Dissertations / Theses on the topic 'AMPK signalling'

AMP-activated protein kinase (AMPK) has been proposed to be a potential therapeutic target for patients with Type 2 diabetes and the metabolic syndrome. While the role of AMPK in muscle and liver is relatively well-characterised, less is known about the role of AMPK in the other principal metabolic tissue, adipose. Obesity is associated with the chronic, sub-clinical inflammation of adipose tissue. Characteristic hypertrophic adipocytes and the elevated infiltration and activation of macrophages stimulate production of cytokines and chemokines, including tumour necrosis factor-alpha (TNF-a), interleukin-1 beta (IL-1beta), interleukin-6 (IL-6) and monocyte chemoattractant protein-1 (MCP-1). These have autocrine, paracrine and endocrine effects which have been suggested to play a key role in the development of peripheral insulin resistance. Increasing evidence suggests that AMPK has anti-inflammatory actions, independent of its effect on carbohydrate and lipid metabolism. Previous work in our laboratory has demonstrated that AMPK inhibits TNF-alpha-stimulated MCP-1 secretion and monocyte adhesion in endothelial cells. The role of AMPK in the regulation of inflammatory signalling in adipocytes is currently poorly characterised. To address this, the effect of AMPK activation on the phosphorylation of TNF-alpha/IL-1beta and IL-6 signalling pathway intermediates was initially assessed in cultured 3T3-L1 adipocytes. Furthermore, the molecular mechanism by which AMPK elicits these effects was investigated. In addition, the effect of AMPK activation on downstream functional consequences of proinflammatory signalling in 3T3-L1 adipocytes and RAW 264.7 macrophages were examined. Finally, the effect of macrophage AMPK activation on inflammation-induced insulin resistance in 3T3-L1 adipocytes was also investigated. A769662 and infection with adenovirus expressing a constitutively active AMPK mutant suppressed IL-1beta-stimulated NFkappaB nuclear translocation in 3T3-L1 adipocytes. Conversely, this was abrogated upon adenoviral expression of a dominant negative AMPK mutant. In line with this, phosphorylation of upstream IkappaBalpha and IKK were also ameliorated upon AMPK activation. In parallel, A769662-mediated AMPK activation inhibited TNF-alpha/IL-1beta-stimulated phosphorylation of JNK, ERK1/2 and p38 MAPKs in 3T3-L1 adipocytes. Furthermore, A769662-mediated inhibition of TNF-alpha/IL-1beta proinflammatory signalling was likely to be independent of endothelial nitric oxide synthase (eNOS) activation and subsequent nitric oxide production. The target of AMPK may be downstream of TAK1, as IKK, JNK and p38 are inhibited in response to both TNF-alpha and IL-1beta; however the mechanism by which AMPK elicits these effects remains to be elucidated. A769662-mediated AMPK activation inhibited phosphorylation of IL-6-stimulated STAT3 (signal transducer and activator of transcription 3) in 3T3-L1 adipocytes independently of phosphatase action, yet A769662 was unable to inhibit constitutive Janus kinase (JAK)-mediated phosphorylation of STAT3, suggesting AMPK may inhibit JAK activity. Inhibition of mTOR was found to suppress STAT3 phosphorylation in a manner mutually exclusive with A769662 stimulation, potentially via activation of T cell protein tyrosine phosphatase (TC-PTP). Adipose tissue from AMPKalpha1-/- mice demonstrated increased basal JNK and STAT3 phosphorylation, further providing evidence for an anti-inflammatory role for AMPK in adipose tissue. In 3T3-L1 adipocytes, A769662 abrogated cytokine-stimulated MCP-1 gene expression, and secretion of chemokines IP-10 (CXCL10), KC (CXCL1) and MCP-1. Furthermore, AMPK activation reduced secretion of IL-5, MCP-1 and MIP-1alpha, but not TNF-alpha, from proinflammatory RAW 264.7 macrophages. Preliminary results indicated that chronic IL-6 and acute TNF-alpha or IL-1beta exposure suppressed insulin-stimulated glucose transport in 3T3-L1 adipocytes. Conditioned medium from activated RAW 264.7 macrophages also inhibited 3T3-L1 adipocyte insulin sensitivity; however, prior AMPK activation failed to attenuate this, potentially as a result of the presence of TNF-alpha. Overall these results suggest that activation of AMPK inhibits activation of multiple distinct proinflammatory signalling pathways in adipocytes and macrophages. AMPK activation may suppress IL-6 signalling via regulation of JAK, while the AMPK-mediated inhibition of IKK and concomitant suppression of MAPKs in response to TNF-alpha/IL-1beta suggests TAK1 as a potential AMPK target. Finally, proinflammatory stimuli induce insulin resistance in adipocytes, however whether this can be rescued by AMPK activation remains to be fully elucidated.

The AMP-activated protein kinase (AMPK) αβγ heterotrimer is a highly conserved serine/threonine protein kinase that acts as a metabolic fuel sensor and is crucial for maintaining cellular energy homeostasis. Mammalian AMPK forms complexes in a 1:1:1 ratio made of unique subunit isoform variations (α1, α2, β1, β2, γ1, γ2, γ3) that allow for 12 distinct AMPK complexes to form, where each complex is subject to a range of modifications such as phosphorylation. Each isoform exhibits distinct tissue-expression signatures, α2 and β2 are expressed in a range of tissues but display high expression in skeletal muscle and γ3 shows the highest selectivity being exclusively expressed in skeletal muscle with small amounts recently found in brown adipose tissue. The majority of direct allosteric AMPK activators bind at a hydrophobic binding pocket formed between the α and β subunits, termed the allosteric drug and metabolite site (ADaM site). AMPK is capable of stimulating glucose uptake independently of insulin signalling, where activating AMPK using ADaM site activators has been shown to improve key hallmarks of type 2 diabetes mellitus (T2DM). Despite this, recent studies show that chronically stimulating all 12 AMPK complexes (pan activation) is detrimental as it can lead to hypertrophic cardiomyopathy. This precludes pan activators from progressing to clinical trials. The current direction for the field is to develop isoform-specific activators that target AMPK expressed in select tissues. For the treatment of T2DM it is beneficial to target skeletal muscle as it is the primary site for glucose disposal, hence, α2, β2, and γ3 isoforms are the most favourable to target. This can be achieved by developing α2β2-specific ADaM site compounds or with novel drugs targeting γ3 directly. Despite this, the current knowledge on isoform-specific AMPK regulation is limited, in particular the regulation by a range of phosphorylation sites across each subunit, the functional role of an NH2-terminal extension (NTE) unique to the γ3 subunit, and the structural mechanism for the isoform specificity of the currently limited range of direct allosteric activators. Therefore, the goal of this thesis is to tackle each of these questions to ultimately aid the field in the development of isoform-specific AMPK activators. Our lab recently characterised the ADaM site activator SC4 as not only α2-selective, but also as a potent β2 activator. In Chapter 2 I perform structure/function analysis of SC4 by substituting the 2-hydroxyphenyl group with polar-substituted cyclohexene-based probes. This resulted in the formation of two compounds, MSG010 and MSG011, that do not display α2-selectivity and are hence classified as pan activators. A crystal structure of MSG011 complexed to AMPK α2β1γ1 revealed a similar binding mode to SC4. Interestingly, it highlighted the absence of an interaction that we saw in the SC4/α2β1γ1 crystal structure between the SC4 2-hydroxyphenyl group and α2K31, which may be important for directing α2-selectivity. These findings will guide future design of α2β2-selective AMPK activators. In addition, MSG010 and MSG011 will serve as important tool compounds in AMPK research as they are most potent pan activators available to date. In Chapter 3 I use a targeted mass spectrometry approach to generated precise phosphorylation stoichiometry profiles of 18 phosphorylation sites across all 12 AMPK complexes. This uncovered important isoform-specific differences, particularly in the basal level of βS108 phosphorylation which is located in the ADaM site and dictates the potency of most activators. Mechanistic target of rapamycin complex 1 (mTORC1) is a nutrient-sensitive protein kinases that governs cell growth and proliferation. It has been known for some time that AMPK inhibits mTORC1 activity by phosphorylation, and our lab recently discovered AMPK is directly phosphorylated by mTORC1 on α2S345 to suppress activity, forming a fundamental negative feedback loop. I found seven phosphorylation sites on AMPK were sensitive to pharmacological mTORC1 inhibition, including four in the unique γ2-NTE and α2S377 which is located in the nucleotide-sensing motif. In particular, β1S182 and β2S184 were found to be mTORC1 substrates in vitro and near-maximally phosphorylated under cellular growth conditions. Lastly, I identify two phosphorylation sites on the γ3-NTE. Despite γ3 being a promising therapeutic target, it is yet to undergo rigorous biochemical characterisation leaving it without a solved crystal or cryoEM structure and the function of its NTE remains unknown. In Chapter 4 I discover that removal of the γ3-NTE results in elevated AMPK activity, suggesting it contains an autoinhibitory region. Furthermore, I detected a direct interaction between the γ3-NTE and the α2 kinase domain which may partly explain the mechanism for AMPK autoinhibition. I characterise one of the γ3-NTE phosphorylation sites I discovered in Chapter 3, γ3S14, identifying it as an autophosphorylation site in vitro and in cellulo with the potential for alternative upstream kinases in cellulo.

Many organisms can execute a dormant state or diapause to survive harsh environmental conditions for extended durations. When Caenorhabditis elegans larvae enter the dauer diapause, they completely arrest development and feeding, but remain active and motile, yet become stress-resistant and extremely long-lived. Entry into dauer is associated with a reduction in insulin-like signalling, the establishment of a generalized cell cycle arrest, the accumulation of nutritive resources and a concomitant global change in metabolism. The precise molecular and physiological processes that induce cell cycle quiescence and enable long-term survival in the absence of caloric intake however remain largely unknown. I show here that the C. elegans orthologs of PTEN, STRAD, LKB1 and AMPK (α1, α2, β1, β2 subunits) cooperate to establish quiescence in the germline stem cell population during dauer development. Interestingly, germline mutations in LKB1 cause predisposition to cancer in humans, while mutations in STRAD or AMPK subunits do not seem to cause cancer. In C. elegans, LKB1 also regulates embryonic polarity, while STRAD and AMPK are dispensable for this process. Thus, my data suggest that LKB1/STRAD regulate cell growth/proliferation through AMPK, while LKB1 also acts independently to regulate polarity, and that this function may be critical for tumor suppression in human. In addition, I show that C. elegans larvae that lack LKB1/AMPK signalling rapidly consume their stored energy and prematurely expire following vital organ failure. This signalling pathway acts in adipose-like tissues to downregulate triglyceride hydrolysis so that these fat reserves are rationed to last the
Plusieurs organismes peuvent entrer en dormance, ou diapause, pour survivre à des conditions environnementales précaires pour une durée prolongée. Lorsque des larves de Caenorhabditis elegans entrent en diapause dauer, elles cessent complètement de se développer ainsi que de se nourrir, cependant elles demeurent actives et mobiles, tout en acquérant une résistance au stress et une longévité extrême. L'entrée en stade dauer est accompagnée d'une réduction de signalisation par l'insuline, de l'établissement d'un arrêt généralisé du cycle cellulaire, de l'accumulation de ressources nutritives et d'un changement global au niveau du métabolisme. Les processus physiologiques et moléculaires précis qui induisent la quiescence cellulaire et permettent la survie prolongée en l'absence de tout apport calorique, demeurent toutefois essentiellement inconnus. Je montre ici que les orthologues de PTEN, STRAD, LKB1 et de AMPK (sous-unités α1, α2, β1, β2) chez C. elegans coopèrent dans l'optique d'établir la quiescence cellulaire dans la population de cellules germinales souches durant le développement de la larve dauer. Il est intéressant de préciser que chez l'humain, une mutation de LKB1 dans la lignée germinale provoque une prédisposition au cancer, tandis qu'une mutation dans une sous-unité de STRAD ou d'AMPK ne semble pas causer de cancer. Chez C. elegans, LKB1 régule aussi la polarité embryonnaire, tandis que STRAD et AMPK sont dispensables pour ce processus. Donc, mes données suggèrent que LKB1/STRAD régulent la croissance et la prolifération des cellules à travers AMPK, tandis que LKB1 fonctionne aussi indépendamment pour contrôler

