Ph.D. (Biochemistry) / Please refer to full text to view abstract

Chapter 1 ---Does the party of government influence the amount and type of inward foreign investment? The results of a number of correlational studies provide inconsistent evidence. However none of these studies - for any level of government or any jurisdiction - have used methods that allow them to speak to causal effects. Regression discontinuity (RD) method is applied to a set of narrow-margin US gubernatorial elections. Over the course of a four-year term the election of a Republican governor causes a 21% boost in the growth of manufacturing-oriented FDI stock, compared to a Democrat. This effect is robust to a series of challenges. However, the same approach provides no evidence that partisanship matters for the overall level of FDI. Chapter 2 ---Does an economic shock open a window of opportunity for reform, and if it does, how does the institution of a state play a role? The paper investigates how economic shocks affect the structural reforms in various institutions. This paper addresses this issue by using the exogenous variation in the international price of large commodity goods to generate the exogenous change in national income. The analysis relies on a unique mapping between new annual data from 1962 to 2005 on economic shocks from commodity prices and structural reforms in 111 countries. I find significant heterogeneous effects across sectors in autocratic countries. In autocracies, positive economic shocks promote reforms in real sectors, but deter reforms in financial sectors. However the impact of economic shocks on structural reform in democratic countries is nil. Chapter 3 ---The deregulation of branch banking across the United States substantially increased the availability of credit to existing borrowers and others who has previously been excluded. Exploiting the staggered timing of changes across states for identification it is estimated that deregulation caused a 3.3% increase in rates of suicide and a 4.7% increase in rates of divorce. This is consistent with a large body of evidence linking excess debt to various measures of individual and relationship distress. Results are in most cases statistically significant at levels much higher than 1%, and prove resilient in a battery of robustness checks and falsification exercises.

We develop a simple one-zone model of the steady-state Crab nebula spectrum encompassing both the radio/soft X-ray and the GeV/multi-TeV observations. By solving the transport equation for GeV-TeV electrons injected at the wind termination shock as a log-parabola momentum distribution and evolved via energy losses, we determine analytically the resulting differential energy spectrum of photons. We find an impressive agreement with the observed spectrum of synchrotron emission, and the synchrotron self-Compton component reproduces the previously unexplained broad 200-GeV peak that matches the Fermi/Large Area Telescope (LAT) data beyond 1 GeV with the Major Atmospheric Gamma Imaging Cherenkov (MAGIC) data. We determine the parameters of the single log-parabola electron injection distribution, in contrast with multiple broken power-law electron spectra proposed in the literature. The resulting photon differential spectrum provides a natural interpretation of the deviation from power law customarily fitted with empirical multiple broken power laws. Our model can be applied to the radio-to-multi-TeV spectrum of a variety of astrophysical outflows, including pulsar wind nebulae and supernova remnants, as well as to interplanetary shocks.

Abstract Habitat selection decisions are crucial in determining fitness. Research indicates that individuals of many taxa are flexible in habitat selection and gather information prior to decision-making in order to control for environmental unpredictability. For time limited migrant birds, cues provide a quick and reliable information source with which to make habitat selection decisions. In this thesis I investigate habitat selection decisions, and their fitness consequences, of boreal passerines using heterospecifics or predators as cues. In support of the heterospecific attraction hypothesis, plots with augmented resident titmice densities attracted increased migrant densities. The predicted negative effects stemming from competition did not occur even at unnaturally high resident densities. This suggests that in the north it may always be beneficial for migrants to use residents as cues in habitat selection decisions. By manipulating habitat selection, I found that great tits (Parus major) had poorer reproductive success when forced to breed in close proximity to pied flycatcher (Ficedula hypoleuca) compared to when breeding alone. Flycatchers, in contrast, did slightly better when breeding close to tits. These results indirectly suggest that heterospecific attraction may not be a mutually positive species interaction. Indeed, flycatchers seem to parasitize the high quality microhabitat indicated by breeding great tits. I also tested if residents provide a reliable cue relative to predation risk. However, willow tit (P. montanus) nest location appeared random relative to avian predator nests. They do not appear to reliably indicate safe breeding habitats to later arriving migrants. In addition, closer proximity to breeding avian predators had a negative impact on willow tit reproductive output. Later arriving migrants may be in a better position to avoid avian predator nests during habitat selection. Pied flycatchers avoid settling in the immediate vicinity of sparrowhawk (Accipiter nisus) nests. However, nest box occupation, laying dates and initial reproductive investment (clutch size) showed a unimodal relationship with distance to sparrowhawk. A unimodal trend in these measures indicates there may be a trade-off between the costs (increased adult predation risk) and potential benefits (decreased nest predation risk) of settling in proximity to avian predator nests. Spatially predictable predation risk gradients that emanate from predator nests are termed a "predation risk landscape". Furthermore, flycatchers nesting closer to sparrowhawks produced fewer and smaller nestlings than those farther away. In addition, measures of maternal physiological stress (body condition and stress protein levels) had a negative linear relationship with distance to sparrowhawk nest. It appears that increased perceived predation risk near avian predator nests results in stressful and poor conditions for adult passerines, which results in lower reproductive output. This thesis highlights the importance of information gathering prior to making habitat selection decisions in order to optimise territory location relative to heterospecifics or predators. These decisions clearly impact individual fitness.

