Multi-scale Modelling of Allostery in Protein Homodimers

Allostery is a form of signalling within biomolecules such that ligand binding to a protein affects its activity at a second site. Allostery was described by early models to be driven by structural changes in the protein. However, more recently there has been increasing evidence that dynamics can contribute to or even drive allostery. The protein studied in this thesis, the Catabolite Activator Protein (CAP), is an allosteric protein homodimer that has been shown to exhibit negatively cooperative binding of the ligand cyclic Adenosine Monophosphate (cAMP) to each of its monomers. Interestingly, CAP is a protein whose allostery is believed to be driven by dynamics rather than a conformational change. In this thesis, a number of coarse grained models are employed to investigate this dynamic allostery in CAP. One family of models, termed Super Coarse Grained (SCG) models explore the global properties of the dynamics of the CAP dimer that cause it to exhibit negatively cooperative allostery. It is shown through these models that changes in protein interactions can provide a basis for changing cooperativity. A second family of coarse grained models called Elastic Network Models (ENM) are studied. These are used to show that adjusting the interactions between specific residues can affect cooperative binding of cAMP to CAP. A number of atomistic approaches are also used to study the cAMP-CAP system, including Molecular Dynamics (MD) and Normal Mode Analysis (NMA). The efficacy of using such approaches for studying the thermodynamics of the allostery in CAP is investigated. The motion observed within the protein is also studied closely to identify potential allosteric pathways. X-ray crystallography and Isothermal Titration Calorimetry (ITC) are finally used to investigate how accurately computational methods can describe the cooperative binding of cAMP to CAP. They are also used to try and determine whether the allostery in CAP can be manipulated experimentally without any observed changes to its structure

Upgrading New Zealand's competitive advantage: a critique and some proposals

The competitve advantage of nations by M. Porter, and Upgrading New Zealand's competitive advantage by G. Crocombe, M. Enright and M. Porter, both contain a number of fascinating - though often unbacked - propositions concerning the optimal set of rules for an economy. In particular, Upgrading New Zealand's competitive advantage seeks to stimulate debate on those rules that will lift New Zealand out of its current recession and generate sustainable economic growth. Using the New-Classical paradigm, this paper constructs a framework within which these propositions can be rigorously analysed. In addition, a small number of modest proposals are made

Multiple lineage specific expansions within the guanylyl cyclase gene family

BACKGROUND: Guanylyl cyclases (GCs) are responsible for the production of the secondary messenger cyclic guanosine monophosphate, which plays important roles in a variety of physiological responses such as vision, olfaction, muscle contraction, homeostatic regulation, cardiovascular and nervous function. There are two types of GCs in animals, soluble (sGCs) which are found ubiquitously in cell cytoplasm, and receptor (rGC) forms which span cell membranes. The complete genomes of several vertebrate and invertebrate species are now available. These data provide a platform to investigate the evolution of GCs across a diverse range of animal phyla. RESULTS: In this analysis we located GC genes from a broad spectrum of vertebrate and invertebrate animals and reconstructed molecular phylogenies for both sGC and rGC proteins. The most notable features of the resulting phylogenies are the number of lineage specific rGC and sGC expansions that have occurred during metazoan evolution. Among these expansions is a large nematode specific rGC clade comprising 21 genes in C. elegans alone; a vertebrate specific expansion in the natriuretic receptors GC-A and GC-B; a vertebrate specific expansion in the guanylyl GC-C receptors, an echinoderm specific expansion in the sperm rGC genes and a nematode specific sGC clade. Our phylogenetic reconstruction also shows the existence of a basal group of nitric oxide (NO) insensitive insect and nematode sGCs which are regulated by O(2). This suggests that the primordial eukaryotes probably utilized sGC as an O(2 )sensor, with the ligand specificity of sGC later switching to NO which provides a very effective local cell-to-cell signalling system. Phylogenetic analysis of the sGC and bacterial heme nitric oxide/oxygen binding protein domain supports the hypothesis that this domain originated from a cyanobacterial source. CONCLUSION: The most salient feature of our phylogenies is the number of lineage specific expansions, which have occurred within the GC gene family during metazoan evolution. Our phylogenetic analyses reveal that the rGC and sGC multi-domain proteins evolved early in eumetazoan evolution. Subsequent gene duplications, tissue specific expression patterns and lineage specific expansions resulted in the evolution of new networks of interaction and new biological functions associated with the maintenance of organismal complexity and homeostasis