Mitochondria are key generators of cellular ATP, vital to complex life. Historically, mitochondrial generation of reactive oxygen species (ROS) was considered to be an unregulated process, produced by dysfunctional mitochondria. More recently, mitochondrial ROS generated by complex I, particularly by the process of reverse electron transfer (RET), has emerged as a potentially biologically relevant signal that is tightly-regulated and dependent on mitochondrial status. ROS production by RET is reported to play a role in the innate immune response and lifespan extension in fruit flies. One way in which mitochondrial ROS may behave as a signal is by altering the activity of AMP-activated protein kinase (AMPK), a key metabolic sensor and regulator of cell metabolism, which is activated when cellular ATP levels decrease during energy demand. Mitochondria can signal to AMPK via the magnitude of the cellular ATP/AMP and ATP/ADP ratios, which alter in response to mitochondrial function. Our view is mitochondria may also signal to AMPK via ROS. Important studies have helped to clarify the role of exogenous or cytosolic ROS in AMPK regulation. However, the effects of mitochondrial ROS on AMPK activity, specifically that generated by complex I, remain unclear and is the main focus of this thesis. I characterized the effects of exogenous H2O2 on cellular AMPK activity, ATP/ADP ratios and cellular redox state in a cell model. I then compounded this with selective mitochondria generated ROS by the mitochondria-targeted redox-cycler, MitoParaquat (MPQ). AMPK activity appeared to correlate with decreasing cell ATP/ADP ratios, indicating that both sources of ROS primarily activate AMPK in an AMP/ADP-dependent mechanism. In parallel, I developed an approach for analyzing the redox state of candidate proteins, an important step in determining if a protein is directly regulated by ROS. I also initiated development of a cell model for studying the downstream effects of mitochondrial ROS production by RET, by expressing alternative respiratory enzymes in a mammalian cell line.

Considerable recent evidence supports the role of AMP-activated protein kinase (AMPK) as an anti-inflammatory mediator, yet the mechanisms of its anti-inflammatory actions are only starting to be unravelled. Inappropriate cytokine stimulated Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signalling is a key feature of many pro-inflammatory events, including atherogenesis. Previous unpublished studies in our group have investigated whether AMPK modifies cytokine stimulation of JAK-STAT signalling in HUVECs. These preliminary investigations demonstrated that pre-treatment of HUVECs with AMPK activator, A769662, significantly inhibits both sIL-6Rα/IL-6 and IFN-α stimulation of STAT3 Tyr705 phosphorylation in HUVECs. IFN-α activates STATs via an IFNα/β receptor 1 (IFNAR1/IFNAR2) complex which is distinct from the sIL-6Rα/IL-6/gp130 complex. The studies in this thesis therefore tested the hypothesis that AMPK was exerting its inhibitory effects at one or more common signalling loci downstream of IFNAR1/IFNAR2 and gp130 at a post-receptor level. First, it was investigated whether AMPK exerts its inhibitory effects on JAK-STAT signalling via a known regulator of JAK or STAT, or an AMPK downstream target known to either directly or indirectly impact on JAK-STAT signalling. A combination of genetic and pharmacological approaches was utilised to assess the role of each of the following AMPK targets: TC-PTP, SHP2, eNOS, PKCλ, SIRT1, CPT1 and mTOR. It was demonstrated that activation of AMPK in HUVECs inhibited sIL-6Rα/IL-6 stimulated STAT3 Tyr705 phosphorylation via a mechanism independent of TC-PTP, eNOS, PKC, SIRT1 and mTOR. Furthermore, inhibition of mTOR and eNOS reduced sIL-6Rα/IL-6 stimulated STAT3 Tyr705 phosphorylation, independent of AMPK activation by A769662. Next, it was investigated whether AMPK acts directly on a signalling component of the JAK-STAT pathway. Specifically, it was hypothesised that AMPK could directly phosphorylate serine or threonine residues within JAK to inhibit IL-6 signalling. siRNA-mediated downregulation of JAK isoforms demonstrated that IL-6 induced STAT3 Tyr705 phosphorylation predominantly via JAK1 in human umbilical vein endothelial cells (HUVECs). In vitro kinase assays of JAK1-derived peptides demonstrated that AMPK can directly phosphorylate two residues, Ser515 and Ser518, within the JAK1 SH2 domain. Subsequently, a GST- 14-3-3 pull down assay of cell lysates produced from A769662 treated JAK1- defcient U4C cells transiently expressing either wild type or S515A/S518A double mutant JAK1 demonstrated that pharmacological activation of AMPK promotes 14-3-3 binding of JAK1 via a mechanism requiring Ser515 and Ser518. Furthermore, mutation of Ser515 and Ser518 abolishes the ability of AMPK to inhibit JAK-STAT signalling by an IL-6 trans-signalling complex and from a constitutively active Val658Phe-mutated JAK1. In this study it is proposed that AMPK phosphorylation of JAK1 at Ser515 and Ser518 inhibits IL-6 stimulated JAK1 phosphorylating STAT3 by interfering with the ability of JAK1 to interact and phosphorylate the GP130 receptor and /or STAT3 and STAT1. Therefore, AMPK phosphorylation of JAK1 could potentially be a novel regulatory mechanism that could be developed as a therapy for treating chronic inflammatory diseases such as atherosclerosis.

Cell signalling determines physiological responses to many cellular stimuli and environmental changes. The transforming growth factor-beta (TGFβ)/bone morphogenetic protein (BMP) signalling pathways begin by binding of ligand to the heterodimeric receptor complex, followed by activation of Smads that translocate to the nucleus to regulate transcription of genes that further mediate cellular physiology. The TGFβ/BMP pathways are very important for proper tissue development and homeostasis, thus precise spatial and temporal regulation of the signalling pathway is required and achieved by many positive and negative signalling regulators. This thesis work identified the liver kinase B1 (LKB1) pathway as a negative regulator of TGFβ/BMP signalling pathways. In the first paper, we established LKB1 as a negative regulator of TGFβ signalling and TGFβ-induced epithelial to mesenchymal transition (EMT). LKB1 impairs Smad4 binding capacity to DNA leading to suppressed TGFβ-activated gene transcription. The second paper describes further the mechanism of LKB1 negative regulation on BMP signalling, by mediating BMP type I receptor degradation resulting in inhibition of BMP-induced cell differentiation. Downstream of LKB1, salt inducible kinase 1 (SIK1) is a TGFβ target gene and its expression is up-regulated by Smad2/3/4-mediated gene transcription. The third paper elucidates the mechanism of SIK1 transcriptional induction via an enhancer element located 3’ of the gene and SIK1-mediated type I TGFβ receptor degradation, which requires the activity of Smad7 and of the Smurf2 ubiquitin ligase. The fourth manuscript finds sucrose non-fermenting (SNF) 1-like kinase 2 (NUAK2) as another TGFβ target gene and its up-regulation results in modification of the mammalian target of rapamycin (mTOR) pathway that controls protein synthesis. NUAK2 cooperates with LKB1 leading to Raptor phosphorylation and inhibition of mTOR-mediated protein synthesis. Collectively, this thesis work has provided a functional link between two important signalling pathways, the metabolic LKB1 pathway and TGFβ/BMP pathway.

In recent years there has been a considerable increase in human obesity levels with an associated increase in the incidence of type 2 diabetes mellitus (T2DM), a disorder of glucose metabolism characterised by insulin resistance. It is unclear why obesity and insulin resistance should frequently exist comorbidly but the current prevalence of polypharmacy suggests the underlying mechanisms may be multifactorial in origin. This project has highlighted plant compounds and extracts with: Insulin-like properties in cell culture experiments in that they induce phosphorylation and therefore inactivation of the transcription factor FOXO1a, which is a major downstream effector insulin and Properties similar to the T2D drugs metformin and pioglitazone, which both activate AMOK signalling and reduce phosphorylation of the ribosomal protein S6. The project began with an analysis of plant extracts which can mediate intracellular cell signalling effects on FOXO1a and AMPK. The pilot data established that one extract (grape seed, GSE) induces regulation of AMPK and FOXO1a much more readily than another (pine bark, PBE). GSE and PBE were subjected to multiple fractionation methods and mass spectrometry to learn more about the active agent(s) in the extracts. Another chapter adopted a candidate-approach, investigating effects of the plant compound gallic acid (GA) on AMPK. Although GA cannot explain the effects of GSE on AMPK, the availability of a variety of analogues of GA allowed investigation of structural requirements for cell responses. Two more extracts, cranberry and lingonberry, were then investigated using the techniques established earlier with GSE and PBE. These studies discovered that fractions containing B-type linkages were more effective at phosphorylating of FOXO1a than those containing A-type linkages, suggesting B-type linkages may be required for these effects. Metformin itself is a synthetic analogue of a plant compound and metformin analogues known as diguanides were synthesised and structure/activity relationships were assessed. Diguanides and biguanides were found ti induce similar responses but diguanides were much more toxic than biguanides, suggesting they may be less specific in their mechanism of action, or alternatively that they have different intramitochondrial targets. The aims of this project were 1. To extract polyphenolic compounds from grape seed, pine bark and berries using chromatography techniques and to characterise these compound using liquid chromatography/mass spectrometry 2. To exclude or confirm insulin-like or metformin-like properties by application to cell culture models to assess effects on regulation of glucose and energy homeostasis by measuring phosphorylation of the transcription factor FOXO1a, AMPK and the ribosomal protein S6.

The autophagy pathway is an essential component of the innate immune response, capable of rapidly targeting intracellular bacteria, which are subsequently degraded by lysosomal enzymes. Recent work has begun to elucidate the regulatory signalling for autophagy induction in response to pathogenic bacteria. However, the initial signalling regulating autophagy induction in response to the detection of pathogens remains largely unclear. Here we report that AMPK, an important upstream activator of the autophagy pathway, is rapidly stimulated upon detection of pathogenic bacteria, prior to bacterial invasion. Bacterial recognition is initially achieved through detection of outer membrane vesicles (OMVs). Additionally, we show that AMPK signalling relieves mTORC1-mediated repression of the autophagy pathway in response to Salmonella infection, positioning the cell for a rapid induction of autophagy. Surprisingly, we found that the activation of AMPK and inhibition of mTORC1 in response to extracellular Salmonella are not accompanied by an induction of bulk autophagy. However, upon Salmonella invasion AMPK signalling is required for efficient and selective targeting of bacteria-containing vesicles by the autophagy pathway through activation of pro-autophagic kinase complexes. Collectively, these results demonstrate a key role for AMPK signalling in coordinating the rapid autophagic response prior to invasion of pathogenic bacteria.