Central to this research, 40 kDa Heat shock proteins (Hsp40s) are known to partner (or cochaperone) 70 kDa Heat shock proteins (Hsp70s), facilitating the selection and transfer of protein substrate to Hsp70 and the stimulation of the protein folding ability of Hsp70. Members of the diverse Hsp70-Hsp40 protein complement of Plasmodium falciparum have been implicated in the cytoprotection of this malaria parasite, and are thought to facilitate the protein folding, assembly and translocation tasks required by the parasite to commandeer the infected human erythrocyte subsequent to invasion. In particular, the parasite has evolved an expanded and specialised 43- member suite of Hsp40 proteins, 19 of which bear an identifiable export motif for secretion into the infected erythrocyte cytoplasm where they potentially interact with human Hsp70. Although type I Hsp40 proteins are representative of typical regulators of Hsp70 activity, only two of these proteins are apparent in the parasite’s Hsp40 complement. These include a characteristic type I Hsp40 termed PfHsp40, and a larger, atypical type I Hsp40 termed Pfj1. Both Hsp40 proteins are predicted to be parasite-resident and are most likely to facilitate the co-chaperone regulation of the highly abundant and stress-inducible Hsp70 homolog, PfHsp70-I. In this work, the co-chaperone functionality of PfHsp40 and Pfj1 was elucidated using in vivo and in vitro assays. Purified recombinant PfHsp40 was shown to stimulate the ATPase activity of PfHsp70-I in in vitro single turnover and steady state ATPase assays, and co-operate with PfHsp70-I in in vitro aggregation suppression assays. In these in vitro assays, heterologous partnerships could be demonstrated between PfHsp70-I and the human Hsp40, Hsj1a, and human Hsp70 and PfHsp40, suggesting a common mode of Hsp70-Hsp40 interaction in the parasite and host organism. The functionality of the signature Hsp40 domain, the Jdomain, of Pfj1 was demonstrated by its ability to replace the equivalent domain of the A. tumefaciens Hsp40, Agt DnaJ, in interactions with the prokaryotic Hsp70, DnaK, in the thermosensitive dnaJ cbpA E. coli OD259 deletion strain. An H33Q mutation introduced into the invariant and crucial HPD tripeptide motif abrogated the functionality of the J-domain in the in vivo complementation system. These findings provide the first evidence for the conservation of the prototypical mode of J-domain based interaction of Hsp40 with Hsp70 in P. falciparum. Immunofluorescence staining revealed the localisation of PfHsp40 to the parasite cytoplasm, and Pfj1 to the parasite cytoplasm and nucleus in cultured intraerythrocytic stage P. falciparum parasites. PfHsp70-I was also shown to localise to the parasite cytoplasm and nucleus in these stages, consistent with the literature. Overall we propose that PfHsp40 and Pfj1 co-localise with and regulate the chaperone activity of PfHsp70-I in P. falciparum. This is the first study to identify and provide evidence for a functional Hsp70-Hsp40 partnership in P. falciparum, and provides a platform for future studies to elucidate the importance of these chaperone partnerships in the establishment and survival of the parasite in the intraerythrocytic-stages of development.

