Hypoxia-inducible factor (HIF) network:insights from mathematical models

Oxygen is a crucial molecule for cellular function. When oxygen demand exceeds supply, the oxygen sensing pathway centred on the hypoxia inducible factor (HIF) is switched on and promotes adaptation to hypoxia by up-regulating genes involved in angiogenesis, erythropoiesis and glycolysis. The regulation of HIF is tightly modulated through intricate regulatory mechanisms. Notably, its protein stability is controlled by the oxygen sensing prolyl hydroxylase domain (PHD) enzymes and its transcriptional activity is controlled by the asparaginyl hydroxylase FIH (factor inhibiting HIF-1).To probe the complexity of hypoxia-induced HIF signalling, efforts in mathematical modelling of the pathway have been underway for around a decade. In this paper, we review the existing mathematical models developed to describe and explain specific behaviours of the HIF pathway and how they have contributed new insights into our understanding of the network. Topics for modelling included the switch-like response to decreased oxygen gradient, the role of micro environmental factors, the regulation by FIH and the temporal dynamics of the HIF response. We will also discuss the technical aspects, extent and limitations of these models. Recently, HIF pathway has been implicated in other disease contexts such as hypoxic inflammation and cancer through crosstalking with pathways like NF?B and mTOR. We will examine how future mathematical modelling and simulation of interlinked networks can aid in understanding HIF behaviour in complex pathophysiological situations. Ultimately this would allow the identification of new pharmacological targets in different disease settings.

A dynamic model of the hypoxia-inducible factor 1α (HIF-1α) network

Activation of the hypoxia-inducible factor (HIF) pathway is a critical step in the transcriptional response to hypoxia. Although many of the key proteins involved have been characterised, the dynamics of their interactions in generating this response remain unclear. In the present study, we have generated a comprehensive mathematical model of the HIF-1a pathway based on core validated components and dynamic experimental data, and confirm the previously described connections within the predicted network topology. Our model confirms previous work demonstrating that the steps leading to optimal HIF-1a transcriptional activity require sequential inhibition of both prolyl- and asparaginyl-hydroxylases. We predict from our model (and confirm experimentally) that there is residual activity of the asparaginyl-hydroxylase FIH (factor inhibiting HIF) at low oxygen tension. Furthermore, silencing FIH under conditions where prolyl-hydroxylases are inhibited results in increased HIF-1a transcriptional activity, but paradoxically decreases HIF-1a stability. Using a core module of the HIF network and mathematical proof supported by experimental data, we propose that asparaginyl hydroxylation confers a degree of resistance upon HIF-1a to proteosomal degradation. Thus, through in vitro experimental data and in silico predictions, we provide a comprehensive model of the dynamic regulation of HIF-1a transcriptional activity by hydroxylases and use its predictive and adaptive properties to explain counter-intuitive biological observations.

Monitoring of transcriptional dynamics of HIF and NFκB activities

Genetic experiments over the last few decades have identified many regulatory proteins critical for DNA transcription. The dynamics of their transcriptional activities shape the differential expression of the genes they control. Here we describe a simple method, based on the secreted luciferase, to measure the activities of two transcription factors NF?B and HIF. This technique can effectively monitor dynamics of transcriptional events in a population of cells and be up-scaled for high-throughput screening and promoter analysis, making it ideal for data-demanding applications such as mathematical modelling.

An actor-network theory study of public sector inter-organisational collaboration