Ca2+ is an important second messenger which modulates a variety of signalling pathways in both excitable and non-excitable cells. In the CNS, Ca2+ plays an important role in neurons both physiologically and pathologically. Ca2+ influx following synaptic activity, is important in development, plasticity, redox balance, as well as in neuroprotection, largely through activation of pro-survival pathways downstream of synaptic NMDAR activation, including upregulation of antioxidant defences. However, excessive Ca2+ influx in neurons leads to neuronal damage and excitotoxicity, in which mitochondrial Ca2+ uptake through the mitochondrial Ca2+ uniporter (Mcu) resulting in mitochondrial dysfunction is a key player. Excitotoxicity occurs due to glutamate efflux from astrocytes following stroke, traumatic brain injury and in chronic neurodegenerative diseases, leading to excessive neuronal NMDAR activation and triggering of its downstream pro-death pathways. This thesis focuses on understanding the pro-survival and pro-death effects of signalling pathways activated by Ca2+ in neurons, as well as the potential effect of neuronal synaptic activity on influencing neuroprotective gene transcription in astrocytes. I investigated the role of AMPK, a master regulator of metabolism, in NMDA excitotoxicity in cortical neurons as a potential downstream effector of Mcu-dependent excitotoxic death; and found the deletion of AMPKα1/2 to be neuroprotective against NMDA-mediated excitotoxicity, however I found AMPK activation to be independent of Mcu. I also investigated the expression pattern of Mcu and other mitochondrial calcium regulatory genes (MCRGs), and found MCRGs to be differentially expressed in different neural cells (primary neurons vs astrocytes), and neuronal subtypes (CA1 vs CA3 region of the hippocampus), suggesting differing dependence on the various MCRGs in mitochondrial Ca2+ handling in these cell types. A better functional understanding of these genes will allow for investigation of their importance in mitochondrial Ca2+ handling, including their potential role in excitotoxicity. I next investigated the neuroprotective effects of synaptic activity induced Ca2+ influx, focusing on antioxidant target genes of Nrf2, a transcription factor and major regulator of antioxidant genes. I found that unlike astrocytes, neurons express very low levels of Nrf2. However, synaptic activity increased the expression of several Nrf2 target genes in neurons, independently of astrocytes and Nrf2. Additionally, I found no effect of synaptic activity on increasing Nrf2 protein levels, despite previous reports in literature of Nrf2 pathway activation following synaptic activity. Finally, using RNA-seq I identified a list of genes strongly upregulated by a known Nrf2 activator in astrocytes, and found no evidence that neuronal activity triggers expression of these genes independently of neurons, providing further evidence that neuronal activity does not activate the Nrf2 pathway in astrocytes. This suggests that synaptic activity via pathways activated by Ca2+ signalling provides neurons with cell-autonomous antioxidant defences, independently of Nrf2; thus providing a distinct pathway for antioxidant defences in neurons from the Nrf2 pathway, which is activated in astrocytes providing neurons with non-cell autonomous antioxidant support. These results give us further insight into the mechanisms that underlie synaptic and non-synaptic Ca2+ signalling pathways mediating neuronal survival and death, which could help in identifying therapeutic targets to combat excitotoxicity and oxidative stress in various neurological diseases.

Ventilatory drive is mediated by respiratory central pattern generators that are located in the brainstem, which are continuously modulated by specialised peripheral and central chemoreceptors to adjust ventilatory patterns according to changes in arterial PO2. These specialised oxygen-sensing chemoreceptors are activated in response to acute reductions in arterial PO2 and ultimately trigger a respiratory response that acts to restore oxygen-levels. However, the molecular mechanism by which mammals are able to regulate their breathing pattern in such a manner during hypoxia remains controversial. Therefore, the studies performed in this thesis aimed to investigate the possibility that this process may be mediated by the liver kinase B 1 (LKB1)/ AMP-activated protein kinase (AMPK) signalling pathway, which is central to cellular adaptations to metabolic stress. This first involved the development of transgenic mice in which Lkb1 or AMPK were deleted. Global knockout of Lkb1 (Sakamoto, 2006) or AMPK activity (Viollet et al., 2009) are embryonic lethal. Thus, the Cre/loxP system was used to develop transgenic mice that had either Lkb1 or both isoforms of the AMPK catalytic α- subunit (α1 and α2) conditionally knocked out in catecholaminergic cells (including therein hypoxia-activated cells of the brainstem and carotid body) by driving Cre expression through a tyrosine-hydroxylase-specific promoter region. The consequent effects on the ventilatory response to hypoxia were then examined using unrestrained whole-body plethysmography. This demonstrated that, in contrast to the hyperventilation evoked in controls, increased ventilation was virtually abolished in the Lkb1 and AMPK α1 and α2 double knockouts during hypoxia. Both knockout mice also exhibited periods of hypoventilation with frequent apnoeas during hypoxia. Additionally, studies on single AMPK α1 and AMPK α2 knockouts identified that the ventilatory dysfunction in AMPK α1 and α2 double knockouts was primarily caused by AMPK α1 deletion. In contrast, the severe ventilatory abnormalities exhibited during hypoxia following the deletion of Lkb1 and AMPK in catecholaminergic cells were mostly reversed upon exposure of mice to hypoxia with hypercapnia. Also, the ventilatory response to hypercapnia alone was without any major effect as a result of Lkb1 deletion or the dual-deletion of AMPK α1 and α2 catalytic subunits in catecholaminergic cells. This thesis therefore demonstrates, for the first time, that the LKB1-AMPK signalling pathway is key to respiratory adaptations during hypoxia, by regulating catecholaminergic oxygen-sensing cells, thus protecting against hypoventilation and apnoeas. The LKB1-AMPK signaling pathway can thereby determine oxygen and energy supply at both a cellular and whole-body level.

Hexavalent Chromium (Cr(VI) induces malignant cell transformation in normal bronchial epithelial (BEAS-2B) cells. Cr(VI)-transformed cells exhibit increased level of antioxidants, are resistant to apoptosis, and are tumorigenic. RNAseq analysis in Cr(VI)-transformed cells showed that expression of transcripts associated with mitochondrial oxidative phosphorylation is reduced, and the expression of transcripts associated with pentose phosphate pathway, glycolysis, and glutaminolysis are increased. Sirtuin-3 (SIRT3) regulates mitochondrial adaptive response to stress, such as metabolic reprogramming and antioxidant defense mechanisms. SIRT3 was upregulated and it positively regulated mitochondrial oxidative phosphorylation in Cr(VI)-transformed cells. Our results suggests that SIRT3 plays an important role in mitophagy deficiency of Cr(VI)-transformed cells. Furthermore, SIRT3 knockdown suppressed cell proliferation and tumorigenesis of Cr(VI)-transformed cells. Nrf2 is a transcription factor that regulates oxidative stress response. This study investigated the role of Nrf2 in regulating metabolic reprogramming in Cr(VI)-transformed cells. We observed that in Cr(VI)-transformed cells p-AMPKthr172 was increased, when compared to normal BEAS-2B cells. Additionally, Nrf2 knockdown reduced p-AMPKthr172. Our results suggest that Nrf2 regulated glycolytic shift via AMPK regulation of PFK1/PFK2 pathway. Furthermore, our results showed that Nrf2 constitutive activation in Cr(VI-transformed cells increased cell proliferation and tumorigenesis. Overall this dissertation demonstrated that Cr(VI)-transformed cells undergo metabolic reprogramming. We demonstrated that Nrf2 constitutive activation plays decisive role on metabolic reprogramming induction, and SIRT3 activation contributing to increased cancer cell proliferation and tumorigenesis.

Chapter 1 Provides an overview of drug discovery with particular emphasis on library selection and hit identification methods using virtual based approaches. Chapter 2 Gives an outline of the bone morphogenetic protein (BMP) signalling pathway and literature BMP pathway modulators. The association between the regulation of BMP pathway and cardiomyogenesis is also described. Chapter 3 Describes the use of ligand based virtual screening to discover small molecule activators of the BMP signalling pathway. A robust cell based BMP responsive gene activity reporter assay was developed to test the libraries of small molecules selected. Hit molecules from the screen were synthesised to validate activity. It was found that a group of known histone deacetylase (HDAC) inhibitors displayed most promising activity. These were evaluated in a secondary assay measuring the expression of two BMP pathway regulated genes, hepcidin and Id1, using reverse transcription polymerase chain reaction (RT-PCR). 188 was discovered to increase expression of both BMP-responsive genes. Chapter 4 Provides an overview of existing cannabinoid receptor (CBR) modulating molecules and their connection to progression of atherosclerosis. Chapter 5 Outlines the identification and optimisation of selective small molecule agonists acting at the cannabinoid 2 receptor (CB₂R). Ligand based virtual screen was undertaken and promising hits were synthesised to allow structure activity relationship (SAR) to be developed around the hit molecule providing further information of the functional groups tolerated at the active site. Subsequent studies led to the investigation and optimisation of physicochemical properties around 236 leading to the development of a suitable compound for in vivo testing. Finally, a CB₂R selective compound with favourable physicochemical properties was evaluated in vivo in a murine inflammation model and displayed reduced recruitment of monocytes to the site of inflammation.

Recent advances in both satellite and terrestrial mobile communications technologies are now leading to the realisation of the dream of the global personal communications within a few years. Satellite systems, as a complement to terrestrial cellular systems, are introduced into the future Personal Communication Networks (S-PCN) to provide global coverage and to allow global roaming. The inter-working and the integration between the satellite and the terrestrial cellular systems (e.g. GSM system) are the key issues in developing the network architecture and designing the control functions and signalling protocols of satellite systems. This thesis focuses on the design of a satellite signalling control system. The coverage and link properties of ICO10 and LE066 satellite constellations, the representatives of low earth orbit (LEO) and medium earth orbit (MEO) satellite systems, are considered. A satellite specific network architecture is proposed to accommodate the requirements of satellite dynamics and resource control function. The physical layer of satellite signalling links are designed to cope with the specific features of LEO or MEO satellite air-interfaces. In order to overcome problems specific to LEO or MEO satellite systems and to provide call set-up control function, three important signalling protocols are proposed for the S-PCNs. The priority based fast access scheme is designed for the satellite random access channel allowing low access delay for the call set-up related access packets, even when the channel load is high. The satellite diversity based paging approach is proposed to optimize the paging performance. The modified selective re-transmission (M-SRT) and Go-Back-N (M-GBN) protocols are proposed to cope with the transaction type transmission on the dedicated control channel. Simulation results have shown siginficant improvement of the M-SRT and M-GBN protocols in call setup delay. Two protocols are also compared in the aspects of implementation complexity and call set-up performance. Finally, the integration scenarios between satellite and GSM system have been examined for S-PCN in the call handling related functions and associated signalling protocols. The GSM higher layer signalling protocols are tailored to provide the call control related functions. The optimum integration scenario is derived under the criterion of minimum modifications to the GSM higher layer signalling protocols and minimum complexities of the control functions.

