Signals via the T cell receptor (TCR) are critical for the development of T cells in the thymus, maintenance of a self-tolerant peripheral T cell repertoire and the activation of T cells in secondary lymphoid organs. A dynamic balance between tyrosine phosphorylation and dephosphorylation is essential for the maintenance of homeostasis and proper regulation of the immune system. The cytoplasmic phosphatase, PTPN22 (protein tyrosine phosphatase non-receptor type 22) is involved in negative modulation of signal transduction through the TCR and plays a central role in regulating lymphocyte homeostasis. Genome wide association studies reveal that point mutations in PTPN22 confer an increased risk of developing multiple autoimmune diseases in humans. The precise function of PTPN22 and how mutations contribute to autoimmunity is controversial. Loss-of-function mutations in PTPN22 are associated with elevated T effector cell expansion and autoreactive B cells in both humans and mice. A thorough dissection of the molecular involvement of PTPN22 and its allelic variant R619W is important to delineate its role in autoimmunity, to this end we utilised the Ptpn22-/- mice generated in our laboratory. In order to address whether R619W allelic variant is a gain- or loss-of-function mutation, we expressed both PTPN22WT and PTPN22R619W constructs in primary activated Ptpn22-/- T lymphocytes using lentiviral transduction. Surprisingly expression of either wild type or variant phosphatase showed no affect on cytokine production. Preliminary results from bone marrow chimeras generated by retroviral expression of PTPN22WT and PTPN22R619W in Ptpn22 deficient mice showed reduced T cell activation compared to Ptpn22-/- T cells. PTPN22WT appeared to be more suppressive of T cell responses than variant PTPN22R619W. Consistent with studies conducted in comparable knock-in mouse models, our data point to the variant PTPN22R619W as being a partial loss of function allele. To elucidate the mechanism of PTPN22 action in context of an autoimmune disease, we investigated the effect of Ptpn22 deficiency on the phenotype of SKG mice. The SKG mouse harbours a point mutation (W163C) within the carboxyl terminal SH2- domain of ZAP-70, which results in decreased TCR signalling and impaired thymocyte development with defective positive and negative selection. These mice are prone to developing CD4+ T cell mediated autoimmune arthritis that closely resembles rheumatoid arthritis in humans. We found that thymus differentiation was partially restored in SKG Ptpn22-/- thymocytes and Ptpn22 deficiency enhanced TCR mediated signalling in SKG Ptpn22-/- thymocytes relative to SKG thymocytes. Consistent with increased signalling observed in the thymocytes, there was improved in vitro proliferation and IL-2 production of CD4+ T lymphocytes from SKG Ptpn22-/- mice compared to SKG mice. By contrast to SKG mice, SKG Ptpn22-/- mice developed less severe mannan-induced arthritis and showed decreased proportions of Th17 and higher numbers of regulatory T cells. These results show that removal of PTPN22 can compensate, at least partially, for the deficient ZAP-70 activity in the SKG mouse, thus linking PTPN22 and ZAP-70 to the same signalling pathway. This study advances our understanding of how manipulating TCR signals impacts on downstream T cell functions, suggesting PTPN22 may be a valuable target for the treatment of autoimmune diseases. Further studies to determine physiological role of the phosphatase and its disease-associated variants could provide insight into mechanism of immune activation, tolerance and autoimmunity.