The anhydrobiotic potential and molecular phylogenetics of species and strains of Panagrolaimus (Nematoda, Panagrolaimidae)

Members of the genus Panagrolaimus are bacterialfeeding nematodes that occupy a diversity of niches ranging from Antarctic and temperate soils to terrestrial mosses. Some members of this genus are able to survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. We have assembled a collection of Panagrolaimus species and strains and have investigated their anhydrobiotic phenotypes. Our data show that within the genus Panagrolaimus there is a continuum of strains ranging from those unable to survive exposure to low relative humidity (RH) without prior preconditioning at high RH (slow desiccation strategists), through strains that have limited ability to survive rapid desiccation but whose anhydrobiotic ability improves upon preconditioning, to strains such as P. superbus that can readily survive immediate exposure to severe desiccation (fast desiccation strategists). Using this panel of nematodes we investigated the effect of preincubation at high RH on the accumulation of trehalose and on the nematodesâ anhydrobiotic potential. We found that there is a strong correlation between trehalose induction and anhydrobiotic survival in Panagrolaimus. Furthermore, the high trehalose levels observed in fully hydrated P.superbus (10% dry mass) suggest that constitutive expression of trehalose pre-adapts this fast dehydration strategist to combat desiccation. All the strains observed, regardless of survival rates, undertook both coiling and clumping, which has the effect of reducing surface area and slowing the rate of water loss during desiccation. Phylogenetic analyses were carried out to investigate whether the observed anhydrobiotic phenotypes were the result of convergent evolution or represented a single phylogenetic lineage. These analyses, derived from alignments of the rDNA ITS and D3 sequences, indicate that the strongly anhydrobiotic strains of Panagrolaimus form a single phylogenetic lineage, which is separate from the weakly anhydrobiotic strains. The weakly anhydrobiotic strains are also phylogenetically divergent from each other. Our data indicate that Panagrolaimus has the potential to be an excellent model system for the investigation of molecular aspects of nematode anhydrobiosis

The anhydrobiotic potential and molecular phylogenetics of species and strains of Panagrolaimus (Nematoda, Panagrolaimidae)

Members of the genus Panagrolaimus are bacterialfeeding nematodes that occupy a diversity of niches ranging from Antarctic and temperate soils to terrestrial mosses. Some members of this genus are able to survive extreme desiccation by entering into a state of suspended animation known as anhydrobiosis. We have assembled a collection of Panagrolaimus species and strains and have investigated their anhydrobiotic phenotypes. Our data show that within the genus Panagrolaimus there is a continuum of strains ranging from those unable to survive exposure to low relative humidity (RH) without prior preconditioning at high RH (slow desiccation strategists), through strains that have limited ability to survive rapid desiccation but whose anhydrobiotic ability improves upon preconditioning, to strains such as P. superbus that can readily survive immediate exposure to severe desiccation (fast desiccation strategists). Using this panel of nematodes we investigated the effect of preincubation at high RH on the accumulation of trehalose and on the nematodesâ anhydrobiotic potential. We found that there is a strong correlation between trehalose induction and anhydrobiotic survival in Panagrolaimus. Furthermore, the high trehalose levels observed in fully hydrated P.superbus (10% dry mass) suggest that constitutive expression of trehalose pre-adapts this fast dehydration strategist to combat desiccation. All the strains observed, regardless of survival rates, undertook both coiling and clumping, which has the effect of reducing surface area and slowing the rate of water loss during desiccation. Phylogenetic analyses were carried out to investigate whether the observed anhydrobiotic phenotypes were the result of convergent evolution or represented a single phylogenetic lineage. These analyses, derived from alignments of the rDNA ITS and D3 sequences, indicate that the strongly anhydrobiotic strains of Panagrolaimus form a single phylogenetic lineage, which is separate from the weakly anhydrobiotic strains. The weakly anhydrobiotic strains are also phylogenetically divergent from each other. Our data indicate that Panagrolaimus has the potential to be an excellent model system for the investigation of molecular aspects of nematode anhydrobiosis

