The HPr proteins were characterized as folding by a two-state folding mechanism. Here, we present a comparison of the equilibrium and kinetic folding for the HPr protein from Bacillus subtilis, E coli and a key variant from these proteins. For the wild-type protein we find that GHX is greater than GUDC, suggesting that the HPr does not fold by a simple two-state mechanism. This discrepancy is revealed by testing the two-state nature of the folding reaction of HPr with mutation. We show that removing a single charge side chain (Asp 69) converts the HPr protein back to a simple two-state mechanism. Ribonuclease Sa and two charge-reversal variants can be converted into amyloidin vitro by the addition of 2,2,2-triflouroethanol (TFE). We report here amyloid fibril formation for these proteins as a function of pH. The pH at maximal fibril formation correlates with the pH dependence of protein solubility, but not with stability, for these variants. Additionally, we show that the pH at maximal fibril formation for a number of ivwell-characterized proteins is near the pI, where the protein is expected to be the least soluble. This suggests that protein solubility is an important determinant of fibril formation.

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Student Number : 9306227A - PhD thesis - School of Molecular and Cell Biology - Faculty of Science / It is well documented that N-capping motifs are stabilising local motifs for -helices. N-capping motifs have been identified within hGST A1-1 at the N-terminal ends of -helix 9 and helix 6. The conservational role of these two motifs in protein stability, folding and function was investigated. -Helix 9 is a unique structural feature to class Alpha GSTs that is important for its catalytic functioning. This amphipathic helix is highly dynamic, where upon ligand binding at the active-site, the delocalised C-terminal region becomes immobilised to form a structured helix forming a “lid” over the active-site. The specific role of the Asp N-cap motif toward the stability and dynamics of helix 9 was determined by substituting the Asp-209 for a Gly. ANS binding and urea-induced activity studies showed that by removing the N-cap motif of helix 9 in hGST A1-1, the helix 9 is destabilised rendering a less hydrophobic binding site compared to that in the wild-type. The helical content of the peptide, corresponding to helix 9 in the C-terminal region of hGST A1-1 (208 -222), decreased significantly upon the removal of the N-cap motif. The explanation for the conservation of the Asp N-cap residue can be found in its stabilising role of the C-terminal region of class Alpha GSTs. This stabilising role was however less apparent in context of the protein compared to that in the peptide. Majority of the atomic contacts owing to the stability of helix 9 appear to be governed by non-local tertiary interactions rather than local interactions, such as the N-cap motif. These tertiary interactions are likely to include short and long range contacts between residues on the surface of the protein that are already known to contribute towards the stability of the C-terminal region. In this study, the ligand displacement-studies and the molecular docking results strongly suggest that 8-aniline-1-napthalene sulfonate binds at the H-site in hGST A1-1. The N-capping motif of helix 6 identified in class Alpha GSTs is located within the core of domain 2. This motif is a common feature found amongst almost all GST-like proteins and is thought to be the folding nucleation site (Stenberg et al. J. Biol. Chem. 275 (2000), 10421-10428). The N-cap (Ser- 154) and N3 (Asp-157) residues were each substituted with an Ala in hGST A1-1 to investigate the role of this motif in the folding of hGST A1-1. Both substitutions resulted in thermal sensitive mutants compared to that of the wild-type. The N3 substitution (D157A) was however too disruptive, where the yields of this mutant were insufficient for any further studies to be carried out. For the N-cap mutant (S154A), the unfolding kinetic studies revealed a significantly destabilised core in domain 2 compared to that of the wild-type. The kinetic folding studies monitored by fluorescence spectroscopy, revealed that the N-cap motif contributes to the efficient folding and dimerisation of the subunits, and to a far lesser extent towards the final tight packing and reorganisation of tertiary interactions in hGST A1-1. Since no changes in the burst-phase of S154A was evident compared to that of the wild-type, it seems unlikely that this motif is a folding nucleation site in hGST A1-1. These results do not exclude the possibility that this motif contributes to the rapid formation secondary structure during the burst-phase of folding. Due to the highly conserved region surrounding helix 6 , the role of this motif contributing to the stability of hGST A1-1 could be a general feature for GSTs and GSTlike proteins. In this study, further insight into the mechanism of folding for hGST A1-1 was gained. The hydrophobic core packing surrounding helix 6 occurred as a late folding event, that is during the final packing and reorganisation of tertiary interactions of the protein. The N-cap motif is an important structural feature for the fast folding of domain 2. This N-cap motif is a unique structural feature important for the efficient folding of the monomers, which is exclusive to its role in stabilising helix 6 in hGST A1-1.

