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Development of methods to monitor maturation and trafficking of Carboxypeptidase Y (CPY) and its G255R mutant (CPY*)
Get PDFProtein folding is a vital biological process which underpins many cellular functions in both eukaryotic and prokaryotic cells. This mechanism is a prime example of macromolecular self-assembly which leads to important biological function, such as molecular trafficking to specific cellular parts and cellular differentiation. However, whilst for the majority of cases proteins fold into their correct 3D structure with long-term stability, there is a propensity for proteins to misfold due to insufficient molecular interactions between the amino acids within the polypeptide chain. Once formed, these misfolded proteins have the potential to aggregate and cause pathological or even neurological diseases. Thus, it is of importance to probe the mechanism(s) of protein misfolding to uncover its molecular origins.
The yeast species known as baker’s yeast (S. Cerevisiae) is a model organism to probe this mechanism, and carboxypeptidase Y (CPY) has been proposed as a suitable model protein, due to its high abundance within the yeast endoplasmic reticulum (ER), to understand this process. Literature has shown that CPY is a widely used model protein in understanding protein sorting events within the ER of S. Cerevisiae. The advantages, and subsequent choice of this protein, are based upon its structure and role. It plays a part in the C-terminal chemistry of polypeptides, and thus may inform on mis-interactions which contribute to misfolding. Furthermore, CPY trafficking from the ER to the Golgi to the vacuole has provided information on sorting signal events which are like mammalian cellular signals, and thus share similar features with other organisms. It is also a preferable model protein due to its unique catalytic triad (active site). Although classified as a serine protease, it has a much greater pH and temperature range than other proteases, and can thus maintain high activity across environmental changes. Its mutated analogue, carboxypeptidase Y* (CPY*), has also been chosen as a model protein to compare its molecular sorting mechanism with CPY. It is characterised by a glycine-arginine mutation at the 255 amino acid position.
The purpose of this project is to uncover the molecular mechanisms of misfolding, namely, post trafficking of CPY & CPY*, whether these misfolded proteins renature and continue trafficking or whether they are degraded by cellular machinery. Alternatively, whether there is evidence of competition between these processes. This would also shed light on the kinetics of these processes and the likelihood of clearance of these misfolded proteins from the ER. To probe these processes, the maturation of pre-cursor forms of both CPY and CPY* have been studied, as they undergo cellular trafficking across the secretory pathway from the ER to the Golgi to the vacuole.
The initial experiments have been used to test whether CPY/CPY* can be detected in a western blot through SDS-PAGE gels, and whether CPY/CPY* can be detected in an immunoprecipitate. The final experiment was used to assess whether the dose-dependent cell-cycle regulator 2 (DCR2) plays a role in ER-induced stress, by specifically affecting CPY* degradation. All such experiments employ classical molecular biology techniques. These findings could shed light on whether degradation, by means of disulphide bond breaking and thus slower migration on SDS-PAGE gels, or renaturation, by means of cellular mechanisms, is the dominant mechanism within the ER
APPLYING ACTIVITY-BASED PROTEIN PROFILING TO IDENTIFY POTENTIALLY DRUGGABLE SERINE HYDROLASE ENZYMES IN LEISHMANIA SPP
Get PDFLeishmaniasis is a poverty related and one of the most important tropical diseases in the world with more than 12 million infected people, 0.9 to 1.6 million new cases each year, between 20000 and 30000 deaths per year, and 350 million people at risk of infection. Available drugs face problems with toxicity, administration and resistance which challenge their effectiveness. Consequently, the identification of new drug targets and the development of new treatments are imperative. Although serine hydrolases have been demonstrated to participate in crucial roles in the life cycle of the parasite and its virulence, these have not been yet characterized and the Leishmania serinome remains surprisingly neglected. This project attempts map and explore therapeutic targets within SHs present in the Leishmania proteome using an activity-based protein profiling (ABPP) strategy in the quest to find new protein drug targets for drug discovery. Initial experiments using commercial fluorophosphonate (FPs) probes revealed significant differences between the SH expression levels throughout their life cycles and between different Leishmania spp. As these probes are only effective for in vitro labelling, a suite of cell permeable probes has been synthesized and applied to study the Leishmania serinome in whole cells. Following proteome labelling and enrichment, the mass spectrometry-based tagging method, iTRAQ, led to the identification of two serine proteases: Carboxypeptidase LmxM.18.0450 and prolyl oligopeptidase (POP) LmxM.36.6750. Using a competitive ABPP approach, we were able to identify small molecule inhibitors for these enzymes which did showed activity against both L. mexicana promastigotes and axenic amastigotes. Collectively, these findings suggest that the serinome is a valuable source of new drug targets and that ABPP is a reliable approach for target discovery
The Monitoring and Control of Government Towards Contracted-out Public Service Providers: The Case Study of Saudi Arabia
Get PDFThe process of controlling contracted-out public services has attracted academic studies, however, very few studies have covered this topic in relation to developing countries, such as Saudi Arabia. Saudi Arabia started Vision 2030 aiming to improve the performance of the public sector. This vision relies in large part on the private sector, especially privatization and contracting-out of public services. As the government retains accountability for the quality of the services, they must monitor public-service contracts in order to achieve their objectives. Therefore, drawing on Structuration Theory (ST), which is novel for this context, this study aims to explore how the relationship between the government and public service providers is associated with controls and monitoring systems put in place. Few studies have examined these areas in relation to the public sector in detail. To bridge these gaps in previous accounting studies, 33 interviews were conducted, and relevant documents were collected. Two public services (public transportation and elderly care) were examined in two Saudi Arabian cities, Riyadh and Dammam, meaning that a total of four comparative case studies were explored. This is another novelty as this study attempts to broaden the understanding of controls and relations by investigating multiple studies. The results showed that control mechanisms were utilized differently by government actors at various levels and connected with the relationships between government and providers. Processual/relational variables played key roles, and they were strictly linked with the control systems. Different perspectives with regard to the enabling and constraining role of controls were revealed. Some patterns between controls and ST variables were discovered