The extant coelacanths have a close resemblance to the coelacanth fossil records dating back to 230mya. Like their predecessors, the extant coelacanths inhabit rocky caves at a depth of 100-300m below sea level. In the Comoros, the water temperature at these depths is estimated to fluctuate between 14-20°C. High-level adaptation to these environment and lack of competition are thought to have led to the morphological uniformity and slow change throughout the history of the coelacanths. Under stress conditions, proteins unfold or misfold leading to the formation of aggregates. Molecular chaperones facilitate the correct folding of other proteins so that they can attain a stable tertiary structure. In addition, molecular chaperones aid the refolding of denatured proteins and the degradation of terminally misfolded protein after cellular stress. Heat shock proteins form one of the major classes of molecular chaperones. Here we show that, despite high-level adaptation to a unique habitat and slow change, the genome of the coelacanth encodes the major and highly conserved molecular chaperone, Hsp70. Latimeria menadoensis and Latimeria chalumnae contain intronless hsp70 genes encoding Hsp70 proteins archetypal of known Hsp70s. Based on the coelacanth codon usage, we have shown that bacterial protein expression systems, particularly Escherichia coli, may not be appropriate for the overproduction of coelacanth Hsp70s and coelacanth proteins in general. Also interesting, was the discovery that like the rat Hsc70, the L. menadoensis Hsp70 could not reverse thermal sensitivity in a temperate sensitive E. coli DnaK mutant strain, BB2362. We also report the successful isolation of a 1.2 kb region of L. menadoensis hsp70 upstream regulatory region. This region contain three putative heat shock elements, a TATA- box and two CAAT-boxes. This regulatory region resembled the Xenopus, mouse, and particularly tilapia hsp70 promoters, all of which have been shown to drive the expression of reporter genes in a heat dependent manner. Taken together, this data is the first to strongly suggest an inducible Hsp70-base cytoprotection mechanism in the coelacanth. It further provides basis to formulate testable predictions about the regulation, structure and function of Hsp70s in the living fossil, Latimeria.

Health-shock can be defined as a health event that causes severe hardship to the household because of the financial burden for healthcare payments and the income loss due to inability to work. It is one of the most prevalent shocks faced by the people of underdeveloped and developing countries. In Pakistan especially, policy makers and healthcare sector face an uphill battle in dealing with health-shock due to the lack of a publicly available dataset and an effective data analytics approach. In order to address this problem, this thesis presents a data analytics and visualization framework for health-shock prediction based on a large-scale health informatics dataset. The framework is developed using cloud computing services based on Amazon web services integrated with Geographical Information Systems (GIS) to facilitate the capture, storage, indexing and visualization of big data for different stakeholders using smart devices. The data was collected through offline questionnaires and an online mobile based system through Begum Memhooda Welfare Trust (BMWT). All data was coded in the online system for the purpose of analysis and visualization. In order to develop a predictive model for health-shock, a user study was conducted to collect a multidimensional dataset from 1000 households in rural and remotely accessible regions of Pakistan, focusing on their health, access to health care facilities and social welfare, as well as economic and environmental factors. The collected data was used to generate a predictive model using a fuzzy rule summarization technique, which can provide stakeholders with interpretable linguistic rules to explain the causal factors affecting health-shock. The evaluation of the proposed system in terms of the interpretability and accuracy of the generated data models for classifying health-shock shows promising results. The prediction accuracy of the fuzzy model based on a k-fold crossvalidation of the data samples shows above 89% performance in predicting health-shock based on the given factors. Such a framework will not only help the government and policy makers to manage and mitigate health-shock effectively and timely, but will also provide a low-cost, flexible, scalable, and secure architecture for data analytics and visualization. Future work includes extending this study to form Pakistan’s first publicly available health informatics tool to help government and healthcare professionals to form policies and healthcare reforms. This study has implications at a national and international level to facilitate large-scale health data analytics through cloud computing in order to minimize the resource commitments needed to predict and manage health-shock.