RESUMO: O corpo carotídeo (CB) é um pequeno órgão sensível a variações na PaO2, PaCO2 e pH. As células tipo I (células glómicas) do corpo carotídeo, as unidades sensoriais deste órgão, libertam neurotransmissores em resposta às variações dos gases arteriais. Estes neurotransmissores atuam quer em recetores pré-sinápticos, localizados nas células tipo I, quer em recetores póssinápticos, localizados nas terminações do nervo do seio carotídeo, ou em ambos. A activação dos recetores pré-sinápticos modula a atividade do corpo carotídeo, enquanto que, a activação dos recetores pós-sinápticos, de carater excitatório, desencadeia um aumento da frequência de descarga das fibras do CSN, com subsequente despolarização dos neurónios do gânglio petroso, e posterior despolarização de um grupo específico de neurónios do centro respiratório central, desencadeando, como resposta final, hiperventilação. Estes recetores pré- e pós-sinápticos podem ser classificados em ionotrópicos ou metabotrópicos, estando os últimos acoplados a adenilatos ciclases transmembranares (tmAC). O mecanismo exato pelo qual as variações dos gases arteriais são detetadas pelo CB não se encontra ainda completamente elucidado, mas tem sido sugerido que alterações nos níveis de cAMP estejam associadas ao mecanismo de deteção de variações de O2 e CO2. Os níveis de cAMP podem ser regulados através da sua via de síntese, mediada por dois tipos de adenilatos ciclases: tmAC sensível aos eurotransmissores e adenilato ciclase solúvel (sAC)sensível a variações de HCO3/CO2, e pela sua via de degradação mediada por fosfodiesterases. A via de degradação do cAMP pode ser manipulada farmacologicamente, funcionando enquanto alvo terapêutico para o tratamento de patologias do foro respiratório (e.g. asma, hipertensão pulmonar, doença pulmonar obstructiva crónica e apneia do sono), que induzem um aumento da actividade do CB.O trabalho descrito nesta dissertação partiu da hipótese de que a actividade do CB é manipulada por fármacos, que interferem com a via de sinalização do cAMP, tendo sido nosso objectivo geral, investigar o papel do cAMP na quimiotransdução do CB de rato, e determinar se a actividade dos enzimas responsáveis pela via de sinalização do cAMP é ou não regulada por variações de O2/CO2. Assim, a relevância deste trabalho é a de estudar e identificar possíveis alvos moleculares (sAC, isoformas de tmAC e PDE) com potencial para serem usados no tratamento de patologias relacionadas com o controlo respiratório. A primeira parte do presente trabalho, centrou-se na caracterização farmacológica da PDE4 no CB e em tecidos não quimiorecetores (e.g. gânglio cervical superior e artérias carótidas), e na observação do efeito de hipóxia aguda na acumulação dos níveis de cAMP, induzidos pelos inibidores de PDE, nestes tecidos. A quantificação de cAMP foi efectuada por técnica imunoenzimática (EIA), tendo sido elaboradas curvas de dose-resposta para os efeitos de inibidores, não específicos (IBMX) e específicos para a PDE2 e PDE4 (EHNA, Rolipram e Ro 20-1724), nos níveis de cAMP acumulados, em situações de normóxia (20%O2/5%CO2) e hipóxia (5%O2/5%CO2). A caracterização das PDE no gânglio cervical superior foi aprofundada, utilizando-se a técnica de transferência de energia de ressonância por fluorescência (FRET) em culturas primárias de neurónios, na presença de inibidores não específicos (IBMX) e específicos para a PDE3 e PDE4 (milrinone e rolipram, respetivamente). Foram igualmente estudadas, através de RT-qPCR, as alterações na expressão de PDE3A-B e PDE4A-D, no gânglio cervical superior, em resposta a diferentes percentagens de oxigénio. Na segunda parte do trabalho investigou-se a via de síntese do cAMP no CB em resposta a variações na concentração de HCO3/CO2. Em concreto, o protocolo experimental centrou-se na caracterização da sAC, dado que a sua actividade é regulada por variações de HCO3/CO2. A caracterização da expressão e regulação da sAC, em resposta a variações de HCO3/CO2 ,foi efectuada no CB e em tecidos não quimioreceptores periféricos (e.g. gânglio cervical superior, petroso e nodoso) por qRT-PCR. A actividade deste enzima foi caracterizada indirectamente através da quantificação dos níveis de cAMP (quantificação por EIA), induzidos por diferentes concentrações de HCO3/CO2, na presença de MDL-12,33-A, um inibidore da tmAC. A expressão das isoformas da tmAC no CB e gânglio petroso foi determinada por RT-qPCR. Adicionalmente, estudámos a contribuição relativa da tmAC e sAC no mecanismo de sensibilidade ao CO2 no CB. Para o efeito foram estudadas as alterações: 1) nos níveis de cAMP (quantificado por EIA) na presença de diferentes concentrações de HCO3/CO2 e ao longo do tempo (5-30 min); 2) na ativação da proteína cinase A (PKA, FRET baseado em sensores) em células tipo I do CB; e 3) na frequência de descarga do CSN (registos) na presença e ausência de ativadores e inibidores da sAC,tmAC e PKA. Por último, foi caracterizada a expressão e actividade da sAC nos quimioreceptors centrais (locus ceruleus, rafe e medula ventro-lateral) através de técnicas de RT-qPCR e EIA. A expressão das isoformas da tmAC foi aprofundada no locus coeruleus através de RT-qPCR. Por fim, comparámos a contribuição da tmAC e sAC nos níveis de cAMP no locus coeruleus em condições de normocapnia e hipercapnia.O nosso trabalho teve os seguintes resultados principais: 1) PDE4 está funcional no corpo carotídeo, artérias carótidas e gânglio cervical superior de rato, embora a PDE2 só se encontre funcional neste último; 2) Os efeitos dos inibidores de PDE nos níveis de acumulação de cAMP foram exacerbados em situações de hipóxia aguda no CB e artérias carótidas, mas foram atenuados no gânglio cervical superior; 3) No gânglio cervical superior, diferentes tipos de células apresentaram uma caracterização específica de PDEs, sugerindo uma subpopulação de células neste gânglio com funções fisiológicas distintas; 4) Embora todas as isoformas de PDE4 e PDE3 estivessem presentes no gânglio, a PDE3a, PDE4b e a PDE4d foram as isoformas mais expressas. Por outro lado, incubações de gânglio cervical superior, em diferentes percentagens de oxigénio, não alteraram (não regularam) significativamente a expressão das diferentes isoformas de PDE neste órgão; 5) a sAC encontra-se expressa e funcional no CB e nos quimiorecetores centrais estudados (locus coeruleus, rafe e medula ventrolateral). A sAC apresenta maior expressão no CB comparativamente aos restantes orgãos estudados, exceptuando os testículos, orgão controlo. Variações de HCO3/CO2 de 0/0 para 24/5 aumentaram os níveis de cAMP no CB e quimiorecetores centrais, tendo sido o aumento mais significativo observado no CB. Concentrações acima dos 24mM HCO3/5%CO2 não induziram alterações nos níveis de cAMP, sugerindo que a actividade da sAC se encontra saturada em condições fisiológicas (normocapnia) e que este enzima não desempenha qualquer papel na deteção de situações de hipercapnia; 6) No CB, a expressão das isoformas tmAC1, tmAC4, tmAC6 e tmAC9 é mais elevada comparativamente à expressão da sAC; 7) Utilizamos diferentes inibidores da tmAC (MDL 12-330A, 500μM, 2’5’-ddADO, 30-300μM, SQ 22536, 200μM) e da sAC (KH7, 10-100μM) para estudar a contribuição relativa destes enzimas na acumulação do cAMP no CB. Tanto a tmAC como a sAC contribuem para a acumulação dos níveis de cAMP em condições de hipercapnia. Contudo, existe um maior efeito destes inibidores nas condições de 12 mM HCO3/2.5%CO2 do que em condições de normocapnia e hipercapnia, sugerindo um papel relevante destes enzimas na atividade do CB em situações de hipocapnia; 8) Não se observaram variações nos níveis de cAMP em resposta a diferentes concentrações de HCO3/CO2 ao longo do tempo (5-30 min). O efeito inibitório induzido por ddADO e KH7 foi sobreponível após 5 ou 30 minutos de incubação em todas as concentrações de HCO3/CO2 estudadas; 9) Por último, verificou-se um aumento na frequência da descarga do nervo do seio carotídeo entre as condições de normocapnia e hipercapnia acídica. Ao contrário do KH7 (10μM), o 2’5’-ddADO reduziu significativamente a frequência de descarga do nervo, quer em condições de normocapnia quer de hipercapnia acídica. Contudo, não se verificou aumento na frequência de descarga do nervo entre normocapnia e hipercapnia isohídrica, sugerindo que a sensibilidade à hipercapnia no CB é mediada por variações de pH. Em conclusão, os resultados decorrentes deste trabalho permitiram demonstrar que, embora os enzimas que medeiam a via de sinalização do cAMP possam ser bons alvos terapêuticos em condições particulares, a sua actividade não é específica para o CB. Os resultados sugerem ainda que o cAMP não é um mediador específico da transdução à hipercapnia neste orgão. Contudo, os nossos resultados demonstraram que os níveis de cAMP são mais elevados em condições fisiológicas, o que sugere que o cAMP possa ter uma função homeostática neste orgão. Por último, o presente trabalho demonstrou que os aumentos de cAMP descritos por outros em condições de hipercapnia, não são observáveis quando o pH se encontra controlado. ------------------ ABSTRACT: The work presented in this dissertation was aimed to establish how specific is cAMP-signaling pathways in the CB mainly in different CO2 conditions and how O2 concentrations alter/drives the manipulation of cAMP signaling in the CB. The experimental studies included in this thesis sought to investigate the role of cAMP in the rat CB chemotransduction mechanisms and to determine whether the enzymes that participate in cAMP signal transduction in the CB are regulated by O2/CO2. We characterized the enzymes involved in the cAMP-signaling pathway in the CB (sAC, tmAC, PDE) under different O2/CO2 conditions. Our results demonstrated that many of these enzymes are involved in CO2/O2 sensing and while they may be useful in treating conditions with alterations in CO2/O2 sensing,they will not be specific to chemoreception within the CB: 1) PDE4 is ubiquitously expressed in CB and non-chemoreceptor related tissues and their affinity to inhibitors change with O2 tensions in both CB and carotid arteries, and 2) sAC and tmAC are expressed in peripheral and central chemo- and non-chemoreceptor tissues and their effect on cAMP levels do not change between normocapnic and isohydric hypercapnic conditions. Our results provide evidence against a specific role of cAMP as a mediator for O2 and CO2 chemotransduction in the rat CB and emphasized the role of pH in CO2 sensitivity of the CB. Furthermore, our results demonstrate that cAMP levels are maintained higher under physiological conditions, supporting recent finding from our lab, which all together suggests that cAMP has a homeostatic function in this organ.

Introduction: Van Gogh-Like 2 (VANGL2) is a scaffolding planar cell polarity protein involved in non-canonical Wnt signalling. It has been shown to have crucial roles in regulating epithelial development and homeostasis. Moreover, VANGL2 has been implicated in human cancers, with increased expression and copy number amplification seen in several cancer contexts. Many related components within this pathway have also been linked to cancer development, with VANGL2 expression known to regulate factors involved in cell migration and extracellular matrix (ECM) remodelling in cell lines. These cellular processes tend to be erroneously activated in cancer. VANGL2 is known to inhibit the classical driver pathway of colorectal cancer (CRC), canonical, or β- catenin dependant, Wnt signalling, in CRC cell lines. The aim of this thesis is to determine the expression of VANGL2 in CRC, and to investigate how VANGL2 may act to regulate intestinal homeostasis and disease. Methods: Transcriptional verification of VANGL2 expression in the mouse intestine was carried out by quantitative Real Time-Polymerase Chain Reaction (qRT-PCR), and transcripts localised within the murine colon using RNA-In Situ Hybridisation (RNAISH). Expression and localisation of the VANGL2 protein and related non-canonical Wnt signalling components was confirmed using immuno-histochemistry (IHC). Furthermore, using a combination of human Tissue Micro-Array (TMA), transcriptional data and genomic data, we determined an association between VANGL2 on tumour grade and disease-free survival. To functionally validate the effects of VANGL2 on colorectal biology, we used a model in which VANGL2 is selectively deleted from the colonic epithelium using Villin-CreERT Vangl2flox mouse lines. Using a combination of molecular biology methods, we identified the ECM as differentially regulated following VANGL2 modulation. To test the role of VANGL2 in colorectal cancer, we used a murine colorectal cancer model in which adenomatous polyposis coli (APC) is deleted from colonic epithelium resulting in the formation of cancer concurrently with deletion of Vangl2. We evaluated survival of these mice as well as tumour number and size. Tumour tissue was analysed using IHC, qRT-PCR and 3-Dimensional organoid culture. Results: Within this thesis I have illustrated that the murine colonic epithelium expresses Vangl2, and other components known to interact with VANGL2 including Vangl1, Wnt5A, and Protein Tyrosine Kinase 7 (Ptk7). I have also shown that VANGL2 is expressed within the human colonic epithelium. I go on to show that 9.2% of human CRC possesses VANGL2 transcriptional alterations which correlates with a worsened disease-free survival (DFS) rate among patients. Using IHC, I also show that higher grade CRC is associated with increased VANGL2 expression. In our murine cancer model, mice with single or dual-copy loss of VANGL2 were found to have a reduced number of colonic tumours, while maintaining similar tumour size. Investigations to identify how VANGL2 may have control of tumour initiation were carried out focussing on the ECM. I found that, contrary to what I have discovered in the healthy murine colon, tumours from VANGL2-deficient mice had increased transcription of the ECM markers Secreted protein acidic and rich in cysteine (Sparc) and Decorin (Dcn), as well as increased expression of the ECM regulators Matrix Metallopeptidase 9 (Mmp9) and Tissue Inhibitor of Metalloproteinases 1 (Timp1). Changes in the ECM was also seen at the protein level, with increases in staining for the ECM components Col1 (Collagen, type I), and Laminin in VANGL2-deficient tissue. The ECM modulator Connective Tissue Growth Factor (Ctgf), is implicated in multiple cancers including CRC and is increased within VANGL2-deficient tumours at both the transcript and protein level, implicating Ctgf in increasing the ECM of these tumours.

