No description available.

No description available.

No description available.

In vitro analysis of osteoblasts deficient in endothelial NO synthase (eNOS) or inducible NOS (iNOS) enzymes, indicated that NO produced via eNOS is required for normal osteoblast growth and differentiation. The transcriptional regulation of two genes, the extracellular matrix protein Tenascin-Cytotactin (Tn-C) and eNOS, which contain strain-related regulatory sites and are known to respond to mechanical stimuli, was investigated in osteoblasts and osteocytes (ob-mix). Stretch was applied via 2 different systems and the transcription level of the gene promoters assayed by dual luciferase reporter assay. It was demonstrated that transcription of Tn-C and eNOS is upregulated in response to physiological levels of tensile and/or shear stress. This signal is mediated via the putative StRE and SSRE in Tn-C promoter and at the proximal end of the eNOS promoter is indicated. A system was built to study calcium (Ca²⁺) fluxes in response to shear stress in primary ob-mix and an immortalised simian virus human foetal osteoblast cell-line (SV-HFO). Studies have shown that pulsating fluid flow (PFF) stimulates a rapid [Ca²⁺]_i increase in primary ob-mix. Rapid transient increases in [Ca²⁺]_i were also recorded in a synchronised culture of SV-HFO when subjected to PFF. Both these observations are consistent with the hypothesis that Ca²⁺ increases contribute to the osteoblastic NO response to shear stress. Taken together, these results confirm the important role NO plays in bone physiology and elucidate aspects of transcriptional regulation and activation of the eNOS enzyme.

Negative regulation of cytokine signalling is essential for maintaining hematopoietic homeostasis. We investigated the role of SH2B1 and CBL in the negative regulation of EPO and GM-CSF signaling, respectively. Erythropoiesis is driven by the cytokine erythropoietin (EPO), which mediates its signal by binding to its cognate receptor, the erythropoietin receptor (EPO-R). Murine knock-in studies have demonstrated EPO-R Tyr343 to play an important role in EPO mediated signalling. We have utilized a Cloning of Ligand Target (COLT) screen to identify the adaptor protein SH2B1 as an interactor of EPO-R pTyr343. We have demonstrated that SH2B1 binds to EPO-R via two mechanisms. The amino-terminus of SH2B1 and the membrane proximal region of EPO-R mediate SH2B1 constitutive binding to EPO-R. SH2B1 binds to EPO-R pTyr343 and pTyr 401 in an SH2 domain-dependent manner. SH2B1 displayed dose- and time- dependent Serine/Threonine phosphorylation in response to EPO stimulation. Knockdown of SH2B1 resulted in enhanced activation of Jak2 and EPO-R. These studies demonstrate SH2B1 as a novel negative regulator of EPO signalling. Mutations in the linker region and the RING finger of CBL have been identified in a number of myeloid malignancies, including juvenile myelomonocytic leukemia. We investigated how linker region mutant, CBL-Y371H, and RING finger mutant, CBL-C384R lead to GM-CSF hypersensitivity. Expression of these CBL mutants in the human hematopoietic cell line, TF-1, showed enhanced stimulation induced phosphorylation of GM-CSFR βc. We also demonstrated that the loss of E3 ligase activity of these CBL mutants results in increased expression of JAK2 and LYN kinases. Assessment of the effects of CBL mutants on downstream signalling revealed enhanced phosphorylation of SHP2, CBL and S6. Dasatinib induced inhibition of SRC family kinases abolished the elevated phosphorylation of CBL mutants, and equalized the phosphorylation of GM-CSFR βc in the wild type and CBL mutant cells.