The main objective of the thesis is to provide a detailed assessment of the performance of four types of Low Reynolds Number (LRN) Eddy Viscosity Models (EVM), widely used for industrial purposes, on flows featuring SWBLI, using experimental and direct numerical simulation data. Within this framework the two-equation linear k-ε of Launder and Sharma (1974) (LS), the two-equation linear k-ω SST, the four-equation linear φ-f of Laurence et al. (2004) (PHIF) and the non-linear k-ε scheme of Craft et al. (1996b,1999) (CLSa,b) have been selected for testing. As initial test cases supersonic 2D compression ramps and impinging shocks of different angles and Reynolds numbers of the incoming boundary layer have been selected. Additional test cases are then considered, including normal shock/isotropic turbulence interaction and an axisymmetric transonic bump, in order to examine the predictions of the selected models on a range of Mach numbers and shock structures. For the purposes of this study the PHIF and CLSa,b models have been implemented in the open source CFD package OpenFOAM. Some results from validation studies of these models are presented, and some explorations are reported of certain modelled source terms in the ε-equation of the PHIF and CLSb models in compressible flows. Finally, before considering the main applications of the study, an examination is made of the performance of different solvers and numerical methods available in OpenFOAM for handling compressible flows with shocks. The performance of the above models, is analysed with comparisons of wall-quantities (skin-friction and wall-pressure), velocity profiles and profiles of turbulent quantities (turbulent kinetic energy and Reynolds stresses) in locations throughout the SWBLI zones. All the selected models demonstrate a broadly consistent performance over the considered flow configurations, with the CLSb scheme generally giving some improvements in predictions over the other models. The role of Reynolds stress anisotropy in giving a better representation of the evolution of the boundary layer in these flows is discussed through the performance of the CLSb model. It is concluded that some of the main deficiencies of the selected models is the overestimation of the dissipation rate levels in the non-equilibrium regions of the flow and the underestimation of the amplification of Reynolds stress anisotropy, especially within the recirculation bubble of the flows. Additionally, the analysis of the performance of the considered EVM's in a normal shock/isotropic turbulence interaction illustrates some drawbacks of the EVM formulation similar to the ones observed in normally-strained incompressible flows. Finally, a hybrid Detached Eddy Simulation (DES) approach is incorporated for the prediction of the transonic buffet around a wing.

The heat shock protein-70 (Hsp70) family of molecular chaperones are ubiquitous highly conserved proteins that are critical for the viability of cellular homeostasis. The ATPase activity of Hsp70 proteins is critical to their function as the affinity of a given Hsp70 for non-native substrate is modulated by ATP binding and hydrolysis. When bound to ATP, Hsp70s possess a low affinity for a given substrate protein, while the hydrolysis of ATP to ADP causes a conformational change that results in a high affinity for substrate proteins. The basal ATPase activity of Hsp70s is too low to facilitate their function in vivo, and co-chaperones are essential to modulate the efficient protein folding by Hsp70. Heat shock protein-40 (Hsp40) heat shock proteins are essential for the in vivo function of Hsp70s by stimulating the ATPase activity of these proteins and facilitating transfer of substrates. The Type III class of Hsp40 proteins have not been well characterised due to their poor levels of conservation at the primary sequence level. This is due to the fact that Type III Hsp40s only contain a J-domain and a poorly conserved C-terminal region. The newly identified Type IV class of Hsp40s, contain an abrogated HPD tripeptide motif in the J-domain and have also not been extensively studied. Trypanosoma brucei (T. brucei) is a unicellular flagellated protozoan parasite. It is the causative agent of Human African Trypansomiasis (HAT) which results in thousands of deaths and devastating agricultural losses in many parts of Africa. T. brucei undergoes a complex lifecycle that is characterised by the transition from an insect vector to a mammalian host in markedly different conditions of temperature, pH, nutrient availability and respiratory requirements. It has been proposed that molecular chaperones may enhance the survival of these parasites due to their cytoprotective effect in combating cellular stress. Due to the fact that T. brucei infection is invariably fatal if left untreated, and that no novel treatment regimens have been developed recently, the identification of potential novel drug targets among proteins essential to the parasite’s survival in the host organism is an attractive aspect of T. brucei research. Because Type III Hsp40s are poorly conserved with respect to Hsp40s found in the human host, the identification of any of these proteins found to be essential to T. brucei survival in humans could potentially make attractive novel drug targets. An in depth in silico investigation into the Type III Hsp40 complement as well as partner Hsp70 proteins in T.brucei was performed. T. brucei possesses 65 Hsp40 proteins, of which 47 were classed as Type III and 6 of which were identified as being putative Type IV Hsp40s. A small but significant number (5) of Type III TbHsp40s contained tetratricopeptide (TPR) domains in addition to the J-domain. The J-domains of the Type III TbHsp40 complement were found to be conserved with respect to those of canonical Hsp40 proteins, although the mutation of certain residues that play a key role in Hsp40-Hsp70 interaction was noted. Potential partnerships of these proteins in the parasite was also investigated. The coding regions of three previously uncharacterised TbHsp40s were successfully amplified from T. brucei TREU927 genomic DNA and cloned into an expression vector. Tbj1, a Tcj1 ortholog, was selected for further study and successfully expressed and biochemically characterised. Tbj1 expressed in E. coli was found to be insoluble, but large amounts were recovered with the aid of a denaturing purification followed by refolding elution strategies, and the bulk of the protein recovered was in compact monomeric form as determined by size-exclusion chromatography fast protein liquid chromatography (SEC-FPLC). The addition of Tbj1 to a thermally aggregated substrate resulted in increased levels of aggregation, although Tbj1 was able to assist two Hsp70 proteins in the suppression of aggregation. Tbj1 proved unable to stimulate the ATPase activity of these same Hsp70s, and could not rescue temperature sensitive cells when replacing E.coli DnaJ and CbpA. It was concluded that Tbj1 does not possess independent chaperone activity, but could display Hsp40 co-chaperone properties under certain circumstances. This could allude to a specialised function in the T. brucei parasite. The lack of human orthologues to Tbj1 could result in the attractiveness of this protein as a novel drug target.