G protein-coupled receptors (GPCRs) comprise the largest group of cell surface receptors, translating environmental signals into cellular responses via cognate G protein partners. Contrary to our initial understanding, most GPCRs do not function in living cells as monomers, but most likely dimers, or even larger arrays of receptors. Standard drug design approaches rely on the notion that drugs binding the two receptors in a given dimer likely function independently of one another. However, this view has been challenged by recent work showing that ligand binding at both receptors can modulate dimeric receptors via allosteric communication. While one receptor may actually be needed to drive signalling, the other acts to control or modulate these signals, without a direct signalling outcome itself. Based on the notion of allosteric modulation within homo- and heterodimers, I tested and compared changes in signalling downstream as well as at the level of the receptor-G protein-effector (RGE) complex in response to different combinations of ligands at each protomer. Using a combination of calcium, cyclic adenosine monophosphate, and mitogen-activated protein kinase signalling assays, I have demonstrated functional interactions for a putative D2 dopamine receptor, oxytocin receptor heterodimer (D2R/OTR), in HEK 293 cells. Immunoprecipitation, bioluminescence resonance energy transfer (BRET) and confocal microscopy experiments reveal D2R and OTR do in fact form a heterodimer in vitro, which may explain the nature of these potential allosteric functional interactions. Using BRET, I assessed the RGE complex conformational dynamics in HEK 293 cells for two other heterodimers, β2-adrenergic receptor with cannabinoid CB1 receptor (β2AR/CB1R) and β2AR/OTR, in order to determine how they manifest in parallel to signalling events themselves. These studies reveal functional interactions can occur in terms of signalling complex conformation. Thus GPCR signalling can be modulated by its partner receptor at the level of downstream effector signalling or at the level of the signalling complex itself. With that said, putative heterodimers need to be reanalyzed in vivo for their allosteric properties, which may explain some of the side effects of so many drugs, and may have implications in drug design.
Les récepteurs couplés aux protéines G (RCPG) constituent le plus grand groupe de récepteurs de la surface cellulaire, qui traduisent les signaux environnementaux en réponses cellulaires via leurs protéines G associées. Contrairement à notre compréhension initiale, la majorité des RCPG ne fonctionnent pas en tant que monomères, mais possiblement en tant que dimères ou même oligomères. Les approches actuelles de conception de médicament estiment que lors de la liaison d'un médicament aux deux récepteurs d'un dimère quelconque, ces derniers fonctionnent potentiellement indépendamment l'un de l'autre. Cependant, cette notion a été reconsidérée par une étude récente montrant que la liaison d'un ligand aux deux récepteurs peut les altérer par voie de communication allostérique. Alors qu'un premier récepteur peut être requis pour initialiser la signalisation, un second peut contrôler ou modifier ces signaux, n'ayant pas nécessairement une signalisation directe comme résultante. Dans l'étude suivante, basée sur la notion de modulation allostérique au sein d'homodimère et d'hétérodimère, les changements de signalisation en aval ainsi qu'au niveau du complexe récepteur/protéine G/effecteur (RGE) ont été étudiés et comparés en réponse à différentes combinaisons de ligands pour chaque protomère. En utilisant une combinaison d'essais de signalisation de calcium, d'adénosine monophosphate cyclique (cAMP) et de protéine kinase activée par des agents mitogènes (MAPK), une interaction fonctionnelle entre le récepteur dopaminergique D2 et le récepteur de l'ocytocine (D2R/OTR) a été démontrée dans les cellules HEK 293. Des expériences d'immunoprécipitation, de transfert d'énergie de résonance par bioluminescence (BRET) et de microscopie confocale ont révélé la présence d'hétérodimère entre le D2R et l'OTR in vitro, ce qui pourrait expliquer la nature des interactions fonctionnelles allostériques. En utilisant la technique de BRET, la dynamique fonctionnelle du complexe RGE dans les cellules HEK 293 a été examinée chez deux autres hétérodimères, soit celui composé du récepteur adrénergique β2 et du récepteur cannabinoïde CB1 (β2AR/CB1R) et l'hétérodimère β2AR/OTR, afin de déterminer comment ils traduisent les évènements de signalisation. Ces études démontrent donc qu'une interaction fonctionnelle peut survenir sur le plan de la conformation du complexe de signalisation. Par conséquent, la signalisation d'un RCPG peut être modulée par son récepteur partenaire au niveau des effecteurs ou au niveau du complexe de signalisation lui-même. Pour cette raison, il serait impératif de réanalyser in vivo les propriétés allostériques d'hétérodimères putatifs, ce qui pourrait expliquer certains effets secondaires d'une multitude de médicaments et ce qui pourrait impliquer des changements majeurs dans la façon de concevoir de nouveaux médicaments.

The endothelium acts to maintain vascular homeostasis, including the regulation of vascular tone, blood fluidity and coagulation. Endothelial dysfunction, a condition largely characterised by reduced NO bioavailability, is an important feature associated with the aetiology of several pathophysiological disorders including type 2 diabetes and cardiovascular disease. AMPK is the downstream component of a protein kinase cascade important in the regulation of cellular and whole body metabolism. AMPK has been demonstrated to mediate a number of physiological responses in the endothelium, including the stimulation of eNOS phosphorylation and NO synthesis; and as such AMPK represents a therapeutic target in the dysfunctional endothelium. VEGF has been established as the prime angiogenic molecule during development, adult physiology and pathology. VEGF stimulates NO production, proposed to be a result of phosphorylation of Ser-1177 on eNOS, a residue also phosphorylated upon AMPK activation in cultured endothelial cells. The present study, utilising HAEC as a model, provides the first demonstration that AMPK is activated by physiological concentrations of VEGF; and furthermore, partially mediates VEGF-stimulated phosphorylation of eNOS on Ser-1177 and subsequent NO production. In addition, the present investigation demonstrates that the upstream AMPK kinase CaMKK is responsible for these VEGF-mediated effects. VEGF is known to increase intracellular calcium levels in endothelial cells via the generation of DAG and IP3. DAG increases Ca2+ influx through a family of non-selective cation channels, whereas IP3 promotes the release of Ca2+ from intracellular stores. High potassium-induced depolarisation, which reduces the driving force for Ca2+ entry through non-selective cation channels in endothelial cells, abolished VEGF-mediated AMPK activation, whereas the IP3 receptor blocker 2-APB was without effect. Exposure of HAEC to a DAG mimetic (OAG) also stimulated AMPK, an effect which was sensitive to the CaMKK inhibitor STO-609 and high potassium induced depolarization. The functional effects of VEGF-stimulated AMPK were also assessed in HAEC. Ablation of AMPK abrogated VEGF-stimulated HAEC migration and proliferation, two key features of the angiogenic process. While AMPK was necessary for VEGF-stimulated endothelial cell proliferation direct activation of the kinase was insufficient to induce this process. AICAR-stimulated AMPK activation has been demonstrated to stimulate fatty acid oxidation in endothelial cells. However, exposure of HAEC to VEGF did not alter fatty acid oxidation in the present study. Together, the current investigation suggests that a VEGF-Ca2+-CaMKK-AMPK-eNOS- NO pathway is present in HAEC, and furthermore, that AMPK is required, albeit insufficient, for the VEGF-stimulated angiogenic response.

Following the discovery of the DISC1 gene in 2000, subsequent research has led to DISC1 becoming one of the most promising candidate genes for psychiatric disorders. Acting as a scaffold protein, DISC1 has a large number of interacting proteins and is involved in a series of intracellular signalling pathways. Amongst these binding proteins are two enzymes, PDE4 and GSK3β, that were originally implicated in psychiatric disease by virtue of their inhibition by psychoactive drugs. PDE4 enzymes are inhibited by rolipram, which possesses anti-depressant and anti-psychotic activity, while GSK3β is one of the major targets of lithium, a potent mood stabiliser. Both these enzymes are intricately involved in the PI3K/AKT, cAMP, and MAPK signalling pathways, all of which have a number of downstream outcomes with potential relevance to psychiatric disorders. The Millar and Porteous laboratory had established that DISC1 modulates PDE4 activity, but this predated awareness of GSK3 as another DISC1 interactor whose binding site overlapped with that of PDE4. Since cAMP is a key regulator of signalling pathways in the brain, I hypothesised that not only DISC1, but also GSK3β may be involved in the regulation of PDE4 activity to control local cAMP levels and gradients. To investigate this hypothesis, I characterised SHSY5Y cells as a model for measuring PDE4 activity, and performed a series of genetic and pharmacological manipulations on this system. Inhibition of GSK3β resulted in a decrease of basal PDE4 activity that was amplified by DISC1 overexpression. Wild type cells that were treated with forskolin exhibited a significant increase in PDE4 activity, which was suppressed by GSK3β inhibition and both overexpression and knockdown of DISC1. Further experiments confirmed that none of these changes were a result of differences in PDE4 mRNA or protein expression. Thus I have provided evidence that suggests tonic activation of PDE4 by GSK3β and evidence for modulation of PDE4 activity by DISC1. I provide evidence for the localisation of PDE4B & PDE4D with key psychiatric associated receptors in structures resembling developing dendritic spines; furthermore, agonism of NMDA receptors results in a significant increase in PDE4 activity in primary neurons. These results are a simple demonstration of an emerging principle in psychiatric research: that none of the signalling pathways implicated in psychiatric disease are acting in isolation. There are likely to be multiple points of integration between these pathways, with the demonstrated DISC1-GSK3β-PDE4 interaction forming one of these points. My results add an important new element to the understanding of how the DISC1 complex may regulate intracellular signalling in response to extracellular cues.

Two-component signalling systems (TCSs) are found in most prokaryotic genomes. They typically comprise of two proteins, a histidine (or sensor) kinase (HK) and an associated response regulator (RR), containing transmitter and receiver domains respectively, which interact to achieve transfer of a phosphoryl group from a histidine residue (of the transmitter domain in the HK) to an aspartate residue (of the partner RR’s receiver domain). An automated analysis pipeline using the NCBI’s RPS-BLAST tool was developed to identify and classify all TCS genes from completed prokaryotic genomes using the PFAM and CDD protein domain databases. A large proportion of TCS genes were found to be simple hybrid kinases (HYs) containing both a transmitter domain and a receiver domain within a single protein, presumably the result of the fusion or combination of separate HK and RR genes. This propensity to consolidate functionality into a single protein was found to be limited in the presence of either a transmembrane sensory/input domain or a DNA binding domain – two spatially separated functions. While HK and RR genes are usually found together in the genome, in some species a large proportion of TCS domains are found as part of complex hybrid kinases (genes containing multiple TCS domains), in isolated or orphaned genes, or in complex gene clusters. In such organisms the lack of paired HK and RR genes makes it difficult to define genome-encoded signalling networks. Identifying paired transmitter and receiver domains from a pan-genomic survey of prokaryotes gives a database of amino acid sequences for thousands of interacting protein-protein complexes. Covariation between columns of multiple sequence alignments (MSAs) identifies particular pairs of residues representing interactions within the docked complex. Using numerical scores, these amino acids pairs were successfully used as explanatory variables in a generalised linear model (GLM) to predict the probabilities of interaction between transmitter and receiver domains.