This thesis presents a linearized technique to determine a risk-based index for dynamic security. The method is an extension to an existing technique in which the risk of steady state security is calculated using the mean and variance of load uncertainty. The proposed method is applied to calculate the risk indices for the New England 39 bus test system. The results obtained from the proposed method are validated against those estimated by Monte Carlo simulation. Both approaches produce virtually the same results for small load deviations.

This thesis presents a linearized technique to determine a risk-based index for dynamic security. The method is an extension to an existing technique in which the risk of steady state security is calculated using the mean and variance of load uncertainty. The proposed method is applied to calculate the risk indices for the New England 39 bus test system. The results obtained from the proposed method are validated against those estimated by Monte Carlo simulation. Both approaches produce virtually the same results for small load deviations.

This dissertation consists of three chapters. In the first chapter, we study the role of financial frictions on the demand side of the economy. In particular, we study the interaction between firm and household credit constraints over the business cycle. We construct a real business cycle model with explicit modeling of price and quantity side of housing. This allows us to include both firm and household financing frictions. The model is estimated for the U.S economy using quarterly data on key macroeconomic variables over the period 1970 - 2006. Household and firm financial accelerators operate primarily through movement in house and capital prices respectively. We find clear evidence of the operation of a financial accelerator mechanism, whereby shocks to the economy are amplified most in the presence of both types of frictions, as opposed to just firm or household frictions. Over the business cycle, total factor productivity shocks in the non-housing sector explain about half of the volatility of GDP and consumption. However, cyclical variations in housing investment and housing prices are predominantly explained by housing preference and housing technology shocks. Finally, spillovers from household financing frictions are mostly concentrated in consumption. However, they also affect business investment via its impact on the demand for capital and consequently its price. The second chapter focuses on financial frictions on the supply side. We study the role of bank capital in the transmission of shocks to the economy. Given the evolutionary change in the financial services industry and the growth of shadow banking in the decades prior to the global recession, we characterize credit intermediation with a heterogeneous banking sector comprised of traditional retail and shadow banking. We approach the shadow banking system from a regulation perspective wherein commercial banks have incentives to transfer loans from on- to off-balance sheet to gain regulatory relief. Since bank capital is costly, banks cover part of their funding needs by loan sale in the secondary market. Furthermore, these transferred loans are bundled together and converted into liquid asset backed securities. Commercial banks’ effective return is subject to their monitoring effort, which is unobservable and hence introduces a moral hazard problem in loan sale. This limits the amount of loan sold in the secondary market. We find that loan sale and securitization enhances credit intermediation in normal times and improves the resilience of the system to productivity shocks. However, it also exposes the economy to shocks emerging in the financial system. In response to financial market shocks, the government via its backstop program, can ameliorate its impact on the economy. Finally, we compare the model economy with Basel I and Basel II capital requirement and find that business cycle fluctuations are amplified under Basel II regime. Furthermore, in response to a negative productivity shock there is a transfer of loans from on to off balance sheet under Basel II rules with procyclical capital constraints. This points towards a need for countercyclical capital requirement as being implemented under Basel III accord. In the third chapter, we focus on the question of trade off between price and financial stability goals for the conduct of monetary policy. The recent crisis has generated renewed interest in Hayekian theory and Minsky’s instability hypothesis, which claims that accommodative monetary policy can be harmful for an economy by promoting excessive risk taking – the so called risk taking channel of monetary policy transmission. Risk Taking Channel has been documented for the U.S and Euro area and we investigate the presence of this in Asia. Using annual and quarterly data on publicly listed banks in Asia, we find that when interest rates are too low - lower than a benchmark - bank risk increases. Furthermore, there is also a case for greater supervision and capital stringency to alleviate risk taking.