Negative regulation of cytokine signalling is essential for maintaining hematopoietic homeostasis. We investigated the role of SH2B1 and CBL in the negative regulation of EPO and GM-CSF signaling, respectively. Erythropoiesis is driven by the cytokine erythropoietin (EPO), which mediates its signal by binding to its cognate receptor, the erythropoietin receptor (EPO-R). Murine knock-in studies have demonstrated EPO-R Tyr343 to play an important role in EPO mediated signalling. We have utilized a Cloning of Ligand Target (COLT) screen to identify the adaptor protein SH2B1 as an interactor of EPO-R pTyr343. We have demonstrated that SH2B1 binds to EPO-R via two mechanisms. The amino-terminus of SH2B1 and the membrane proximal region of EPO-R mediate SH2B1 constitutive binding to EPO-R. SH2B1 binds to EPO-R pTyr343 and pTyr 401 in an SH2 domain-dependent manner. SH2B1 displayed dose- and time- dependent Serine/Threonine phosphorylation in response to EPO stimulation. Knockdown of SH2B1 resulted in enhanced activation of Jak2 and EPO-R. These studies demonstrate SH2B1 as a novel negative regulator of EPO signalling. Mutations in the linker region and the RING finger of CBL have been identified in a number of myeloid malignancies, including juvenile myelomonocytic leukemia. We investigated how linker region mutant, CBL-Y371H, and RING finger mutant, CBL-C384R lead to GM-CSF hypersensitivity. Expression of these CBL mutants in the human hematopoietic cell line, TF-1, showed enhanced stimulation induced phosphorylation of GM-CSFR βc. We also demonstrated that the loss of E3 ligase activity of these CBL mutants results in increased expression of JAK2 and LYN kinases. Assessment of the effects of CBL mutants on downstream signalling revealed enhanced phosphorylation of SHP2, CBL and S6. Dasatinib induced inhibition of SRC family kinases abolished the elevated phosphorylation of CBL mutants, and equalized the phosphorylation of GM-CSFR βc in the wild type and CBL mutant cells.

[Truncated abstract] Cells need to be able to detect changes in their surrounding environment and transduce these signals into the appropriate cellular compartments. One of the major ways that the cell achieves this signal transduction is through the process of phosphorylation. Protein kinases are the enzymes responsible for catalysing this transfer of phosphate groups from ATP to amino acid residues of their specific substrates. A subfamily of serine/threonine kinases known as the Mitogen-Activated Protein Kinases (MAPKs) is essential in a diverse range of cell processes including growth, metabolism, differentiation and death. The first identified MAPKs, the Extracellular Signal-Regulated Kinases (ERKs), were found to be activated in response to mitogenic stimuli such as growth factors. However, since the discovery of the ERKs, other pathways leading to the activation of related kinases have been recognised. These kinases are preferentially activated in response to stress, and are thus termed “Stress-Activated Protein Kinases” or SAPKs. They consist of the c-Jun N-terminal kinase isoforms 1, 2 and 3 (also called SAPK1γ, SAPK1α and SAPKβ respectively) and the p38 MAPKs, p38α, p38β, p38γ and p38δ (also called SAPK2a, SAPK2b, SAPK3 and SAPK4 respectively). A major challenge in this field has been to identify the substrates and functions of the SAPKs. This has been partly achieved by the development of inhibitors for the JNK MAPKs and SAPK2a/b. However, no inhibitors currently exist that specifically inhibit SAPK3 and SAPK4. Therefore, elucidating the function of these SAPKs has proved more difficult. Recent studies suggest that SAPK3 may play a unique role in the cell compared to other members of the p38 MAPK family. For example, several signalling proteins appear to specifically activate SAPK3 in certain circumstances while not activating other members of the p38 MAPK family. In addition, SAPK3 contains a unique sequence motif that allows it to bind to specialised domains known as PDZ domains. The interaction of SAPK3 with proteins containing these domains may regulate its subcellular localisation and interactions with other proteins in the cell. This project was undertaken to expand the knowledge on the expression, localisation, substrate specificity and binding partners of SAPK3. In Chapter 3 of this thesis, a SAPK3 monoclonal antibody was evaluated for its ability to specifically recognise endogenous SAPK3 protein. SAPK3 was found to be expressed in immortalised cell lines and primary cultures of neonatal rat myocytes, and to be colocalised with the mitochondria of these cells. This co-localisation remained unaltered in response to treatment with the nuclear export inhibitor Leptomycin B, and with exposure to osmotic shock, suggesting that SAPK3 substrates may be localised at the mitochondria

