This thesis investigates the potential for bamboo to be a feedstock for the production of lignocellulosic biofuels. This was assessed from the perspectives of technology, economics and policy, and discusses the main drivers that could help or hinder the viability of such a process at the commercial scale. Three pretreatment technologies (liquid hot water, soaking in aqueous ammonia and dilute acid) and various enzyme loadings were explored as potential processing routes for converting bamboo into bioethanol; these conditions were compared based on the criteria of maximising sugar release and thus total ethanol production. Aspen Plus modelling software was used to simulate the conversion process at a scaled up level of 2,000 dry metric tonnes of bamboo per day. The generated mass and energy balances were used in a discounted cash flow analysis to yield the minimum production price for bamboo-derived bioethanol based on a reference year of 2011. Two case study scenarios (China and Colombia) were modelled as potential locations for establishing a bamboo to bioethanol process. Bioethanol from bamboo in China and Colombia was also compared with bioethanol derived from other lignocellulosic feedstocks (wheat straw and short-rotation coppice poplar) in various locations, and its competitiveness with fossil-based fuels at the pump was assessed. This research demonstrated that bioethanol from bamboo can be just as, if not more competitive than bioethanol from other lignocellulosic resources provided that certain criteria are fulfilled. The main determinants responsible for establishing a future in the alternative fuel market included maintaining a balance between the major factors relating to technology (achieving relatively high sugar yields with low enzyme usage), economics (obtaining low costs for feedstock and high credits for electricity generation), and policy (establishing these conditions in a location with strong policy support for bioethanol).

Mismatch uracil DNA Glycosylase (MUG) from Escherichia coli is an initiating enzyme in the base excision repair (BER) pathway and is responsible for the removal of 3,N4-ethenocytosine and uracil from DNA during the stationary phase of E.coli cell growth. As with other DNA glycosylases, the abasic product is potentially more harmful than the initial lesion. MUG is widely regarded as a 'single turnover' enzyme because it still remains tightly bound to its abasic product after cleavage, thus impeding its catalytic turnover. This may be a general protective mechanism to protect the abasic BER intermediate, whereby coordination of enzyme activity in BER is achieved through displacement of the DNA glycosylase by the downstream apurinic-apyrimidinic (AP) endonuclease. Numerous DNA glycosylases have now been cited as having an enhanced turnover in the presence of an AP endonuclease. The aim of this project is to investigate enzyme coordination between MUG and its both downstream AP endonucleases, Exonuclease III (ExoIII) and Endonuclease IV (EndoIV), in the initial steps of BER. We show here that MUG binds its substrate, abasic DNA and non-specific DNA in the differential modes. A 2:1 cooperative binding stoichiometry with abasic DNA is demonstrated to be of functional significance in both product binding and catalysis via fluorescence anisotropy assays, band shift assays and loss-of-function site-directed mutagenesis methods. The effects of the ExoIII and EndoIV on the MUG turnover kinetics with a U·G containing substrate was investigated. Both ExoIII and EndoIV greatly enhance the turnover of MUG. Furthermore, the analysis of both ExoIII catalytic activity dependent and concentration dependent on MUG turnover demonstrate ExoIII may employ a product scavenging mechanism to enhance MUG turnover. These combined results constitute a new concept that MUG has a pre-catalytic discrimination ability to coordinate its reactivity behavior with the other enzymes.

Roads are a distinctive feature in any landscape, with many countries giving 1-2% of their land surface over to roads and roadsides (Forman 1998). However, the ecological effects of roads spread beyond the physical footprint of the network and may impact 15-20% of the land or more (Forman & Alexander 1998). The Brazilian Amazon contains approximately one third of the world's remaining rainforest, covering an area of 4.1 million km2. The region is highly biodiverse with 10-20 percent of the planet's known species, it is also one of the three most bioculturally diverse areas in the world (Loh & Harmon 2005), and it provides many valuable ecosystem services. However, the Brazilian Amazon is rapidly undergoing extensive development with widespread land-use conversion. Road development is often perceived as the initial stage of development, opening access to remote areas for colonisation, agriculture development, resource extraction, and linked with these; deforestation (Chomitz & Gray 1996, Laurance et al. 2001, Perz et al. 2007, Laurance et al. 2009, Caldas et al. 2010). As such roads are a key spatial determinant of land use conversion in the Amazon region, dictating the spatial pattern of deforestation and biodiversity loss (Fearnside 2005, Kirby et al. 2006, Perz et al. 2008). Given that roads are a key spatial determinant of land use conversion and that they have extensive impacts on rates and patterns of habitat loss, it is important that we know how much, how fast and where road networks are developing in this globally important ecosystem. In this thesis, I aim to construct models of road network development to help better understand and predict the impacts of economic development in the Brazilian Amazon.