This work focuses on the development of a spray dried adenoviral vector for its application as a thermally stable and inhalable vaccine against tuberculosis. / As the global public health community continues to strive for more equitable vaccine access, thermal instability of liquid vaccines continues to be a significant challenge due to strict cold-chain temperature requirements. Dry powder vaccines offer a favourable alternative, with the ability to retain vaccine efficacy at ambient temperature conditions. In the form of dry powder, vaccines against respiratory diseases can also be administered via inhalation for targeted delivery to the lung tissue. A processing technique known as spray drying is particularly promising for the development of thermally stable and inhalable dry powder vaccines, offering a method of continuous and scalable production. Spray drying is widely used in the pharmaceutical industry and can effectively encapsulate and immobilize labile biologics, like adenoviral vectors, within a glassy carbohydrate matrix to help retain biologic function. However, pulmonary delivery of a thermally stable, viral vectored dry powder vaccine has yet to be demonstrated. This thesis focuses on improving the formulation of a carbohydrate excipient blend of mannitol and dextran encapsulating a human serotype 5 adenovirus (AdHu5), with the goal of producing an inhalable vaccine with sufficient viral potency for in vivo murine testing. First, the impact of cryoprotective agents used for frozen storage of the stock adenovirus was investigated with respect to viral activity retention, thermal stability and inhalation properties of the dry powder after spray drying. Trehalose was considered a preferred cryoprotective agent, compared to glycerol traditionally used for adenoviral cryo-storage, allowing for the preparation of a high potency viral dry powder with 1.5 log loss of viral titre after processing and thermal aging. Further investigation of the dextran mass ratio and dextran molecular weight used within the excipient blend revealed that incorporating mannitol in a 1:3 ratio with 500 kDa dextran can further improve viral activity to achieve 0.8 log loss of viral titre after aging. Through controlled drying dynamics, this formulation led to improved activity retention and thermal stability, in addition to desirable aerosolization properties for pulmonary delivery. Using this optimized formulation, custom-made intratracheal dosator devices were evaluated for pulmonary powder delivery in mice. The method of powder loading in the device was found to be a significant factor of device performance in vivo when determining if the critical powder mass dosage could be delivered. Successful intratracheal delivery of the AdHu5-vectored dry powder was achieved with a pipette-tip loading dosator and led to a strong bioactive response. Overall, this work indicates the feasibility of murine pulmonary delivery and immunological testing of a thermally stable, adenoviral-vectored vaccine in dry powder form. / Thesis / Master of Applied Science (MASc) / Most vaccines currently available on the market must be stored and transported at temperatures ranging from 2-8 ⁰C to properly maintain their function, with some vaccine requiring temperatures as low as -80 ⁰C. The equipment required to maintain such temperatures is costly and is a significant limitation for developing nations trying to secure vaccine access. As an alternative to traditional liquid vaccine formulations, dry powder vaccines offer stability at room temperature without the need for expensive equipment and can also be administered through inhalation. Using a processing method called spray drying, an active vaccine component can be encapsulated in a carefully selected sugar formulation which forms a protective coating as the particles dry to provide stabilization. Since the efficacy of such dry powder vaccines must be first evaluated with mouse models, the focus of this work was to improve an existing blend of sugars to produce a dry vaccine powder that contains high enough dosage for mouse testing. Processing losses from spray drying were minimized through careful selection of vaccine cryoprotective agents, in addition to optimizing the blend ratio and molecular weight of sugars used for encapsulation. Successful delivery of the optimized powder to the lungs of mice was also accomplished after analyzing the suitability of a variety of custom-made handheld devices. This work shows that inhalable dry powder vaccine delivery is a promising solution to help improve temperature stability and achieve more equitable access to vaccines globally.