Phosphate (Pi) deficiency is a global problem for food production. Plants have evolved complex mechanisms to adapt to low Pi. We focused on the initial aspects of adaptation to low Pi - perception and immediate-early responses to changes in external Pi. To examine whether a labile repressor controls expression of the high affinity Pi transporter, Pht1;1, we performed electrophoretic mobility shift assays (EMSA) but observed only weak protein-DNA binding activity using extracts from Arabidopsis suspension cultures or seedlings. The regulatory role of different regions in Pht1;1 promoter was dissected by promoter deletion analysis, using uidA as a reporter. We identified two domains important for regulation: sequences between -1898 bp and - 932 bp are important for induction of Pht1;1 in low Pi; the intron in the 5’UTR impacts Pht1;1 expression in the young part of both primary and lateral root apices. A complementary approach to identify repressors of Pi starvation responses was pursued: We identified ZAT18, a putative transcription factor, as a candidate repressor. ZAT18 contains an EAR motif, a repressor domain in plants; the expression of ZAT18 responds to Pi starvation. Using transgenic lines with promoter::ZAT18-VENUS constructs, we studied its expression, localization and abundance in different levels of Pi availability: ZAT18 is mainly expressed in the nucleus of Arabidopsis root hair cells. Its accumulation was induced by 4 day Pi starvation. We also performed a microarray analysis to examine global gene expression levels during Pi starvation and rapid recovery. Our data indicated that 258 genes were induced and 188 genes were suppressed during Pi starvation. For most of these genes, responses were reversed after 4 hour Pi recovery. Further study of these genes will help to define targets of the early Pi starvation-signalling pathway.

A proteína colibistina (CB), uma Rho GEF neuro-especifica, apresenta papel importante na formação e funcionamento das sinapses inibitórias do sistema nervoso central por interagir com a proteína scaffold gefirina e com receptores GABAA e promover o agrupamento e transporte dessas proteínas para a membrana pós-sináptica. Recentemente, nosso grupo de pesquisa identificou interação de CB com um complexo proteico que controla o início da tradução em eucariotos (complexo eIF3), o que sugeriu pela primeira vez que essa proteína pode estar envolvida também na regulação da tradução em células neurais. Ainda, já havia sido descrito que gefirina, parceira funcional de CB, interage com mTOR, uma quinase que desempenha papel fundamental no controle do início da tradução. Contudo, até o momento não havia estudos adicionais investigando o papel de CB neste cenário. Assim sendo, o presente trabalho teve como objetivo investigar o envolvimento da proteína CB no controle do início da síntese proteica mediada pela via de sinalização mTORC1. Foram utilizados dois modelos experimentais: i) um sistema de expressão heteróloga - superexpressão de CB em células HEK293T, e ii) um modelo endógeno de expressão - células neuroprogenitoras derivadas de células-tronco pluripotentes induzidas (iNPCs) provenientes de indivíduos controles e de um paciente com deleção no gene da CB. Por meio de experimentos de coimunoprecipação nós verificamos que CB interage fisicamente com mTOR nos dois modelos experimentais utilizados. Ainda, nossos resultados mostraram que CB parece modular a atividade da via mTORC1, e nas iNPCs derivadas do paciente a ausência de CB leva a um aumento na ativação desta via de sinalização. Em concordância com esses resultados, nós observamos aumento em neo-síntese proteica nas iNPCs provenientes do paciente, o que pode ser um mecanismo patofisiológico contribuindo para as alterações cognitivas e comportamentais observadas no paciente. Embora estudos adicionais sejam necessários para melhor entender os mecanismos moleculares deste controle de início de tradução mediado por CB, nós sugerimos um modelo no qual CB, por interagir fisicamente com mTOR e eIF3, sequestra estas proteínas e impede que mTOR ative seus alvos e desencadeie a formação do complexo de inicio de tradução. Em conclusão, nossos resultados oferecem novas evidências do envolvimento de CB no controle da síntese proteica / Collybistin (CB), a neural specific RhoGEF, plays key roles in inhibitory synapse formation and function that cluster and localize the scaffold protein gephyrin and GABAA receptors to the neural postsynaptic membrane. We have recently reported that CB interacts with a protein complex that controls translation initiation in eukaryotic cells (eIF3 complex), which suggested for the