No description available.

Barley (Hordeum vulgare L.) is an important crop grown throughout the world. Quality and quantity of barley is compromised by abiotic stresses, salinity in particular which is already widespread in many regions. Mitogen Activated Protein Kinase (MAPK) cascades are signal transduction pathways involved in biotic and abiotic plant defence mechanisms. Here, the function of the barley gene HvBWMK1 has been investigated. Transgenic barley plants that overexpress HvBWMK1, or in which HvBWMK1 has been down-regulated by antisens, were generated, as well as empty vector control plants. The transformation efficiency using Agrobacterium was 10.4% independent transgenic plant per embryo. Northern blot analysis of HvBWMK1 primary overexpression lines showed up-regulation of mRNA level, conversely the antisense lines of HvBWMK1 showed clear down-regulation. Real-time PCR analysis in the T1 generation revealed that two over-expresser lines showed higher expression as compared with wild type (by 79% and 35%) while the HvBWMK1 antisense construct reduced endogenous gene expression partially (by 14%, 23% and 39% compared with wild type). Barley seedlings were subjected to two weeks of salt stress (150 mM NaCl) then biochemical and physiological parameters were measured. The overexpression lines showed an increase in tolerance to salinity stress compared to antisense lines and controls. Tolerance was accompanied with increasing endogenous proline and chlorophyll levels and a reduction in hydrogen peroxide content after salinity exposure. Overall these results suggest that the barley MAPK HvBWMK1 acts as a positive regulator in barley tolerance to salinity stress.

Cell growth, the addition of mass and volume, is required for the development and homeostasis of all organisms. In mammalian cells, cell growth and size homeostasis usually requires an instructive signal in the form of a growth factor with loss of this signal resulting in cell atrophy. The primary Schwann cell has proven a powerful system to study cell growth in vitro – underlining the requirement of growth factors for mammalian cells biogenesis. Using this system, IGF-1 was identified as a Schwann cell growth factor that drives cell volume addition. In contrast, NRG-1 has no effect on Schwann cell volume, but does drive mitochondrial biogenesis - highlighting that cell growth can be non-uniform, diverging from the simple coordinate addition of volume and organelles. In this thesis, I demonstrate that the addition of mass and volume can be uncoupled during cell growth. IGF-1 drives coordinate addition of cell mass and volume, whereas sustained Raf/MEK/ERK activation drives addition of protein mass and specific organelles in the absence of an increase in cell volume. Furthermore, the addition of volume can be uncoupled from mass accumulation, downstream of IGF-1. This demonstrates that factors other than protein mass limit volume addition. To investigate the regulation of cell volume I took two approaches: Firstly, a cellular approach - comparing plasma membrane dynamics in growing and growth factor-starved cells. I show that membrane turnover is extremely fast with the whole membrane internalised ~ three times an hour. Moreover, this rapid and ATP-dependent rate is maintained in starved, autophagic cells - indicating that it is an essential cellular process. The speed of membrane turnover however, precluded using this approach to identify how cells deliver membrane to the cell surface to drive plasma membrane expansion. Secondly, a biochemical approach - to identify important signalling pathways. I identify a critical role for de novo lipogenesis for both the addition and maintenance of cell volume and SREBP-2 as an essential transcription factor mediating this process.

This project aims at understanding how cell fate decision emerges from the overall intracellular network connectivity and dynamics. To achieve this goal both small paradigmatic signalling-gene regulatory networks and their generalization to highdimensional space were tested. Particularly, we drew special attention to the importance of the effects of time varying parameters in the decision genetic switch with external stimulation. The most striking feature of our findings is the clear and crucial impact of the rate with which the time-dependent parameters are changed. In the presence of small asymmetries and fluctuations, slow passage through the critical region increases substantially specific attractor selection by external transient perturbations. This has strong implications for the cell fate decision problem since cell phenotype in stem cell differentiation, cell cycle progression, or apoptosis studies, has been successfully identified as attractors of a whole network expression process induced by signalling events. Moreover, asymmetry and noise naturally exist in any integrative intracellular decision network. To further clarify the importance of the rate of parameter sweeping, we also studied models from non-equilibrium systems theory. These are traditional in the study of phase transitions in statistical physics and stood as a fundamental tool to extrapolate key results to intracellular network dynamics. Specifically, we analysed the effects of a time-dependent asymmetry in the canonical supercritical pitchfork bifurcation model, both by numerical simulations and analytical solutions. We complemented the discussion of cell fate decision with a study of the effects of non-specific targets of drugs on the Epidermal Growth Factor Receptor pathway. Pathway output has long been correlated with qualitative cell phenotype. Cancer network multitargeting therapies were assessed in the context of whole network attractor phenotypes and the importance of parameter sweeping speed.

