No description available.

No description available.

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.

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.

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. / Science, Faculty of / Botany, Department of / Graduate

yes / Inflammation of adipose tissue in obesity is associated with increased IL-1β, IL-6 and TNF-α secretion and proposed to contribute to insulin resistance. AMP-activated protein kinase (AMPK) regulates nutrient metabolism and is reported to have anti-inflammatory actions in adipose tissue, yet the mechanisms underlying this remain poorly characterised. The effect of AMPK activation on cytokine-stimulated proinflammatory signalling was therefore assessed in cultured adipocytes. AMPK activation inhibited IL-1β-stimulated CXCL10 secretion, associated with reduced interleukin-1 receptor associated kinase-4 (IRAK4) phosphorylation and downregulated MKK4/JNK and IKK/IκB/NFκB signalling. AMPK activation inhibited TNF-α-stimulated IKK/IκB/NFκB signalling but had no effect on JNK phosphorylation. The JAK/STAT3 pathway was also suppressed by AMPK after IL-6 stimulation and during adipogenesis. Adipose tissue from AMPKα1−/− mice exhibited increased JNK and STAT3 phosphorylation, supporting suppression of these distinct proinflammatory pathways by AMPK in vivo. The inhibition of multiple pro-inflammatory signalling pathways by AMPK may underlie the reported beneficial effects of AMPK activation in adipose tissue. / British Heart Foundation

No description available.

ABSTRACT Metformin is the most common drug used against type 2 diabetes mellitus. However, it was only recently shown, in human and rat hepatocytes, that metformin-like 5-aminoimidazole-4-carboximide ribonucleoside (AICAR), acts via activation of the AMP-activated protein kinase (AMPK), an enzyme that plays a central role in lipid metabolism. Although it is well known that metformin is used in the treatment of type 2 diabetes and results in significant fat loss, no study has investigated the effects of this drug on adipocytes. In this report I studied the effects of metformin on the formation of fat deposits in mouse 3T3-L1 preadipocytes, as well as its effects on the activation of AMPK in these cells. Our results suggested that metformin significantly inhibits the transformation of pre-adipocytes into adipocytes. This is achieved via the inhibition of intracellular lipid accumulation during adipogenesis. In addition to its inhibition of intracellular lipid accumulation, metformin induced a significant increase in the phosphorylation of AMPK. It has been shown that AMPK activation with AICAR results in the inhibition of the nuclear factor-κB (NF-κB) induced gene expression. Since NF-κB is the key nuclear factor used by HIV-1 during the initiation of its gene transcription, I investigated the possibility of inhibiting HIV-1 replication in U1 cells with metformin and AICAR. I observed that AICAR and metformin inhibit HIV-1 replication in U1 cells. This inhibition wasparalleled by the accumulation of NF-κB in the cytoplasm of AICAR and metformin treated cells, and at the same time by a significant decrease in the concentration of this nuclear factor in the nucleus of these cells. However, I failed to observe any phosphorylation of AMPK by metformin and AICAR in U1 cells. In conclusion, metformin inhibits adipogenesis in mouse adipocytes and this inhibition is likely to take place via the activation of AMPK. AICAR and metformin have inhibitory properties against HIV-1 replication. However, this inhibition does not seem to be by the activation of AMPK.

Ethylene is a plant growth regulator and plays a key role in leaf senescence. Its signaling, however, remains mostly unclear in sweet potato. Ethephon, an ethylene releasing compound, induced sweet potato detached leaf senescence and associated gene expression, and the effects were repressed by mitogen-activated protein kinase (MAPK) kinase inhibitor PD98059. These data suggest that MAPK cascade is likely involved in ethylene signaling leading to leaf senescence and associated gene expression. With gene-specific primers and RT-PCR methods, a full-length cDNA, SPMAPK, was isolated from ethephon-treated sweet potato leaves. SPMAPK contained 1098 nucleotides (365 amino acids) in the open reading frame. Sweet potato SPMAPK also exhibited high amino acid sequence identities (ca. 79.8% to 83.4%) with plant MAPKs, and was most close to Arabidopsis MPK3 and MPK6 in phylogenetic tree analysis. RT-PCR analysis showed that SPMAPK gene expression was detected in roots, stems, and leaves. The mature and partial yellowing leaves expressed higher amount. SPMAPK gene expression was also inducible and significantly enhanced by ethephon. Results from studies with inhibitors or effectors showed that ethephon treatment resulted in acceleration of leaf senescence in detached sweet potato leaves, promotion of leaf chlorophyll content reduction and decrease of photochemical Fv/Fm, and induction of associated gene expression. These ethephon-mediated effects were all delayed or repressed by pretreatment with ethylene receptor inhibitor 1-methylcyclopropene (1-MCP), MAPK kinase inhibitor PD98059, NADPH oxidase inhibitor diphenyleneiodonium (DPI), antioxidant reduced glutathione, calcium ion chelator EGTA, and de novo protein synthesis inhibitor cycloheximide, respectively. Based on these results we conclude that an ethylene-inducible mitogen-activated protein kinase SPMAPK was isolated from sweet potato leaves, and expressed higher amount in mature and partial yellowing leaves. Ethephon-induced sweet potato SPMAPK expression was significantly repressed by 1-MCP, PD98059, DPI, reduced glutathione, EGTA and cycloheximide. These data also suggest that the possible signal components in ethephon-mediated leaf senescence and associated gene expression in sweet potato leaves likely include ethylene receptor, MAPK cascade, elevated H2O2 , external calcium influx, and de novo synthesized proteins. A possible ethylene signaling model leading to sweet potato leaf senescence and associated gene expression was also proposed.

Computational and mathematical models of cellular processes promise great benets in important elds such as molecular biology and medicine. Increasingly, researchers are incorporating the fundamentally discrete and stochastic nature of biochemical processes into the mathematical models that are intended to represent them. This has led to the formulation of models for genetic networks as continuous-time, discrete state, Markov processes, giving rise to the so-called Chemical Master Equation (CME), which is a discrete, partial dierential equation, that governs the evolution of the associated probability distribution function (PDF). While promising many insights, the CME is computationally challenging, especially as the dimension of the model grows. In this thesis, novel methods are developed for computing the PDF of the Master Equation. The problems associated with the high-dimensional nature of the Chemical Master Equation are addressed by adapting Krylov methods, in combination with Finite State Projection methods, to derive algorithms well-suited to the Master Equation. Variations of the approach that incorporate the Strang splitting and a stochastic analogue of the total quasi-steady-state approximation are also derived for chemical systems with disparate rates. Monte Carlo approaches, such as the Stochastic Simulation Algorithm, that simulate trajectories of the process governed by the CME have been a very popular approach and we compare these with the PDF approaches developed in this thesis. The thesis concludes with a discussion of various implementation issues along with numerical results for important applications in systems biology, including the gene toggle, the Goldbeter-Koshland switch and the Mitogen-Activated Protein Kinase Cascade.