first time that this protein may also act as regulator of protein synthesis in neural cells. Moreover, it has been previously described that gephyrin, the CB functional partner, interacts with mTOR, a kinase that plays a pivotal role in the control of translation initiation. However, until now there were no further studies investigating the role of CB in this scenario. The purpose of this study was to investigate if CB is involved in the control of translational initiation mediated by the mTORC1 signaling pathway. Two experimental models were used: i) a heterologous expression system - overexpression of CB in HEK293T cells, and ii) an endogenous expression model - neural progenitor cells derived from induced pluripotent stem cells (iNPCs) from control individuals and a patient with a deletion of the entire CB gene. We performed coimmunoprecipitation experiments and verified that CB physically interacts with mTOR both in 293T cells and in control iNPCs. In addition, our results show that CB appears to modulate the activity of mTORC1 pathway, and the absence of CB leads to increased mTORC1 signaling activation in patient\' iNPCs. In agreement with these results, we observed increased de novo cap-dependent translation in patient cells, which could be a pathophysiological mechanism contributing to cognitive and behavioral abnormalities observed in the patient. Although further studies are needed to better understand the molecular details of CB-mediated translational control, we suggest a model whereby CB, by physically interacting with mTOR and eIF3, sequesters these proteins, thereby preventing both the ability of mTOR to activate its targets and the formation of the translational initiation complex. In conclusion, our results offer new insights into the role of CB in protein synthesis control

Calcitonin gene-related peptide (CGRP) and adrenomedullin (ADM) receptors are heterodimeric complexes composed of the calcitonin receptor-like receptor (CLR) and a receptor activity-modifying protein (RAMP). Association with RAMP1 gives a high affinity CGRP receptor, whereas association with RAMP2 or RAMP3 gives high affinity ADM receptors. CGRP and ADM are widely distributed throughout the body and play important roles and are implicated in many diseases including migraine, heart failure and sepsis. Recently, CGRP has been shown to promote nitric oxide (NO) production and inducible NO synthase (iNOS) expression in trigeminal ganglion glial cells via ERK activation. CGRP is known to induce iNOS/NO production in thoracic artery smooth muscle cells (TA-SMC) pretreated with interleukin-1b. However, the molecular mechanism of CGRP-induced iNOS/NO production in TA-SMC is unknown. Therefore, in order to determine if CGRP induces iNOS/NO production via ERK activation, I first investigated the exact mechanisms through which CGRP activates ERK1-2 in HEK cells. By using different inhibitors I showed that CGRPinduced ERK activation is mainly activated through two major pathways. I showed for the first time that CGRP induces ERK activation through transactivation of ErbB1 and as expected through the cAMP/PKA pathway. Then, in order to characterise a suitable model to study CGRP-induced iNOS expression, I used primary TA-SMC and I showed that CGRP induces iNOS upregulation, which is reduced when cells are incubated with U0126, a MEK inhibitor. Thus, these results suggest that CGRP induces iNOS expression via ERK activation in TA-SMC. However, further experimentation is required to determine the exact ERK pathway responsible for iNOS induction. Compared to CLR•RAMP1 and CLR•RAMP3, little is known about the postendocytic sorting of CLR•RAMP2. Using HEK cells stably expressing CLR•RAMP2, I investigated the molecular mechanisms regulating the ADM receptor. I first showed that, unlike CLR•RAMP1, even transient stimulation of CLR•RAMP2 with ADM promotes degradation of both CLR and RAMP2, indicating that this ADM receptor does not recycle to the cell-surface. Moreover, I showed that CLR, not RAMP2, is constitutively ubiquitinated, which was further enhanced upon ADM stimulation. In order to elucidate the role of ADM-mediated ubiquitination of CLR, I made a lysine-less mutant of CLR, named CLRD9KR. I showed that ubiquitination of CLR did not affect ADM-induced trafficking of CLR•RAMP2 to lysosomes, nor did it affect the degradation or the ERK signalling of CLR•RAMP2. However, I showed that ubiquitination of CLR regulated the rate of degradation of the receptor. Together, these results indicate that CLR•RAMP2 does not recycle and is degraded via a molecular mechanism that is accelerated by ADM-induced ubiquitination of CLR.