Combinatorial Domain Hunting (CDH) is a recently introduced technology that addresses the problem of obtaining solubly expressed protein domains, or more correctly polypeptide truncates, from a larger target. CDH relies upon enzymatic fragmentation of a template gene, non-directional cloning of these fragments into a set of bespoke expression vectors, and screening of the resulting library of clones for the expression of soluble, affinity-tagged, protein fragments. Prior to the work described in this thesis the CDH concept had only been described in outline. The thesis describes various aspects of the application of CDH to a model target, incorporating modifications to the CDH pipeline that were adopted on the basis of this application, as well as development of an extension to the CDH concept that introduces the potential to screen for soluble expression of high-affinity protein-protein complexes. CDH was applied to Hsp90, a well-characterised molecular chaperone with predicted modular structure. Following fragmentation of the Hsp90 cDNA, a library of 15,000 clones was produced and 11,500 colonies were screened for expression of soluble Hsp90 truncates, resulting in 107 preliminary hits, of which seven were duplicates. Assessment of a subset of these hits indicated a significant potential for soluble protein aggregates to survive the affinity purification/ filtration stage of CDH. Implementation of an additional centrifugation step, although initially at some cost to the overall timescale of a CDH screen, was shown to reduce the number of putative false-positive, soluble protein aggregates identified (by 45% in the case of Hsp90, resulting in 55 unique hits). The advantage derived from this modification is that fewer preliminary hits would be taken forward for biochemical and structural validation. The original CDH concept invoked the use of NMR spectroscopy for the assessment of truncate foldedness. In the context of the real world application of CDH to Hsp90, it was found that such assessment was inefficient in terms of the amount of material required per preliminary hit. The thesis describes the application of differential scanning fluorimetry (DSF, also known as ‘Thermofluor’) analysis to the collection of Hsp90 CDH hits, demonstrating significant efficiency savings in the assessment of foldedness, most dramatically in that much smaller amounts of protein were required than for NMR. For the Hsp90 screen, DSF analysis indicated that four out of the 41 (14 of the 55 did not express protein) preliminary CDH hits corresponded to folded truncates (validated by 1D 1H NMR). The 41 hits were also characterised by size exclusion chromatography, where 12 eluted mainly in the void volume, and 11 others with greater than 40% in the void volume, possibly indicating that the centrifugation step applied at small scale, does not reliably eliminate those truncates with the potential to self-associate at a preparative scale. A CDH screen, including the modifications derived in the Hsp90 example, was applied to the Mycobacterium tuberculosis histidine kinase DosS. 52,000 colonies were screened of a 100,000 clone library which resulted in 23 preliminary hits. Bioinformatic analysis indicated that one of the hits covered the putative tandem histidine kinase and histidine kinase acceptor domains. The thesis describes biochemical and biophysical validation of the function and foldedness of this DosS truncate, and enumerates initial attempts at crystallisation and NMR analysis. For a given protein target, identification of folded domains may require the presence of an obligate partner chain: with this in mind, this thesis introduces a novel adaptation of the CDH paradigm, dubbed 2C-CDH, in which the fragment library of one partner protein isolated with one affinity tag, is screened on the basis of immunodetection of an orthogonal tag appended to the other partner. The design and implementation of an appropriate two plasmid orthogonal expression-and detection system is described. A proof-of-principle application of the 2C-CDH concept is provided, using the complex formed between Hsp90 and Cdc37 proteins. Two screens were performed, using a known soluble fragment of either Hsp90 or Cdc37 (as bait) screened against a fragment library (as prey) derived from Cdc37 and Hsp90 respectively. In each case the 2C-CDH procedure identified one or more truncates in the prey library with amino acid sequence boundaries consistent with the known characteristics of the of the Hsp90-Cdc37 structure. Moreover, for each hit, size exclusion chromatography analysis validated maintenance of a well-behaved 1:1 complex at preparative scale. This thesis concludes with a commentary upon the CDH concept in the context of related current and possible future paradigms for soluble protein expression.