EMBARGOED The literature on inter-organisational collaboration, although wide-ranging, offers little guidance on collaboration as process. It focuses in the main on human attributes like leadership, trust and agency, but gives little consideration to the role of objects in the development of inter-organisational collaborations. A central aim of this thesis is to understand the interaction of objects and humans in the development of a particular health and social care partnership in the North East of England. This socio-material perspective was achieved through actor-network theory (ANT) as a methodology, in which the researcher is equally sensitised to the role of human and non-human entities in the development of a network. The case study is that of the North East Lincolnshire Care Trust Plus (CTP). This was a unique health and social care collaboration arrangement between North East Lincolnshire Council and North East Lincolnshire Primary Care Trust, setup to address heath inequalities in the region. The CTP was conceived and developed at a local level by the respective organisation’s decision makers in the face of considerable opposition from regional policy makers and national regulators. However, despite this opposition, the directors eventually achieved their goal and the CTP became operational on 1st September 2007. This study seeks to understand how the CTP was conceived and developed, in the face of this opposition. The thesis makes a number of original contributions. Firstly, it adds to the current body of literature on collaboration by identifying how objects can help problematize issues and cement inter-organisational collaborations. Secondly it provides a novel account describing how two public sector organisations created a unique collaboration, despite pressing resistance from the regulatory authorities; and thirdly it extends Callon’s (1996) notion of problematization to examine how, what is rather vaguely described as ‘context’ in the literature, becomes enmeshed in decisions to collaborate. UNTIL 03/02/2016 THIS THESIS IS ONLY AVAILABLE FOR CONSULTATION AT ASTON UNIVERSITY LIBRARY WITH PRIOR ARRANGEMENT

Pichia pastoris regulates its gene-specific response to different carbon sources at the transcriptional, rather than the translational, level

Background: The methylotrophic, Crabtree-negative yeast Pichia pastoris is widely used as a heterologous protein production host. Strong inducible promoters derived from methanol utilization genes or constitutive glycolytic promoters are typically used to drive gene expression. Notably, genes involved in methanol utilization are not only repressed by the presence of glucose, but also by glycerol. This unusual regulatory behavior prompted us to study the regulation of carbon substrate utilization in different bioprocess conditions on a genome wide scale. Results: We performed microarray analysis on the total mRNA population as well as mRNA that had been fractionated according to ribosome occupancy. Translationally quiescent mRNAs were defined as being associated with single ribosomes (monosomes) and highly-translated mRNAs with multiple ribosomes (polysomes). We found that despite their lower growth rates, global translation was most active in methanol-grown P. pastoris cells, followed by excess glycerol- or glucose-grown cells. Transcript-specific translational responses were found to be minimal, while extensive transcriptional regulation was observed for cells grown on different carbon sources. Due to their respiratory metabolism, cells grown in excess glucose or glycerol had very similar expression profiles. Genes subject to glucose repression were mainly involved in the metabolism of alternative carbon sources including the control of glycerol uptake and metabolism. Peroxisomal and methanol utilization genes were confirmed to be subject to carbon substrate repression in excess glucose or glycerol, but were found to be strongly de-repressed in limiting glucose-conditions (as are often applied in fed batch cultivations) in addition to induction by methanol. Conclusions: P. pastoris cells grown in excess glycerol or glucose have similar transcript profiles in contrast to S. cerevisiae cells, in which the transcriptional response to these carbon sources is very different. The main response to different growth conditions in P. pastoris is transcriptional; translational regulation was not transcript-specific. The high proportion of mRNAs associated with polysomes in methanol-grown cells is a major finding of this study; it reveals that high productivity during methanol induction is directly linked to the growth condition and not only to promoter strength.

Dialogue, partnership and empowerment for network and information security:the changing role of the private sector from objects of regulation to regulation shapers

The protection of cyberspace has become one of the highest security priorities of governments worldwide. The EU is not an exception in this context, given its rapidly developing cyber security policy. Since the 1990s, we could observe the creation of three broad areas of policy interest: cyber-crime, critical information infrastructures and cyber-defence. One of the main trends transversal to these areas is the importance that the private sector has come to assume within them. In particular in the area of critical information infrastructure protection, the private sector is seen as a key stakeholder, given that it currently operates most infrastructures in this area. As a result of this operative capacity, the private sector has come to be understood as the expert in network and information systems security, whose knowledge is crucial for the regulation of the field. Adopting a Regulatory Capitalism framework, complemented by insights from Network Governance, we can identify the shifting role of the private sector in this field from one of a victim in need of protection in the first phase, to a commercial actor bearing responsibility for ensuring network resilience in the second, to an active policy shaper in the third, participating in the regulation of NIS by providing technical expertise. By drawing insights from the above-mentioned frameworks, we can better understand how private actors are involved in shaping regulatory responses, as well as why they have been incorporated into these regulatory networks.