Murine stress-inducible protein 1 (mSTI1) is a co-chaperone that is homologous with the human heat shock protein 70 (Hsp70)/heat shock protein 90 (Hsp90)-organizing protein (Hop). The two proteins are homologues of the highly conserved stress-inducible protein 1 (STI1) family of co-chaperones. The STI1 proteins interact directly and simultaneously at some stage, with Hsp70 and Hsp90 in the formation of the hetero-multi-chaperone complexes that facilitate the folding of signal transducing kinases and functional maturation of steroid hormone receptors. The interactions of mSTI1 with both Hsp70 and Hsp90 is mediated by a versatile structural protein-protein interaction motif, the tetratricopeptide repeat (TPR). The TPR motif is a degenerate 34-amino acid sequence a-helical structural motif found in a significant number of functionally unrelated proteins. This study was aimed at characterizing the structural and functional determinants in the TPR domains of mSTI1 responsible for binding to and discriminating between Hsp70 and Hsp90. Guided by data from Hop's crystal structures and amino acid sequence alignment analyses, various biochemical techniques were used to both qualitatively and quantitatively characterize the contacts necessary for the N-terminal TPR domain (TPR1) of mSTI1 to bind to the C-terminal EEVD motif of heat shock cognate protein 70 (Hsc70) and to discriminate between Hsc70 and Hsp90. Substitutions in the first TPR motif of Lys⁸ or Asn¹² did not affect binding of mSTI1 to Hsc70, while double substitution of these residues abrogated binding. A substitution in the second TPR motif of Asn⁴³ lowered but did not abrogate binding. Similarly, a deletion in the second TPR motif coupled with a substitution of Lys⁸ or Asn¹² reduced but did not abrogate binding. Steady state fluorescence and circular dichroism spectroscopies revealed that the double substitution of Lys⁸ and Asn¹² resulted in perturbations of inter-domain interactions in mSTl1. Together these results suggest that mSTI1-Hsc70 interaction requires a network of electrostatic interactions not only between charged residues in the TPR1 domain of mSTI1 and the EEVD motif of Hsc70, but also outside the TPR1 domain. It is proposed that the electrostatic interactions in the first TPR motif collectively made by Lys⁸ and Asn¹² define part of the minimum interactions required for successful mSTI1-Hsc70 interaction. In the first central TPR domain (TPR1A), single substitution of Lys³°¹ was sufficient to abrogate the mSTI1-Hsp90 interaction. Using a truncated derivative of mSTI1 incapable of binding to Hsp90, residues predicted by crystallographic data to determine Hsp70 binding specificity were substituted in the TPR1 domain. The modified protein had reduced binding to Hsc70, but showed significant binding capacity for Hsp90. In contrast, topologically equivalent substitutions on a truncated derivative of mSTI1 incapable of binding to Hsc70 did not confer Hsc70 specificity on the TPR2A domain. These data suggest that binding of Hsc70 to the TPR1 domain is more specific than binding of Hsp90 to the TPR2A domain. In addition, residues C-terminal of helix A in the second TPR motif of mSTI1 were shown to be important in determining specific binding to Hsc70. Binding assays using surface plasmon resonance spectroscopy showed that the affinities of binding of mSTI1 to Hsc70 and Hsp90 were 2 μM and 1.5 μM respectively. Preliminary in vivo studies revealed differences in the dynamics of binding of endogenous and exogenous recombinant mSTI1 with Hsc70 and Hsp90. The outcome of this study poses serious implications for the mechanisms of mSTI1 interactions with Hsc70 and Hsp90 in the cell.