Cell death within an epithelium could cause a loss of barrier function and damage to surrounding tissues. Mechanisms have therefore evolved to remove dead or dying cells, whilst maintaining epithelial integrity. This study has used monolayers of MDCK (Madin Darby Canine Kidney) cells as a model system to investigate these processes. It has been reported that shape changes in the surrounding cells, combined with junctional remodelling, are responsible for preserving the barrier function at sites of cell death. The aim was to identify and characterise the mechanisms that generate these shape changes; a particular focus was to establish the involvement of dying cells and their healthy neighbours in driving the epithelial repair. 3D time-lapse imaging was performed of UV-exposed MDCK cells, stably transfected with fluorescent reporters. This revealed distinct phases of actin and myosin activity at sites of cell death. To test the functional involvement of actin-myosin contraction, experiments were performed using a myosin inhibitor and also a Rho-defective cell line. The use of cells plated in expression mosaics made it possible to isolate the actin and myosin activity in dying cells and their neighbours. As an alternative means of inducing cell death, live imaging was also performed of cells killed via laser-ablation. The experimental results are complemented by a simple computational model, which simulates the forces acting at sites of cell death in MDCK monolayers. The results show that in UV-treated monolayers, the removal of apoptotic cells is a two stage process, the first of which has not previously been described. During this first stage, an actin ring within the dying cell constricts via actin-myosin contraction over a period of 15 to 30 minutes. This pulls the surrounding cells into a multi-cellular rosette in the apical plane, to cover the space that was occupied by the apoptotic cell. Simultaneously, the ring closure scissions its surface and encloses the cell body within the monolayer. The second stage of the repair process is driven by an actin ring in the surrounding cells, which moves towards the basal plane to extrude the cell remnants over 30 to 40 minutes. Crawling of the neighbour cells then heals the basal area before the membrane of the dying cell permeabilises. The computational modelling supports the experimental findings, predicting that the observed cellular shape changes must arise due to additional actin-myosin activity, and cannot arise passively due to network forces. In contrast to the UV-treated cells, those killed via laser ablation did not exhibit the first phase of the repair process. These necrotic cells remained in situ, constituting a leakage point while the neighbouring cells healed the wound and extruded the remnants. This identifies a difference in the removal of apoptotic and necrotic cells, since the active participation of the former allows a continuous monolayer to be maintained. This study reveals that epithelial repair cell due to apoptosis involves two phases, the first of which is driven by the dying cell and the second by its neighbours. In combination, this activity ensures that epithelial integrity is maintained and apoptotic cells are removed without damage to surrounding tissues.

AMP-activated protein kinase (AMPK) serves as an energy-sensing protein that is activated by a variety of metabolic stresses. Recent studies suggest that AMPK is also regulated by hormones and by nutrients such as glucose and fatty acids. In skeletal muscle it was previously shown that AMPK activity was decreased with increasing glucose concentration. In the present study both the activity and the Threonine-172 phosphorylation of AMPK in incubated rat ventricular cardiac myocytes were found to be decreased by increasing glucose in the presence and absence of palmitate. Glucose also caused a decrease in the AMPK-driven phosphorylation of acetyl-CoA carboxylase. Measurements of the myocyte contents of ATP, ADP, AMP and glycogen showed that the effect of glucose on AMPK activity could not be secondary to changes in the levels of these metabolites. The decrease in AMPK activity with glucose was additive to and distinct from the effect of insulin which is mediated through protein kinase B (PKB). Increasing glucose concentration had no effect on the phosphorylation of Threonine-308 and Serine-473 in PKB. AICAR, a pharmacological activator of AMPK, had no effect on the ability of glucose to inactivate AMPK. The myocyte content of the pentose phosphate pathway (PPP) metabolite xylulose 5-phosphate (Xu5P), a known allosteric activator of PP2A, was increased with increasing glucose concentration such that AMPK activity was inversely related to Xu5P content. The glucose 6-phosphate dehydrogenase inhibitor diehydroepiandeosterone (DHA) and thiamine, the precursor of the coenzyme for transketolase, both increased AMPK activity whereas the NADPH oxidant phenazine methosulphate (PMS) decreased AMPK activity. DHA and PMS respectively decreased and increased flux through the PPP. The findings suggest that inactivation of AMPK by glucose may be mediated by the activity of the PPP which sets the level of Xu5P. Two other findings were also made during the course of this project. First, the activity of phosphofructokinase-2 (PFK-2) in the perfused heart was previously shown to be activated through phosphorylation by AMPK. It was therefore expected that increasing glucose concentration would decrease myocyte PFK-2 activity. However PFK-2 activity was found to be increased by glucose. Second, the phosphorylation of Threonine-308 in PKB in response to insulin was appreciably increased in the presence of AICAR suggesting that there may be crosstalk between AMPK and the insulin signalling pathway at the level of PKB.