The ability of cells to react to changes in their environment is critical to their survival. Effective decision making strategies leading to the activation of specific intracellular pathways hinge on cells sensing and processing extracellular variation. We will only be able to understand and manipulate how cells make decisions if we understand the “design” of the mechanisms that enable them to make such decisions, in terms of how they function, and in terms of their limitations and architecture. In this thesis, using mathematical modelling, I revisited classical signal sensing and transduction mechanisms in light of recent developments in methodological approaches and data collection. I studied the sensing characteristics of one of the simplest of sensors, the allosteric sensor, to understand the limits and effectiveness of its “design”. Using the classical Monod-Wyman-Changeux model of allostery, I defined and evaluated six engineering-inspired characteristics as a function of the parameters and number of sensors. I found that specifying one characteristic strongly constrains others and I determined the trade-offs that follow from these constraints. I also calculated the probability distribution of the number of input molecules that maximizes information transfer and, as a consequence, the number of environmental states a given population of sensors can discriminate between. Next, I proposed a new general model of phosphorylation cycles that can account for experimental reports of ultrasensitivity occurring in regimes that are far away from Goldbeter and Koshland’s zero-order saturation, the classical ultrasensitivity-generating mechanism. The new model exhibits robust ultrasensitivity in “anti-zero-order” regimes. The degree of ultrasensitivity, its limits, and its dependence on the parameters of the system are analytically tractable. The model can, additionally, explain in an intuitive way some puzzling experimental observations. Finally, I addressed the problem of integrating different types of signals from multiple sources, and performed some preliminary exploration of how cells can “learn” to associate and dissociate correlated signals in non-evolutionary time-scales. This work provides insights into the function and robustness of signal sensing and transduction mechanisms and as such is applicable to both the study of endogenous systems and the design of synthetic ones.

Adaptive immune responses are highlyco-ordinated and rely upon efficient intracellular communication to orchestratecell function. Phosphoinositide 3-kinase (PI3K) signalling is a well-studiedand important positive mediator in T lymphocyte function; however the role for SH2-domaincontaining inositol phosphatase 1 (SHIP-1), a negative regulator of PI3Ksignalling, has not been so thoroughly investigated. The use of knockout mousemodels has given an insight into the role of SHIP-1 in murine T cells, butthese are compromised by loss of function during development which impingesupon mature T cell function and by the loss of non-catalytic functions ofSHIP-1. Recent work has indicated a clear rolefor reactive oxygen species (ROS), specifically hydrogen peroxide (H2O2),in immune cell signalling and functional responses including migration. Howeverlike SHIP-1, the functional roles of ROS are poorly understood in human Tlymphocytes, particularly the mechanisms by which ROS signals to alter Tlymphocyte biology. ROS has been previously shown to activate PI3K, Mitogenactivate protein kinase (MAPK) and Src family tyrosine kinase (SFK) signallingin a number of different cell types. In addition, ROS have been shown to inactivatephosphatase and tensin homology (PTEN), another negative regulator of PI3K, andare postulated to inactive SHIP-1 signalling. A pharmacological approach wasutilised to manipulate the catalytic activity of SHIP-1 and the cellularaccumulation of ROS in primary human T lymphocytes. Remarkably, it wasdetermined that both SHIP-1 activation and inhibition reduced the ligand-mediatedfunctions of human T lymphocytes, including signalling, proliferation, adhesionand migration. Furthermore, H2O2 selectively inhibited directionalmigration to chemokine CXCL11, enhanced F-actin polymerisation and decreasedactin polarisation to CXCL11. H2O2 required SFKsignalling to induce the phosphorylation/catalytic activation of SHIP-1 and todecrease the surface expression of CXCR3, both of which could be mechanismsunderlying the deficiency in migration observed with H2O2.