Mediation of biological functions occurs via tightly regulated signal transduction pathways. These complex cascades often employ crosstalk with other signalling pathways to exert strict control to allow for correct cellular responses. The cyclic AMP signalling pathway is involved in a wide range of cellular processes which require tight control, including cell proliferation and differentiation, metabolism and inflammation. Protein Kinase C (PKC) signalling is also involved in the regulation of many biological functions, due to the wide range of PKC isoforms, and there is emerging evidence that there are critical points of crosstalk between these two central signalling pathways. The aims of this research, therefore, are to identify the molecular basis underlying this pivotal cross-communication. The identification of the complex formed by Receptor for activated C Kinase 1 (RACK1), a scaffold protein for PKC, and the cyclic AMP-specific phosphodiesterase PDE4D5 demonstrated a potential area of crosstalk between the cyclic AMP and PKC signalling pathways although the function of the complex remained largely unknown. In this thesis I have outlined a role for RACK1 binding to PDE4D5 to control the enzymatic activity of the phosphodiesterase. Although RACK1 does not affect the intracellular localisation of PDE4D5, it does afford structural stability to PDE4D5, providing protection against denaturation. Furthermore, interaction with RACK1 facilitates high affinity binding of PDE4D5 to cyclic AMP and increases phosphodiesterase sensitivity to inhibition by rolipram, a PDE4-specific inhibitor that is a therapeutic treatment for depression and Alzheimer’s disease. Additionally, RACK1-bound PDE4D5 was found to be activated by PKCα, providing a route of negative regulation by PKC on cyclic AMP in HEK293 cells. The discovery of EPAC (Exchange Protein directly Activated by Cyclic AMP) has opened up the field of cyclic AMP research, providing an alternative route for the cyclic AMP signalling originally thought to occur solely through Protein Kinase A (PKA). Recent investigations have linked cyclic AMP signalling via EPAC to the control of inflammation, through the induction of Suppressor of Cytokine Signalling 3 (SOCS-3) to inhibit IL-6 signalling. Here I have further delineated this pathway in COS1 to show that induction of SOCS-3 by EPAC requires phospholipase C (PLC) ε. Investigation into downstream effectors of PLC action lead to the identification of PKCα and PKCδ as essential components of this pathway, further elucidating a mechanism by which cyclic AMP can affect inflammation and revealing a point of crosstalk between the two signalling pathways. Further elaborating on the identification of PKC isoforms α and δ as crucial components in the control of cytokine signalling by cyclic AMP via EPAC, investigations into the effect of cyclic AMP on PKC α and δ activation and autophosphorylation, and on downstream effectors, were carried out. It was revealed that cyclic AMP had no influence on PKCδ activity, although a role for cyclic AMP signalling through EPAC on the activation and autophosphorylation of PKCα was identified. Additionally, phosphorylation of the downstream kinase ERK was found to occur independently of PKC activation and required the presence of EPAC1 in COS1 cells. The work presented in this thesis therefore begins to delineate a novel pathway in which the cyclic AMP and PKC pathways work together to afford cell regulation, including the regulation of gene expression, through novel areas of crosstalk.

During the growth phase of oogenesis, mammalian oocytes increase several hundred-fold in volume. Although it is known that ovarian granulosa cells send growth promoting signals, neither these external signals nor the transduction pathways that become activated in the oocyte are known. Therefore, the presence and the activity of candidate signaling pathways in growing murine oocytes were investigated. By immunoblotting, the MAP kinases, ERK1 and ERK2, as well as their activating kinase MEK, were detected in oocytes at all stages of growth. However, using a phospho-specific anti-ERK antibody, no immunoreactive species were detectable in isolated granulosa cells or oocytes at any stage of growth, except metaphase II. Phosphorylated ERK was also present, although in smaller quantities, in oocyte-granulosa cell complexes at the later stages of growth. Furthermore, when ovarian sections were stained with an anti-ERK antibody, the protein was found to be highly concentrated in the cytoplasm of oocytes at all stages of growth, with lower levels in the nucleus. Another member of the MAP kinase family, Jun kinase (JNK), was investigated. By immunoblotting, JNK was detected in growing oocytes. Experiments using an anti-JNK antibody on ovary sections revealed the protein to be uniformly distributed in non-growing and growing oocytes with no evidence of preferential nuclear localization. These results imply that an interaction between the oocyte and the granulosa cells may be required to generate phosphorylated ERK. They also imply that growth signals probably are not relayed through ERK, but do not exclude a role for Jun kinase in mediating oocyte growth.

Earlier studies on temporal lobe epilepsy (TLE), by focusing on the anatomical and electrophysiological abnormalities of the hippocampus, have attributed a major role to this limbic structure in the process of epileptogenesis and seizure generation. Recently however, there has been increasing evidence from both animal and human studies that other limbic structures, including the subiculum, the entorhinal cortex (EC, perirhinal cortex (PC) as well as the amygdala, are possibly involved in the process of epileptogenesis. With the help of both acute and chronic models of limbic seizures, I have used an electrophysiological approach to gain more insight into the mechanisms through which these structures could participate in the establishment of hyperexcitable neuronal networks. Particularly, my investigations have focused on assessing the role played by the subiculum, the amygdala and the PC in epileptiform synchronization in vitro. My findings demonstrate that seizure-induced cell damage in chronically epileptic mice results in a change in limbic network interactions whereby EC ictogenesis is sustained via a reverberant EC-subiculum pathway ( Chapter 1). Furthermore, I have discovered that the subiculum, which holds an anatomically strategic position within the hippocampus, is capable of gating hippocampul output activity via a GABAA-receptor mediated mechanism (Chapter 2). My investigations in the amygdala have confirmed that this limbic structure contributes to epileptiform synchronization (Chapter 3). Moreover, using a chronic rat model of TLE, I have found novel evidence suggesting that alterations in inhibitory mechanisms play a role in the increased excitability of the lateral amygdalar nucleus (Chapter 4). Finally, my studies in chronically epileptic rats have also led to preliminary data signifying hyperexcitability of the PC as well alterations in the interactions between the amygdala and this cortical structure (Chapter 5).

The aim of this study was to investigate the involvement of phospholipid-derived second messengers in protein synthesis and satellite cell proliferation and to assess the suitability of the mouse C2C12 satellite cell line as a model for skeletal muscle. The protein kinase C (PKC) activator, phorbol-12-myristate-13-acetate (PMA) increased translation and phospholipase D (PLD) activity in both C 2C12 myoblasts and isolated extensor digitorum longus (EDL) muscle. Exogenous PLD also stimulated translation implying a link between the two events. Insulin increased translation in both models. The response in EDL muscle was mediated through PKC and cyclo-oxygenase metabolites, whilst that in myoblasts was regulated by phosphatidylinositol-3-kinase (PI 3-kinase). However, insulin, PMA and PLD had no effect on the rate of translation (or transcription) in C2C12 cells after they had fused to form multinucleated myotubes. C2C12 myoblasts responded to basic Fibroblast Growth Factor (bFGF), Epidermal Growth Factor (EGF), PMA and exogenous PLD with increases in RNA accretion and DNA synthesis However, bFGF and EGF had no effect on PLD demonstrating the presence of both PLD-dependent and-independent pathways in these cells. The data suggest that although C2C12 cells may be of use in the study of satellite cell hyperplasia, they do not appear to be a good model to investigate the insulin mediated signalling pathways regulating protein synthesis in skeletal muscle.

Hedgehog proteins (Hh) are morphogens and major mediators in many developmental processes. Hh signalling is significant for many aspects of embryonic development, whereas dysregulation of this pathway is associated with several types of cancers. Hh proteins require dual lipidation and Heparan Sulfate Proteoglycans (HSPGs) for their proper distribution and signalling activity. My first aim was to study the role of HSPGs in human (h) Sonic Hedgehog (Shh) signalling and clarify the biological function of hShh/HSPGs complexes in hShh signalling, by investigating the interaction between human hShh and HSPGs. I used DNA mutagenesis and heparin affinity chromatography to determine key residues in hShh involved in heparin binding (K37/38 and K178). The activity of these mutants was tested by detecting induced Alkaline Phosphatase activity in C3H10T1/2 cells and hShh-inducible gene expression in PANC1 human pancreatic carcinoma cells. I examined the biological function of mutated hShhs (K37/38S, K178S and K37/38/178S) that cannot interact with heparin efficiently and showed that they had reduced signalling activity compared to wild type hShh and a control mutation (K74S). Also, I showed that mutant hShh proteins mediate reduced proliferation and invasion of PANC1 cells following hShh RNAi knockdown (KD), and this correlated with reduced Shh multimeric complex formation. Structurally, Shh proteins are unusual in being dually lipid-modified to be fully active. During the post-translational modifications of Shh, N-terminal palmitoylation is facilitated by the product (Hhat) of the hedgehog acyltransferase gene. I have carried out a thorough analysis of Hhat in PANC1 cells. First, I characterised an antibody prepared in the lab to hHhat. I confirmed the specificity of the antibody by immunoblotting using a self-constructed hHhat-EGFP clone, and a control mGup1-EGFP clone. By subcellular fractionation and Western blotting I found Hhat to be a membrane protein. In addition, I used the hHhat antibody to determine the intracellular localisation of hHhat in PANC1 cells by confocal microscopy and showed that hHhat localised in ER mainly but not in Golgi apparatus. I confirmed this using the hHhat-EGFP clone for fluorescence microscopy in transfected cells. To illuminate the biological function of palmitoylation of hShh in production of active hShh and in the formation of hShh multimeric complex I optimised hHhat RNAi knockdown (KD) in PANC1 cells and confirmed this by a cell-based palmitoylation assay. Using semi-quantitative RT-PCR and immunoblot analyses, I showed that hHhat KD caused decreased signalling through the Shh pathway due to reduced production of active hShh. In addition, I investigated the effect of the addition of palmitate to hShh on its association with cells by comparing hHhat KD cells with control cells. Immunoblotting suggests that palmitoylation of Shh improves its ability to associate to cell membranes. Using hHhat KD, gel filtration of high molecular weight complexes of hShh and immunoblotting of hShh I characterised the role of palmitoylation of hShh in multimeric complex formation. Lastly, I investigated the effect of hHhat KD on PANC1 proliferation and invasion, showing that it represses PANC1 proliferation and invasion. These studies provide a firm basis for understanding the functional roles of hShh palmitoylation and its interactions with HSPGs, and provide proof-of-principle for targeting these aspects of hShh biology in tumour cell therapeutics, specifically in the pancreatic carcinoma context.

Status epilepticus (SE) describes a state of persistent seizures which are unrelenting. First- line treatment for status epilepticus uses a group of drugs, the benzodiazepines, that promote the action of the major inhibitory neurotransmitter within the brain, gamma (γ)-aminobutyric acid (GABA). In a subset of patients however, benzodiazepines prove to be ineffective in terminating SE. Previous data from in vitro models has demonstrated that during single seizures, instead of being inhibitory, activation of the GABAA receptor can have an excitatory effect on neurons. To date, it is unknown whether this shift in GABAergic function contributes to SE, nor how it may modulate the anticonvulsant properties of benzodiazepines. In this thesis I explore the role of excitatory GABAergic signaling in an in vitro model of SE and how this may affect the anticonvulsant efficacy of the benzodiazepine, diazepam. Firstly, I confirm that benzodiazepine-resistant SE is prevalent in a South African paediatric population. Secondly, consistent with its established mechanism of action, I show that diazepam enhances GABAAR synaptic currents. Thirdly, using the in vitro 0 Mg²⁺ model of status epilepticus I show that whilst early application of diazepam has anticonvulsant properties, this is lost when the drug is applied during prolonged epileptiform activity. Fourthly, to investigate this phenomenon I use optogenetic activation of GABAergic interneurons to show that interneurons can drive epileptiform discharges during SE-like activity in vitro. Finally, I confirm that during seizure-like events there is a transient shift in GABAergic signaling that is caused by activity driven changes in the transmembrane Cl⁻ gradient. This thesis provides insight into how excitatory GABAergic signaling during prolonged seizures may contribute towards benzodiazepine resistance in SE. I believe that these results are relevant for understanding of the pathophysiology of SE and may help inform optimal treatment protocols for this condition.