<p>This objective of this study was to determine the impact of antimicrobial carcass washes on beef trim quality in the production of frankfurters. Twenty-four beef carcasses were randomly applied a different antimicrobial wash treatments (TRT) during the harvest procedure: 82° C water (CON), peroxyacetic acid (PAA), or lactic acid (LA). Beef carcasses were analyzed for microbial counts and carcass pH. Frankfurters were produced using carcass trim at two different batter temperature processes (PROC): 4°C (CP) or 21°C (HP). Frankfurters were analyzed for cook loss, emulsion stability (ES), color (Minolta L*, a*, b*) over 60-day storage, purge loss, texture, and sensory analysis. LA carcass had a lower pH (6.36; <i>P</i><0.001) 30 min post wash compared to other wash treatments. Frankfurters produced from CON trim had the highest ES water (<i>P</i><0.0001) and ES fat (<i>P</i><0.0001) separation where the LA and PAA treatments were not significantly different (<i>P</i>>0.05). The HP frankfurters had less ES water (<i>P</i><0.0001) and ES fat separation (<i>P</i><0.0001) when compared to CP. However, the CP had a higher cook yield (<i>P</i>=0.002). The HP frankfurters had higher internal and external L* values (<i>P</i><.0001; <i>P</i><.0001, respectively). The CP frankfurters had a higher a* (redness) internal color values (<i>P</i><.0001). However, the HP frankfurters had a higher external a* value (<i>P</i><.0001). The HP frankfurters displayed higher internal and external b* (yellowness) values (<i>P</i><.0001). Sensory results displayed the CP frankfurter to have an increase in hardness (<i>P</i>=0.004), a decrease in cohesiveness (<i>P</i>=0.03) and an increase in juiciness (<i>P</i><.0001). Texture analysis hardness (<i>P</i>=0.009) and chewiness (<i>P</i>=0.01) results showed the CON frankfurters were significantly harder than PAA (<i>P</i><0.05), while LA were not different from CON or PAA frankfurters (<i>P</i>>0.05). The CP frankfurters were found to have a decrease in springiness (<i>P</i>=<.001) and cohesiveness (<i>P</i>=0.03). There was a significant difference in microbial reduction of pre to post wash petri film counts for all treatments (Log₁₀CFU/mL) of aerobic plate count (<i>P</i>= <0.0001), E.<i>coli</i> coliform (<i>P</i>= 0.0002), yeast (<i>P</i>=0.04) and mold (<i>P</i>= <0.001). TRT was found to be significant for APC (<i>P</i>=0.06) and yeast (<i>P</i>=0.004). Overall, our research indicated antimicrobial wash treatments have little effect on frankfurter quality and displayed viable methods for reducing microbial growth on beef carcasses. </p> <br>

Growth and Stability Pact is the primary tool of the coordination of economic policy. This thesis deals with the historical development of the Pact, epmhasising its modification and furthermore its eligibility as means of enforcing the fiscal discipline of member states.

Mutation within the human ALS2 gene, which encodes the protein Alsin, causes a number of recessive motor neuron diseases. The ALS2 gene encodes a 180kDa protein, which has been shown to localize to early endosomes. The Alsin protein comprises three predicted guanine exchange factor (GEF) domains, the best characterised of which is the VPS9 domain for Rab5 GTPase, which is involved in the endocytosis membrane trafficking pathway, particularly in the docking and fusion of early endosomes. Furthermore, Alsin contains a Rho-GEF domain which specifically interacts with Rac-1 GTPase in the PI3K/AKT signal transduction pathway. This pathway has been implicated in numerous biological processes, including control of protein translation, via the mTOR branch of the pathway. To date, most work on the human ALS2 disease phenotypes has focused on the role of alsin in membrane trafficking, and neglected alsin’s potential role in signalling via its Rho-GEF domain. The focus of this project was to study the role of alsin in signalling during early Xenopus development, a period rich in well-characterised cell-cell signalling. I have shown that alsin is maternally loaded and zygotically expressed in the early Xenopus embryo. In cell culture, alsin is localised to early endosomes. Knockdown of alsin protein through the use of mopholinos (MO), resulted in a gastrulation defect, in particular, failure to close the blastopore caused by disrupted mesoderm induction and convergent extension movements. An animal cap assay was used to study mesoderm induction in the presence of als2-MO and activin protein, a potent mesoderm inducer. These animal caps extended normally, indicating proper mesoderm induction. By contrast, als2-MO animal caps failed to extend when co-injected with activin mRNA suggesting that alsin is important for the production and/or secretion of the activin ligand in the source cell. Subsequently it was determined that knockdown of alsin reduced the precursor protein levels of TGF-β family members activin and Xnr-2. These results suggest a novel role for alsin in mRNA stability, translational regulation or post-translational control of specific mesoderm-inducer mRNAs.