Toll-like receptors (TLR) are a family of pattern recognition receptors crucial for pathogen pattern recognition. Upon activation, TLRs induce innate immune responses such as cytokine production. However irregular TLR activities can provide fatal, hence fine tuning of the TLR induced responses are necessary. The TLR mediated immune responses are controlled by the positive/negative regulation of TLR signalling pathways, relocation of TLR proteins and modulation of TLR transcription. Systematic analyses of the agonist-induced transcriptional changes of TLRs were shown for the first time in my thesis. In my experiments, I have shown that each agonist induced a unique pattern of TLR transcription. Following PAM stimulation, mRNA levels of the cognate TLR1/2 increased whereas mRNA levels of the cross-regulating TLR4, 7/8/9 reduced in both cell lines and splenic macrophages from different mice strains. Through investigation of the signalling pathways responsible for mediating such TLR transcriptional changes, I then discovered the balancing effect between NFÎoB and MAPK signalling pathways. PAM induced TLR transcriptional changes were controlled by the additive and/or antagonistic interference between MAPK signalling cascades, ERK, JNK, P38 and NFÎoB signalling pathways. This was the first time that signalling synergy between MAPK and NFÎoB pathways were shown. Furthermore, PAM induced transcription of TLR1 and TLR8 may be partially regulated by the indirect feedback mediated by protein production. Importantly, the maintenance of the basal TLR mRNA expression also required activation of both MAPK and NFÎoB signalling pathways. In addition, signalling control for TLR transcription induced by different agonists (PAM vs. LPS) or in different species (chicken vs. mice) was compared. LPS induced transcriptional changes of the cross-regulating TLR1/2 and 3 but not the cognate TLR4 in RAW cells. The LPS induced TLR transcriptional changes required activation of a combination of MAPK and NFÎoB signalling pathways which shared both similarities and differences to the PAM induced signalling activation. In chicken, PAM induced more potent signalling activation, regulating the TLR transcriptional changes at a lower concentration than in mice. Overall, this thesis demonstrates that the transcriptional regulation of TLRs is complex, mediated by the coordination between MAPK and NFÎoB signalling pathways. These studies have significant implications in providing detailed insight of TLR transcriptional regulation which plays an important role in the regulation of TLR mediated innate immune responses. Please watch the following videos that I made for: A short introduction about TLR regulation - https://youtu.be/LTDdEZ3S97o A short explanation about TLR signalling - https://youtu.be/51IY5XhdJR8.

Simulated evolution is used as a tool for investigating the selective pressures that have influenced the design of animal signalling systems. The biological literature on communication is first reviewed: central concepts such as the handicap principle and the view of signalling as manipulation are discussed. The equation of “biological function” with “adaptive value” is then defended, along with a workable definition of communication. Evolutionary simulation models are advocated as a way of testing the coherence of a given theory. Contra some ALife enthusiasts, simulations are not alternate worlds worthy of independent study; in fact they fit naturally into a Quinean picture of scientific knowledge as a web of modifiable propositions. Existing simulation work on the evolution of communication is reviewed: much of it consists of simple proofs of concept that fail to make connections with existing theory. A particular model (MacLennan & Burghardt, 1994) of the evolution of referential communication in a co-operative context is replicated and critiqued in detail. Evolutionary simulations are then presented that cover a range of ecological scenarios; the first is a general model of food- and alarm-calling. In such situations signallers and receivers can have common or conflicting interests; the model allows us to test the idea that a conflict of interests will lead to an arms race of ever more costly signals, whereas common interests will result in signals that are as cheap as possible. The second model is concerned with communication during aggressive interactions. Many animals use signals to settle contests, thus avoiding the costs associated with fighting. Conventional game-theoretic results suggest that the signalling of aggression or of strength will not be evolutionarily stable unless it is physically unfakeable, but some recent models imply that cost-free, arbitrary signals can be reliable indicators of both intent and ability. The simulation, which features continuous-time perception of the opponent’s strategy, is an attempt to settle the question. The third model deals with sexual signalling, i.e., elaborate displays that are designed to persuade members of the opposite sex to mate. The results clarify the question of whether such displays are the pointless result of runaway sexual selection, or whether they function as honest and costly indicators of genetic quality. The models predict the evolution of reliable communication in a surprisingly narrow range of circumstances; a serious gap remains between these predictions and the ethological data. Future directions for simulation work are discussed.

Intracellular signalling molecules create the signalling cascades which enable the transfer of the signal to the cell. In this work we have studied the influence of tick saliva on the cytokine production and the activation of signalling pathways in ionomycin stimulated murine mast cells. We found out that tick saliva inhibits production of several cytokines and affects two important signalling pathways in mast cells possibly involved in the regulation of cytokine induction.