The regulation of systemic immune responses is dependent on individual cell responses that will concur to induce a coherent response against a stimulus. In turn, cell response is dependent on the processing of intracellular signals generated at the cell membrane and transmitted through successive protein modifications to the nucleus in order to activate gene transcription. This is referred to as intracellular signalling. Tight control of these mechanisms is required to generate an appropriate cell response to environmental stimulations and globally to establish an appropriate immune response. Among protein modifications used to transmit a signal to the nucleus, protein tyrosine phosphorylation represents a pivotal method used by immune cells to rapidly induce signalling. While protein tyrosine kinases (PTKs) phosphorylate proteins, protein tyrosine phosphatases (PTPs) regulate the signalling by removing the phosphate group. The goal of this study was to better characterize intracellular signalling events involved in allergic asthma, a chronic inflammatory disease involving a Th2 immune response. In a first time, we investigated the role of PTPs in the development of asthma. We show that inhibition of global PTP activity in mice, during either the allergen sensitization or the allergen challenge phase, reduces asthma development and is linked to an increased Th1 response in the spleen and lung. Secondly, we revealed that TC-PTP inhibition reduces asthma development, while PTP-1B inhibition exacerbates inflammatory cells recruitment to the lung. Inhibition of either SHP-1 or PTP-PEST activity did not significantly modulate asthma development in our model. In a third set of experiments, we got interested in the signalling pathways triggered by the pro-inflammatory molecules myeloid-related proteins (MRPs) 8 and 14. MRPs are small cytosolic proteins recently described to have extracellular functions. MRP8 expression is resistant to corticosteroid treatment, and potentially promotes inflammation in corticosteroid-treated patients. We identified that MRPs induce signal through the action of TLR-4 and trigger the activation of MEK/ERK and JNK pathways that lead to NF-kappaB translocation. Collectively, our data provide a new characterization of signalling pathways engaged in allergic asthma. This should be helpful in the elaboration of new therapeutic approaches targeting precise pathways to inhibit mechanisms of inflammation.

We have identified a novel Cyclin, called Cyclin O, which is able to bind and activate Cdk2 in response to intrinsic apoptotic stimuli. We have focused on the study of Cyclin Oα and Cyclin Oβ, alternatively spliced products of the gene. Upon treatment with different stress stimuli, transfected Cyclin Oα accumulates in dense aggregations in the cytoplasm compatible with being Stress Granules (SGs). Furthermore, we have seen that Cyclin Oβ and a point mutant of the N-terminal part of the protein constitutively localize to the SGs. Although both alpha and beta isoforms are proapoptotic, only Cyclin Oα can bind and activate Cdk2. On the other hand, we have demonstrated that Cyclin O is upregulated by Endoplasmic Reticulum (ER) stress and is necessary for ER stress-induced apoptosis. Cyclin O activates specifically the PERK pathway and interacts with the PERK inhibitor protein p58IPK. Moreover, Cyclin O participates in the activation of other eIF2α kinases. We have also observed that a pool of Cyclin O is located in active mitochondria, suggesting a function of the protein linked to oxidative metabolism.

Hemos identificado una nueva Ciclina, llamada Ciclina O, que es capaz de unirse y activar Cdk2 en respuesta a estímulos apoptóticos intrínsecos. Nos hemos centrado en el estudio de la Ciclina Oα y la Ciclina Oβ, productos de splicing alternativo del gen. En respuesta a diferentes tipos de estrés, la Ciclina Oα se acumula en agregaciones citoplásmicas densas que podrían corresponder a Gránulos de Estrés (SGs). Además, hemos visto que la Ciclina Oβ y un mutante puntual de la parte N-terminal de la proteína se localizan constitutivamente en los SGs. Aunque las dos isoformas alfa y beta son proapoptóticas, solo la Ciclina Oα es capaz de unirse y activar Cdk2. Por otro lado, hemos demostrado que los niveles de Ciclina O se incrementan en respuesta al estrés de Retículo Endoplásmico (RE) y que esta proteína es necesaria para la inducción de apoptosis dependiente de estrés de RE. La Ciclina O activa específicamente la vía de PERK e interacciona con la proteína inhibidora de PERK p58IPK. Además, la Ciclina O participa en la activación de otras quinasas de eIF2α. La Ciclina O se localiza en mitocondrias activas, lo que sugiere una función de la proteína ligada al metabolismo oxidativo.

I investigated the desensitization mechanisms of the serotonin 1A (5-HT$ sb{ rm 1A}$) and 5-HT$ sb{ rm 1B}$ receptors at the molecular level. Receptor phosphorylation by second messenger kinases and G-protein coupled receptor kinases (GRK) is an initial step in the functional desensitization of G-protein coupled receptor receptors (GPCRs). Evidence suggested that PKC preactivation desensitizes the 5-HT$ sb{ rm 1A}$ receptor. In Ltk-cells, PKC preactivation is pathway-selective, blocks receptor-mediated calcium mobilization but not cAMP inhibition. My first objective was to identify if the prime phosphorylation target of PKC was the receptor itself. Four PKC phosphorylation sites on the 5-HT1A receptor were eliminated using site-directed mutagenesis. The signalling profile of single mutants, and third loop double and triple mutants were examined in Ltk-cells. I determined that the prime target of PKC was the receptor since multiple PKC phosphorylation sites in the third loop mediated pathway-selective uncoupling of the receptor from the calcium mobilization pathway and not the adenylyl cyclase. The PKC site in the second loop of the 5-HT$ sb{ rm 1A}$ receptor was shown to be a critical G-protein-contact site in both neuroendocrine and mesenchymal cells.
My second objective was to investigate if GRK-mediated phosphorylation could enhance homologous desensitization of the endogenously expressed 5-H$ sb{ rm 1B}$ receptor in the opossum kidney (OK) cell line. To define the role of endogenous GRK in desensitization, I cloned the OK-GRK$ sb2$ from the OK cell Line and generated a kinase inactive mutant. The GRK$ sb2$ was shown to phosphorylate an epitope tagged 5-HT$ sb{ rm 1B}$ receptor in vitro. However, in intact cells the OK-GRK$ sb2$ did not enhance agonist-induced desensitization of the 5-HT1B receptor, but enhanced the desensitization of the $ alpha$2C receptor. The kinase-inactive mutant or reduction in OK-GRK2 protein levels using antisense GRK2 cDNA both attenuated the desensitization of the $ alpha$2C receptor but not the 5-HT$ sb{ rm 1B}$ receptor. These results suggest that phosphorylation mediated by GRKs in vitro may not mimic in vivo receptor desensitization. Processes other than those mediated by GRKs may be more important for the desensitization of the 5-HT$ sb{ rm 1B}$ receptor in the OK cells.
In conclusion, I have identified two possible mechanisms by which two related receptors, 5-HT$ sb{ rm 1A}$ and 5-HT$ sb{ rm 1B}$ receptors, are regulated by protein kinases: receptor phosphorylation by PKC and GRK. My results suggest that receptor phosphorylation by PKC plays a role in pathway selective desensitization of the 5-HT$ sb{ rm 1A}$ receptor, while phosphorylation the 5-HT$ sb{ rm 1B}$ receptor by GRK, observed in vitro, does not play an important role in the homologous desensitization of the 5-HT$ sb{ rm 1B}$ receptor.

NGF acting through the TrkA tyrosine kinase receptor promotes survival and axon growth of developing sympathetic neurons in vivo and in vitro. This thesis examines the signalling pathways and proteins used by Trk receptors to mediate neuronal survival, local axon growth, and growth cone regulation in neonatal rat sympathetic neurons of the superior cervical ganglion (SCG).
First, I examined the involvement of key signalling motifs on the Trk receptor in mediating neurotrophin-dependent survival and local axon growth. Using recombinant adenoviruses, I expressed the BDNF receptor TrkB, which is not endogenously expressed in SCG neurons, either in wild-type form or mutated at defined effector binding sites. Ectopically expressed wild-type TrkB activated signalling pathways similarly to endogenous TrkA, and supported both neuronal survival and local axon growth in compartmented Campenot chambers. However, TrkB mutated at the Shc-binding site was impaired in its ability to activate PI3-kinase/Akt and MEK/ERK, and was a poor mediator of both neuronal survival and local axon growth. Furthermore, by using pharmacological inhibitors, I found that TrkB-mediated survival and local axon growth required both PI3-kinase and MEK/ERK signalling.
Next, I investigated the contribution of Trk signalling to growth cone maintenance in sympathetic neurons. Acute inhibition of NGF/TrkA signalling rapidly collapsed growth cones, as did inhibition of either PI3-kinase or MEK/ERK. I then asked whether the chemorepellent Sema3F collapsed sympathetic growth cones by inhibiting TrkA-mediated growth signals. Sema3F did not disrupt NGF-induced activation of TrkA, Shc, or PLC-gamma but significantly reduced PI3-kinase and MEK/ERK activation. Furthermore, sustained hyper-activation of PI3-kinase and MEK/ERK partially blocked Sema3F-induced collapse, suggesting that Sema3F acts, at least in part, by inhibiting Trk-dependent pathways.
Together, these data describe the role of Trk signalling in survival, local axon growth, and growth cone maintenance in cultured sympathetic neurons. The Shc-binding site was found to be critical for TrkB-mediated survival and local axon growth. The PI3-kinase and MEK/ERK pathways were found to be important for survival, local axon growth and growth cone maintenance. Furthermore, the chemorepellent Sema3F collapses sympathetic growth cones, at least in part, by interfering with key neurotrophin-induced signalling pathways.

Three experiments used an appetitive Pavlovian conditioning procedure to investigate the ability of the context to generate excitation. Discrete stimuli (CSs) signalling food unconditioned stimuli (USs) were used to decrease contextual conditioning. Detailed observations of the behaviours of rats during and immediately preceding the presentation of CSs, and in an event-free period were analysed. Experiment 1 showed that a discrete visual CS was able to interfere with contextual conditioning because it was a more efficient cue for food than the context. Experiment 2 found that an auditory CS could reduce contextual conditioning in a similar manner but the topography of responding during the event-free period was specific to the modality of the CS. Experiment 3 demonstrated that signal-appropriate responding during the event-free period occurred only if the CS was a signal for reinforcement.

