, as a surrogate measure of BCR-ABL kinase activity, a large interpatient variation in the degree of imatinib induced kinase inhibition achieved in-vitro, was observed in previously untreated CP-CML patients. The observed in-vitro sensitivity was a good predictor of molecular response in patients treated with 600mg imatinib as front line therapy. Furthermore, analysis of the in-vivo reduction in p-Crkl mediated measured in blood cells in response to imatinib over the first 28 days of therapy, revealed that patients with higher % reductions respond significantly better over a two year period, than those with lower % reductions. Using 14-C labelled imatinib, it was demonstrated that this intrinsic sensitivity correlated to the amount of drug which was retained within the target haemopoietic cell, and furthermore, that a critical determinant of the active influx of imatinib, was the functional activity of the human organic cation transporter -1 (OCT-1), as determined by a prazosin (potent inhibitor of OCT-1) inhibition assay. Patients with high OCT-1 Activity had superior molecular responses when compared to those with low OCT-1 Activity, but in those patients who could tolerate increased imatinib dose, these inferior responses could be largely overcome. In contrast, Nilotinib, a more potent second generation tyrosine kinase inhibitor, is not dependent on OCT-1 for influx, making it a possible treatment choice for patients with low OCT-1 Activity. Both imatinib and nilotinib interact with the efflux transporters ABCB1, and ABCG2. In combination studies imatinib results in a significantly increased intracellular concentration of nilotinib, most likely through interaction with these efflux transporters. Furthermore, commonly used therapies such as proton pump inhibitors also interact with ABCB1 and ABCG2, and demonstrable changes in intracellular drug concentrations were observed in-vitro with concomitant administration of these agents and imatinib or nilotinib at clinically relevant concentrations. In conclusion, these data demonstrate that the degree of kinase inhibition mediated in-vitro and in-vivo by imatinib, is a critical determinant of subsequent molecular response. This intrinsic sensitivity to imatinib induced kinase inhibition is related to the activity of the OCT-1 protein. This protein is not involved in the transport of nilotinib, suggesting it as a possible treatment alternative in those patients with low OCT-1 Activity. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1319077 / Thesis (Ph.D.) -- University of Adelaide, School of Medicine, 2008

Copy of author's previously published article inserted into back cover pocket. / Bibliography: leaves 157-214. / iv, 214, [131] leaves, [19] leaves of plates : ill. (some col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / The function of receptor tyrosine kinase, c-Kit is examined in relation to the role of receptor levels in factor dependence and cell transformation and the function of several naturally occurring isoforms of the human c-Kit receptor were analyzed by expressing cDNA encoding these isoforms in murine cells. / Thesis (Ph.D.)--University of Adelaide, Dept. of Microbiology and Immunology, 1996

B lymphocytes constitutes an important arm of the immune system, and their response to antigen is largely dependent upon signal transduction through the B cell receptor (BCR). Such a potent receptor, however, needs to be further balanced by positive and negative regulators to prevent harmful effects that may arise from inappropriate stimulation. Src family protein tyrosine kinase Lyn is involved in both positive and negative regulation, since the both gain-of-function Lyn and loss-of-function Lyn mutations caused autoimmunity in mice. The exact signalling pathway(s) regulated by Lyn in B cells, however, are still not clear. Work presented in this thesis attempts to elucidate the biochemical mechanisms that underline the double-edged nature of Lyn in BCR signalling. (For complete abstract open document)