Regulation of early steps of GPVI signal transduction by phosphatases: a systems biology approach

We present a data-driven mathematical model of a key initiating step in platelet activation, a central process in the prevention of bleeding following Injury. In vascular disease, this process is activated inappropriately and causes thrombosis, heart attacks and stroke. The collagen receptor GPVI is the primary trigger for platelet activation at sites of injury. Understanding the complex molecular mechanisms initiated by this receptor is important for development of more effective antithrombotic medicines. In this work we developed a series of nonlinear ordinary differential equation models that are direct representations of biological hypotheses surrounding the initial steps in GPVI-stimulated signal transduction. At each stage model simulations were compared to our own quantitative, high-temporal experimental data that guides further experimental design, data collection and model refinement. Much is known about the linear forward reactions within platelet signalling pathways but knowledge of the roles of putative reverse reactions are poorly understood. An initial model, that includes a simple constitutively active phosphatase, was unable to explain experimental data. Model revisions, incorporating a complex pathway of interactions (and specifically the phosphatase TULA-2), provided a good description of the experimental data both based on observations of phosphorylation in samples from one donor and in those of a wider population. Our model was used to investigate the levels of proteins involved in regulating the pathway and the effect of low GPVI levels that have been associated with disease. Results indicate a clear separation in healthy and GPVI deficient states in respect of the signalling cascade dynamics associated with Syk tyrosine phosphorylation and activation. Our approach reveals the central importance of this negative feedback pathway that results in the temporal regulation of a specific class of protein tyrosine phosphatases in controlling the rate, and therefore extent, of GPVI-stimulated platelet activation.

Direct observation by time-resolved infrared spectroscopy of the bright and the dark excited states of the [Ru(phen)2(dppz)]2+ light-switch compound in solution and when bound to DNA

The [Ru(phen)2(dppz)]2+ complex (1) is non-emissive in water but is highly luminescent in organic solvents or when bound to DNA, making it a useful probe for DNA binding. To date, a complete mechanistic explanation for this “light-switch” effect is still lacking. With this in mind we have undertaken an ultrafast time resolved infrared (TRIR) study of 1 and directly observe marker bands between 1280–1450 cm-1, which characterise both the emissive “bright” and the non-emissive “dark” excited states of the complex, in CD3CN and D2O respectively. These characteristic spectral features are present in the [Ru(dppz)3]2+ solvent light-switch complex but absent in [Ru(phen)3]2+, which is luminescent in both solvents. DFT calculations show that the vibrational modes responsible for these characteristic bands are predominantly localised on the dppz ligand. Moreover, they reveal that certain vibrational modes of the “dark” excited state couple with vibrational modes of two coordinating water molecules, and through these to the bulk solvent, thus providing a new insight into the mechanism of the light-switch effect. We also demonstrate that the marker bands for the “bright” state are observed for both L- and D enantiomers of 1 when bound to DNA and that photo-excitation of the complex induces perturbation of the guanine and cytosine carbonyl bands. This perturbation is shown to be stronger for the L enantiomer, demonstrating the different binding site properties of the two enantiomers and the ability of this technique to determine the identity and nature of the binding site of such intercalators.

The evolution and heterogeneity of outward foreign direct investment from Latin America

The PhD dissertation investigates the rise of emerging country multinationals (EMNEs), a phenomenon that has opened up a series of research themes and debates. The main debate in this field is the extent to which the theories/frameworks on foreign direct investment (FDI), which have been developed from investigations on multinationals from developed countries, is relevant in explaining outward FDI from EMNEs. This debate is sparked by research suggesting that EMNEs supposedly do not hold the characteristics that are seen as a prerequisite to engaging in FDI. The underlying theme in this PhD is that the field should move away from a one size fit all categorisation of EMNEs, and explore the heterogeneity within EMNEs. Collecting data through various databases, archival articles and annual reports, there was an examination of the internationalisation process of 136 Latin American Multinationals (LAMNEs). The research explores the differences in internationalisation trajectories and global strategies and classifies firms into one of four categories. The four categories that LAMNEs fall into are: Natural-Resource Vertical Integrator, which are firms that are in resource seeking sectors; Accelerated Global, which depict firms that have become global over a very short period of time; Traditional Global, which are EMNEs that have internationalised at the same pace as developed country MNEs and Local Optimisers that only acquire or internationalise to developing countries. The analysis also looks at which decade LAMNEs engaged in FDI, to see if LAMNEs that internationalised during the 1970s and 1980s, during a time when Latin America had a closed economy, was different to LAMNEs that internationalised during the Washington consensus era of the 1990s or to firms that have only just internationalised within the last decade. The findings show that LAMNEs that internationalised before 1990 were more likely to adopt Local Optimiser strategies. However, more LAMNEs that started to internationalise during the 1990s started to adopt Traditional Global strategies, although Local Optimisers were the most prominent strategy. From 2002, there was more prominence of Accelerated Global strategies and a lot more heterogeneity among LAMNEs. Natural-Resource Vertical Integrator LAMNEs, tended to start to internationalisation process during the 1970s/1980s. Despite the rise of EMNEs, and by extension LAMNEs opting to use cross border merger and acquisitions (M&A), there is little research on whether this entry mode has been successful. Contrary to the argument that EMNEs are “internationalising successfully” through this strategy, the findings show that these firms are highly geared and are running less efficiently against their Western competitors. In comparison, LAMNEs internationalising through a more gradual approach, are outperforming their Western competitors on efficiency and are not highly geared- i.e. do not hold a lot of debt. The conclusion of the thesis is the emphasis of moving away from evaluating firms from their country or region of origin, but rather through the global strategy they are using. This will give a more a robust firm level of analysis, and help develop the understanding of EMNEs and international business theory.

Estrogen regulation of chicken riboflavin carrier protein gene is mediated by ERE half sites without direct binding of estrogen receptor

Estrogen is an important steroid hormone that mediates most of its effects on regulation of gene expression by binding to intracellular receptors. The consensus estrogen response element (ERE) is a 13 bp palindromic inverted repeat with a three nucleotide spacer. However, several reports suggest that many estrogen target genes are regulated by diverse elements, such as imperfect EREs and ERE half sites (ERE 1/2), which are either the proximal or the distal half of the palindrome. To gain more insight into ERE half site-mediated gene regulation, we used a region from the estrogen-regulated chicken riboflavin carrier protein (RCP) gene promoter that contains ERE half sites. Using moxestrol, an analogue of estrogen and transient transfection of deletion and mutation containing RCP promoter/reporter constructs in chicken hepatoma (LMH2A) cells, we identified an estrogen response unit (ERU) composed of two consensus ERE 1/2 sites and one non-consensus ERE 1/2 site. Mutation of any of these sites within this ERU abolishes moxestrol response. Further, the ERU is able to confer moxestrol responsiveness to a heterologous promoter. Interestingly, RCP promoter is regulated by moxestrol in estrogen responsive human MCF-7 cells, but not in other cell lines such as NIH3T3 and HepG2 despite estrogen receptor-alpha (ER-�) co transfection. Electrophoretic mobility shift assays (EMSAs) with promoter regions encompassing the half sites and nuclear extracts from LMH2A cells show the presence of a moxestrol-induced complex that is abolished by a polyclonal anti-ER� antibody. Surprisingly, estrogen receptor cannot bind to these promoter elements in isolation. Thus, there appears to be a definite requirement for some other factor(s) in addition to estrogen receptor, for the generation of a suitable response of this promoter to estrogen. Our studies therefore suggest a novel mechanism of gene regulation by estrogen, involving ERE half sites without direct binding of ER to the cognate elements.