Growth factor receptor tyrosine kinases (RTKs) are critical initiators of signal transduction pathways necessary for cell growth, differentiation, migration and survival. Many of these signals are coordinated through scaffold proteins that are phosphorylated upon their recruitment to the activated receptor complex. This provides binding sites for multiple proteins to activate and generate distinct biological responses. The amplitude and duration of a signal is regulated via dephosphorylation and degradation of target proteins. Signal regulation in this manner acts to promote the formation and disassembly of multi-protein complexes and diversify and localize signals downstream from RTKs.
The Met RTK and its ligand, hepatocyte growth factor (HGF), are positive regulators of epithelial morphogenesis, scatter, and survival. However little was known regarding the proteins responsible for attenuating Met receptor activation. In Chapter II, I demonstrated that the Met receptor was hyperphopshorylated in PTP1B-null mice in response to Fas-induced liver damage. Inhibition of Met signaling with PHA665752, removed protection from liver failure in PTP1B-null hepatocytes, demonstrating that PTP1B was a negative regulator of the Met RTK and its removal promoted cell survival against Fas-induced hepatic failure.
In response to Met receptor stimulation, the Gab1 scaffold protein is the prominent protein recruited and phosphorylated downstream from Met and is critical in mediating Met-dependent biological responses. In chapters III and IV, I identified the serine/threonine kinase Pak4 and the microtubule-bound guanine nucleotide exchange factor GEF-H1 as novel proteins recruited to Gab1 following Met receptor activation. I demonstrate that Gab1 and Pak4 synergize to enhance migration and invasion following HGF stimulation. Furthermore, the recruitment of Pak4 to Gab1 is important for its subcellular localization to lamellipodia and critical for epithelial cell dispersal and morphogenesis downstream from Met. In addition, GEF-H1 is important in focal adhesion formation and turnover and this correlates with the ability of GEF-H1 to promote epithelial migration and invasion downstream from Met.
Overall, these studies investigate molecular mechanisms regulating Met-dependent signals and demonstrate for the first time that the Met receptor is a substrate for PTP1B and identify Pak4 and GEF-H1 as key integrators of Met dependent cellular migration and invasion.
Les récepteurs tyrosine kinase aux facteurs de croissance sont des initiateurs critiques des voies de signalisation nécessaires à la croissance, la différentiation, la migration et la survie cellulaire. Beaucoup de ces signaux sont coordonnés par des protéines d'échafaudage qui sont phosphorylées au cours de leur recrutement au complexe de récepteurs activés. Ceci fournit des sites de liaison à de multiples protéines permettant l'activation et la génération de différentes réponses biologiques. L'amplitude et la durée d'un signal est régulée via la déphosphorylation et la dégradation des protéines cibles. De cette façon, la régulation du signal agit pour promouvoir la formation et le désassemblage de complexes protéiques et pour diversifier et localiser les signaux en aval des récepteurs tyrosine kinase.
Le récepteur Met et son ligand HGF (Hepatocyte Growth Factor) sont des régulateurs de la morphogenèse, la dispersion et la survie des cellules épithéliales. Toutefois, peu d'informations sont disponibles sur les protéines responsables de l'extinction des signaux issus du récepteur Met. Dans le chapitre II, je démontre que le récepteur Met est hyperphosphorylé dans les souris knock-out pour PTP1B en réponse aux dommages induits par Fas. L'inhibition par le composé PHA665752 de la signalisation par Met, relève la protection contre les crises hépatiques dans les souris KO pour PTP1B. Ceci démontre que PTP1B est un régulateur négatif de Met et son retrait permet la survie cellulaire contre les crises hépatiques induites par Fas.
En réponse à la stimulation du récepteur Met, la protéine d'échafaudage Gab1 est la plus importante des protéines recrutées et phosphorylées en aval de Met et cette protéine est critique dans la médiation des réponses biologiques dépendantes de Met. Dans les chapitres III et IV, j'ai identifié la kinase Ser/Thr Pak4 et le facteur d'échange de guanine lié aux microtubules (GEF-H1) en tant que nouvelles protéines recrutées à Gab1 suite à l'activation de Met. Je démontre que Gab1 et Pak4 agissent de façon synergique pour promouvoir la migration et l'invasion suite à la stimulation par HGF. De plus, le recrutement de Pak4 à Gab1 est important pour sa localisation cellulaire dans les lamellipodes et est critique pour la dispersion et la morphogenèse des cellules épithéliales en aval de Met. En outre, GEF-H1 est important pour la formation et le roulement des points d'adhésion focaux ce qui est en corrélation avec la capacité de GEF-H1 de promouvoir la migration et l'invasion épithéliale en aval de Met.
Ces études ont pour but d'investiguer les mécanismes moléculaires régulant les signaux dépendants de Met et démontrent pour la première fois que le récepteur Met est un substrat pour PTP1B. Finalement, Pak4 et GEF-H1 sont identifiés comme des intégrateurs clés de la migration et l'invasion cellulaire dépendante de Met.

G protein coupled receptors, G proteins and their downstream effectors adenylyl cyclase (ACs) were thought to transiently interact at the plasma membrane by random collisions following agonist stimulation. However a growing number of studies have suggested that a major revision of this paradigm was necessary to account for signal transduction specificity and efficiency. The revised model suggests that signalling proteins are pre-assembled as stable macromolecular complexes together with modulators of their activity prior to receptor activation. How and where these signalling complexes form and the mechanisms governing their assembly and maintenance are not completely understood yet. Initially, we addressed this question by exploring AC2 interaction with beta2-adrenergic receptors (beta2ARs) and heterotrimeric G proteins as parts of a pre-assembled signalling complex. Using a combination of biophysical and biochemical techniques, we showed that AC2 interacts with them before it is trafficked to the cell surface in transfected HEK-293 cells. These interactions are constitutive and do not require stimulation by receptor agonists. Furthermore, the use of dominant-negative Rab/Sar monomeric GTPases and dominant-negative heterotrimeric G protein subunits proved that AC2/beta2AR and AC2/Gbetagamma interactions occurred in the ER as measured using both BRET and co-immunoprecipitation experiments, while interaction of the Galpha subunits with the above complexes occurred at a slightly later stage. Both Galpha and Gbetagamma played a role in stabilizing these complexes. Our data also demonstrated that stimulation of AC was still possible when the complex remained on the inside of the cell but was reduced when the GalphaS/AC2 interaction was blocked, suggesting that the addition of the GalphaS subunit was required to render the nascent complexes functional prior to trafficking to proper sites of action. Next, we tackled the issue of higher order assembly of effectors and G proteins, using two different AC isoforms and GalphaS as a model. We demonstrated that AC2 can form heterodimers with AC5 through direct molecular interaction in unstimulated HEK-293 cells. AC2/5 heterodimerization resulted in a reduced total level of AC2 expression, which affected cellular accumulation of cAMP upon forskolin stimulation. The AC2/5 complex was stable in presence of receptor or forskolin stimulation. We provided evidence that co-expression with GalphaS increased the affinity of AC2 for AC5 as monitored by BRET. In particular, the complex formed by AC2/5 lead to synergistic accumulation of cAMP in presence of GalphaS and forskolin, with respect to either of the parent AC isoforms themselves. Finally, we also showed that this complex can be detected in native tissues, as AC2 and AC5 could be co-immunoprecipiated from lysates of mouse heart. Taken together, we provided evidence for stable formation of signalling complexes involving receptor/G proteins/adenylyl cyclase or G proteins/heterodimeric adenylyl cyclases and that G proteins play a crucial role for their assembly and function.

Angiopoietin-1 (Ang-1), the main ligand for the endothelial cell (EC)-selective Tie-2 receptors, promotes survival, proliferation, migration and differentiation of these cells. Despite its importance in various aspects of vascular biology, the mechanisms of action of the Ang-1/Tie-2 receptor pathway have not been fully explored.
To identify the downstream modulators of Ang-1, we evaluated changes in the transcriptome of human umbilical vein endothelial cells (HUVECs) treated with Ang-1 protein for four hours by employing the oligonucleotide rnicroarray technology. Eighty-six genes were significantly upregulated by this treatment and forty-nine genes were significantly downregulated. These genes are involved in the regulation of cell cycle, proliferation, apoptosis, transcription and differentiation. Furthermore, we found that the Erk1/2, PI3-Kinase and mTOR pathways are implicated in promoting gene expression in HUVECs in response to Ang-1. Analysis of the microarray data employing the Ingenuity Pathways analysis software to place the regulated genes in the context of biological networks revealed several highly connected nodes including the chemokine Interleukin-8 (IL-8) and the transcription factor Early growth response-1 (Egr-1). Due to the importance of these genes in promoting angiogenesis, we decided to evaluate their roles in Ang-1/Tie-2 receptor signaling and biological effects.
Ang-1 induced IL-8 expression in a time- and dose-dependent manner in ECs through both transcriptional and post-transcriptional mechanisms. To study the functional role of Ang-1-induced IL-8, we generated HUVECs that overexpress Ang-1. In these cells, neutralizing IL-8 significantly reduced EC proliferation and migration. IL-8 promoter activity experiments and gel shift assays revealed the involvement of the transcription factor AP-1 in Ang-1-induced IL-8. Ang-1 stimulated the phosphorylation of c-Jun through activation of Erk1/2, JNK and PI-3 kinase pathways. Similarly, Ang-1 provoked the expression and DNA binding of Egr-1 in HUVECs. Employing siRNA and DNAzyme to specifically knock-down Egr-1, we found that Ang-1-induced Egr-1 also promotes EC proliferation and migration.
We conclude that Ang-1 provokes a coordinated response intended to promote EC survival, proliferation, and angiogenesis and to inhibit EC apoptosis. Ang-1 induces EC proliferation and migration in part through the secretion of the soluble mediator Interleukin-8 and through induction of the transcription factor Egr-1.

Cyclic-3',5'-guanosine monophosphate (cGMP) is a fundamental intracellular signalling molecule that regulates vascular homeostasis through the tight control of vascular smooth muscle cell (VSMC) reactivity (i.e. vasoconstriction/relaxation) and proliferation. Aberrant VSMC growth and sustained vasoconstriction are hallmarks of cardiovascular disease, exemplified by pulmonary hypertension (PH). Multidrug resistance proteins (MRPs) are membrane bound transporters that facilitate cGMP cellular export thereby representing a potential mechanism that regulates intracellular cGMP-driven signalling. C-type natriuretic peptide (CNP) is an important vasoactive peptide released from the endothelium that maintains vascular homeostasis. CNP binds to natriuretic peptide receptor-B (NPR-B), generating cGMP, and NPR-C, which acts as a clearance receptor removing CNP from the circulation and a signalling pathway regulating vascular function via a cGMP-independent mechanism. Herein, I investigated two separate hypotheses: that MRPs play an important role in maintaining vascular homeostasis, and that endothelium-derived CNP and its cognate receptor, NPR-C, protects against the development of PH. The role of MRPs in regulating vascular homeostasis was investigated using organ bath pharmacology, human VSMC (hVSMC) proliferation and measuring mean arterial blood pressure (MABP) in conscious and anaesthetised mice. To investigate the role of endothelium-derived CNP and NPR-C in PH, male and female CNP and NPR-C knockout (KO) mice were used in two experimental models of PH: hypoxia plus Sugen5416 (SU5416) and bleomycin-induced. The severity of PH was measured using right ventricular systolic pressure (RVSP), MABP, right ventricular hypertrophy (RVH) and pulmonary vascular remodelling. MRP inhibition resulted in concentration-dependent vasorelaxation of mouse aorta per se and increased the potency of cGMP-dependent vessel relaxation in response to activation of both particulate and soluble guanylate cyclases (pGC and sGC). MRP inhibition alone also caused concentration-dependent attenuation of hVSMC proliferation, and enhanced cGMP-mediated attenuation of hVSMC growth via pGC and sGC activation. MRP inhibition per se did not decrease MABP in either anaesthetised or telemeterised mice. However, MRP inhibition did dose-dependently enhance reductions in MABP due to pGC activation in anaesthetised mice. Deletion of endothelial cell-derived CNP (ecCNP KO) in male and female mice did not result in any significant differences in RVSP, RVH or pulmonary vascular remodelling between WT and KO in the hypoxia plus SU5416 model of PH. However, global deletion of NPR-C in both male and female mice caused a significant increase in RVH but not RVSP or vascular remodelling when compared to WT. Both male and female NPR-C KO mice developed significantly increased RVSP compared to WT in the bleomycin-induced model of PH. However, only females exhibited a significant increase in RVH and lung weight in addition to RVSP. In conclusion, MRP inhibition demonstrates potential therapeutic utility to treat cardiovascular diseases by potentiating the vasodilatory and VSMC antiproliferative actions of natriuretic peptides and nitric oxide. Endothelial cellderived CNP is not essential to host protection against PH, whereas its cognate receptor NPR-C demonstrates a cardioprotective capacity. NPR-C attenuates bleomycin-induced PH in both males and females, with a greater effect observed in females. Overall, NPR-C agonism could potentially be used to ameliorate the cardiac and vascular pathology associated with PH.