Political strategies of external support for democratization

Political strategies of external support to democratization are contrasted and critically examined in respect of the United States and European Union. The analysis begins by defining its terms of reference and addresses the question of what it means to have a strategy. The account briefly notes the goals lying behind democratization support and their relationship with the wider foreign policy process, before considering what a successful strategy would look like and how that relates to the selection of candidates. The literature's attempts to identify strategy and its recommendations for better strategies are compared and assessed. Overall, the article argues that the question of political strategies of external support for democratization raises several distinct but related issues including the who?, what?, why?, and how? On one level, strategic choices can be expected to echo the comparative advantage of the "supporter." On a different level, the strategies cannot be divorced from the larger foreign policy framework. While it is correct to say that any sound strategy for support should be grounded in a theoretical understanding of democratization, the literature on strategies reveals something even more fundamental: divergent views about the nature of politics itself. The recommendations there certainly pinpoint weaknesses in the actual strategies of the United States and Europe but they have their own limitations too. In particular, in a world of increasing multi-level governance strategies for supporting democratization should go beyond preoccupation with just an "outside-in" approach

High resolution magnetic microscopy based on semi-encapsulated graphene Hall sensors

The realization of quantitative, noninvasive sensors for ambient magnetic imaging with high spatial and magnetic field resolution remains a major challenge. To address this, we have developed a relatively simple process to fabricate semi-encapsulated graphene/hBN Hall sensors assembled by dry transfer onto pre-patterned gold contacts. 1 lm-sized Hall cross sensors at a drive current of 0.5 lA exhibit excellent room temperature sensitivity, SI 700 V/AT, and good minimum detectable fields, Bmin ¼ 0.54 G/Hz0.5 at a measurement frequency of 1 kHz, with considerable scope for further optimization of these parameters. We illustrate their application in an imaging study of labyrinth magnetic domains in a ferrimagnetic yttrium iron garnet film

Aid Allocation of the Emerging Central and Eastern European Donors

The paper examines the main characteristics of the (re)emerging foreign aid policies of the Visegrád countries (the Czech Republic, Hungary, Poland, Slovakia), concentrating on the allocation of their aid resources. We adopt an econometric approach, similar to the ones used in the literature for analyzing the aid allocation of the OECD DAC donors. Using this approach, we examine the various factors that influence aid allocation of the Visegrád countries, using data for the years between 2001 and 2008. Our most important conclusion is that the amount of aid a partner county gets from the four emerging donors is not influenced by the level of poverty or the previous performance (measured by the level of economic growth or the quality of institutions) of the recipients. The main determining factor seems to be geographic proximity, as countries in the Western-Balkans and the Post-Soviet region receive much more aid from the Visegrád countries than other recipients. Historical ties (pre-1989 development relations) and international obligations in the case of Afghanistan and Iraq are also found to be significant explanatory factors. This allocation is in line with the foreign political and economic interests of these new donors. While there are clear similarities between the four donors, the paper also identifies some individual country characteristics

Synthesis of a series of novel 3,9-disubstituted phenanthrenes as analogues of known <i>N</i>-methyl-D-aspartate receptor allosteric modulators

9-Substituted phenanthrene-3-carboxylic acids have been reported to have allosteric modulatory activity at the NMDA receptor. This receptor is activated by the excitatory neurotransmitter L-glutamate and has been implicated in a range of neurological disorders such as schizophrenia, epilepsy and chronic pain and neurodegenerative disorders such as Alzheimer’s disease. Herein, the convenient synthesis of a wide range of novel 3,9-disubstituted phenanthrene derivatives starting from a few common intermediates is described. These new phenanthrene derivatives will help to clarify the structural requirements for allosteric modulation of the NMDA receptor