Since the dividend irrelevance theory of Miller and Modigliani (1961), academics and practitioners still have little understanding of the managerial incentives underpinning dividend policy. Black (1976) observed, “The harder we look at the dividend picture, the more it seems like a puzzle, with pieces that just don’t fit together.” <br /> <br /> This thesis aims to shed additional light on the dividend puzzle. Accordingly, two theoretical models have been developed to help explain why firms pay dividends or repurchase their own shares. The models consider the case in which the managers of a high-quality firm (firm H) and a low-quality firm (firm L) choose to use corporate cash flows to pay dividends, repurchase shares, or invest in a real project from which they can earn private benefits. I focus on the case in which firm H has a positive NPV project whereas firm L has a negative NPV project. <br /> <br /> In the first model, developed in spirit of Isagawa (2000), I show that paying dividends is a dominated strategy for firm H, regardless of the managerial weight parameter. If the manager is myopic, firm L will choose to repurchase shares at the detriment of existing shareholders. If the manager is farsighted, on the other hand, firm L will choose to pay dividends. I also consider the case in which investors are irrational in that they do not update their beliefs upon observing one firm repurchasing shares while the other firm paying dividends. The model shows that, in inefficient market, firm L will not mimic given that firm H repurchases shares since it cannot obtain any benefit from doing so. <br /> <br /> In the second model, built on Fairchild and Zhang’s (2005) work, in which the managerial payout decisions depend on the relative magnitudes of dividend and repurchase catering premia, I demonstrate that a myopic manager of firm H may pass up a positive NPV project in order to cater to investor demand for dividends or share repurchases (an adverse selection problem). In addition, I show that the agency cost of free cash flow can be mitigated if the dividend-catering premium is sufficiently high. That is, firm L’s manager will have a strong incentive to return excess cash to shareholders rather than invest it in a negative NPV project. <br /> <br /> Then, I investigate dividend changes in Thailand over the period 2002-2005. To test the signalling and free cash flow hypotheses, I first analyse profitability changes around dividend changes and benchmark them with control firms, and examine the relation between dividend changes and the past and future profitability. Consistent with Benartzi et al.’s (1997) evidence in the U.S., dividend changes in Thailand do not signal future profitability but rather the past performance. Then, I examine the determinants of dividend changes and firm’s decision to change dividends. I also investigate the short-run and long-run stock price performance of dividend-changing firms, and the relation between announcement returns and hypothesised independent variables. Finally, I examine firms’ investment behaviour following dividend changes. The results do not support the view that dividend changes signal future profitability. Overall, the findings are broadly consistent with the free cash flow hypothesis rather than the signalling hypothesis. <br /> <br /> Additionally, I provide preliminary evidence on open-market share repurchases (OMRs) in Thailand over the period December 2001 to January 2007. I find that stock prices react positively to OMR announcements and continue to increase in the longer term, suggesting that stock market underreacts to the signal conveyed by the managers of repurchasing firms. Comparing the actual repurchase cost with the costs of benchmark portfolios, I find that the actual repurchase cost is the lowest. This finding suggests that the managers of repurchasing firms have substantial ability to time the market.

The PI3K/mTOR signalling pathway plays a major role in biology and disease. Therefore, effective inhibitors that target proteins of this pathway have been developed. However, acquired resistance of cancer cells is a prevalent phenomenon that limits the durable response of these compounds. It is becoming apparent that experimental approaches for comprehensive biochemical analysis contribute to understand the complex mechanisms that confer drug resistance, and advances in largescale technologies including genomic sequencing and proteomics allow unprecedented molecular coverage without being biased for specific genes/cellular pathways. Initially, the phenotypic response of sensitive and resistant cells to the absence or presence of a PI3K inhibitor (PI3Ki), as well as other kinases, was examined. This study revealed that PI3Ki-resistant cells experience extensive phenotypic changes upon withdrawal of the PI3Ki from the culture media. The regulation of the proteome and phosphoproteome of sensitive and PI3Ki-resistant cells grown with or without the PI3Ki was analysed by shotgun mass spectrometry-based label-free quantitative technology. This analysis demonstrated that the proteomes and phosphoproteomes of drug-resistant cells are remodelled conditional to the presence of PI3Ki, and that the levels of enzymes with metabolic roles are modulated in resistant cells. Functional analysis of the metabolism of cells capable to survive in absence of PI3K/mTOR activity demonstrated that the bioenergetic activity of these cells is contingent on the presence of the selection drug. The complete set of protein-coding regions of the genome (exome) of sensitive and PI3Ki-resistant cells was then sequenced. This study unveiled common alterations in exome regions across PI3Kiresistant cell lines, as well as a degree of genomic heterogeneity between them. Lastly, the impact of lactic acid, a metabolic product, on a defined signalling network of the MCF7 breast cancer cell line was analysed. This study described the capacity of this metabolite to change the activity of signalling network branches.

Many species use visual features to avoid predation by several methods, such as concealing themselves, deceiving predators and hindering capture. One of the most striking strategies is aposematism, or warning coloration, in which prey use conspicuous visual signals to advertise chemical or physical defences, and thereby deter predators from attacking. My thesis focuses on the form of these warning signals, namely which elements of visual patterns might be most effective in generating predator avoidance, as well as how these different visual features relate to defence levels and ultimately to prey survival in the wild. To address these issues, I studied the warning signals of Lepidoptera and in particular burnet moths (Zygaenidae: Zygaeninae), day-flying moths with distinctive red and black wings and the remarkable ability to both synthesise defensive compounds and sequester them from their host plants. Technological advances and a growing understanding of animal vision mean that animal signals can be studied in an increasingly precise and ecologically-relevant way. Throughout this thesis, I use sophisticated methods to quantify both the defensive chemicals and wing coloration of burnet moths, as perceived by their avian predators. I examine the key features of day-flying defended Lepidoptera, then focus on the potential for quantitative signal honesty in burnet moths. I explore the relationship between defence levels and measures of coloration, both within the six-spot burnet moth, Zygaena filipendulae, and across species in the Zygaenidae, then test the effects of variation in warning signals on predation risk for artificial burnet-like prey in the field. My work highlights some of the complicating factors that should be accounted for in the study of warning coloration, especially when investigating the potential for quantitative signal honesty. I hope my thesis will provide a basis for future research on the defensive strategies of day-flying moths and inspire others to pursue investigations into aposematism in the Zygaenidae.

The NF-κB transcription factor is expressed in the majority of mammalian cells and regulates a large number of genes with important functions in a variety of cellular processes including cell growth, division, apoptosis and inflammatory responses. Perturbation of NF-κB response has been implicated in a variety of diseases such as asthma and inflammatory bowel disease, in addition to various forms of cancer. Through experiments at the single cell level it has been shown that NF-κB displays complex temporal activation, notably including nucleo-cytoplasmic oscillations. It has been observed that these oscillations occur in a heterogeneous manner; as such they are masked when measured at the population level. In contrast, pulsed TNFα treatment at 100 min intervals produces regular and synchronous nuclear peaks of NF-κB. Such pulsatile stimulation may reflect more accurately physiological conditions. The work in this project uses a Systems Biology approach consisting of bioinformatic, mathematical, and experimental methodologies to investigate how NF-κB can regulate such a diverse set of gene responses. Previously published studies have proposed that target gene expression levels following NF-κB activation (continuous TNFα) can be explained by a combination of key parameters, including transcript degradation rate, transcript structure, and transcription initiation rate. Initial work in this project highlighted that these explanatory factors are not sufficient to describe the observed temporal order of gene transcription. The roles of miRNAs and NF-κB subunit phosphorylation in regulation were additionally explored. A large set of genes was identified that are activated more strongly by pulsed TNFα than by continuous TNFα treatment. This suggests a new unreported mechanism of gene regulation, the possible causes of which are examined in this thesis. The gene list was refined by altering pulse frequency, which revealed an enrichment of NF-κB targets correlated with the regularity of these pulses. Temperature shift and anti-inflammatory drug treatment (Diclofenac) were shown to have a profound effect on NF-κB oscillation frequency. These perturbations provide an alternative method to study the effects of NF-κB oscillation frequency on specific target genes, independent of a pulse regime. Integration and analysis of these datasets suggested that a core, frequency-encoded set of genes regulated by NF-κB might exist. It is proposed that such genes may respond optimally to specific frequencies of NF-κB activation, implying a potential frequency threshold. The presence of such genes may explain the need for the complex systems that control NF-κB timing. It was noted that there was an enrichment of genes encoding transcription factors within the frequency encoding set, in addition to proteins which are known to be involved in the control of inflammation.