Musical Conservatism: Victorian Composers and the Philosophy of Precedent
Get PDFMusical conservatism is a concept that is regularly invoked in both musicological literature and popular discourse, but is almost never defined or explained. The words ‘conservative’ or ‘reactionary’ are also often used as derogatory terms to denote insincerity, naivety, or lack of artistic depth. Edward German (1862-1936), Charles Villiers Stanford (1852-1924), and Edward Elgar (1857-1934), the primary case studies of this thesis, have each been identified – and often self-identified – with both musical conservatism and conservatism more broadly. Each of these composers also acted as a political activist for conservative causes, which they supported through their music and writings. Through an in depth study of a large volume of primary materials and the discourse surrounding the music of these three composers, this thesis provides an examination of the claim that the music and politics of conservative composers are intrinsically linked because they emerge from the same philosophical foundations. Their writings and music are also analysed through an assessment of their adherence to the first principles of conservatism, newly theorised here as a four part series of philosophical statements. Conservatism, in this conceptualisation (drawn from the writings of conservative and anticonservative theorists from Burke to Scruton) is to be understood as belief in the primacy of precedent, flawed and imperfectible human nature, the acceptability of inequality, and the importance of the pursuit of beauty in art. It addresses previously unanswered definitional questions on the subject of musical conservatism, its problems as a term, and its intrinsic nature. It also explores the relationship between musical conservatism and the pervading philosophical and political conservatism of late Victorian and Edwardian England
Perturbative integrability in 1+1 dimensions and affine Toda theories
Get PDFIn this thesis, the perturbative integrability of 1+1 dimensional bosonic massive quantum field theories is investigated. Starting from a theory with a generic polynomial-like potential, the constraints on the masses and Lagrangian couplings emerging by requiring purely elastic amplitudes at the tree level are obtained. It is observed that theories satisfying these constraints are completely determined by their mass ratios and 3-point couplings, while all the higher-order couplings can be obtained recursively in terms of them by imposing the absence of production for higher numbers of external legs. By exploiting different root system properties, it is shown that all the bosonic affine Toda field theories universally satisfy the constraints of purely elasticity at the tree level: a complete proof of their tree-level integrability is therefore provided. Subsequently, the higher-order poles observed in the bootstrapped S-matrices of the ADE series of affine Toda models are studied in perturbation theory. These singular points have been explained in the past in terms of anomalous threshold singularities in certain Feynman diagrams, where multiple propagators go on-shell simultaneously in loop integrations. Networks of Feynman diagrams contributing to these higher-order poles are found and residues at the poles are obtained through perturbation theory, showing agreement with the bootstrapped results. We show that the residues are generated by suitably cutting the loop diagrams into products of tree-level graphs, which will be called ‘atoms’. Most of these atoms simplify between one another and only a small number of them survive matching the bootstrapped results. The simplification mechanism between atoms inside networks is reminiscent of Gauss’s theorem in the space of Feynman diagrams
Direct Nonparametric Predictive Inference Classification Trees
Get PDFClassification is the task of assigning a new instance to one of a set of predefined categories based on the attributes of the instance. A classification tree is one of the most commonly used techniques in the area of classification. In recent years, many statistical methodologies have been developed to make inferences using imprecise probability theory, one of which is nonparametric predictive inference (NPI). NPI has been developed for different types of data and has been successfully applied to several fields, including classification. Due to the predictive nature of NPI, it is well suited for classification, as the nature of classification is explicitly predictive as well.
In this thesis, we introduce a novel classification tree algorithm which we call the Direct Nonparametric Predictive Inference (D-NPI) classification algorithm. The D-NPI algorithm is completely based on the NPI approach, and it does not use any other assumptions. As a first step for developing the D-NPI classification method, we restrict our focus to binary and multinomial data types. The D-NPI algorithm uses a new split criterion called Correct Indication (CI), which is completely based on NPI and does not use any additional concepts such as entropy. The CI reflects how informative attribute variables are, hence if the attribute variable is very informative, it gives high NPI lower and upper probabilities for CI. In addition, the CI reports the strength of the evidence that the attribute variables will indicate regarding the possible class state for future instances, based on the data. The performance of the D-NPI classification algorithm is compared against several classification algorithms from the literature, including some imprecise probability algorithms, using different evaluation measures. The experimental results indicate that the D-NPI classification algorithm performs well and tends to slightly outperform other classification algorithms.
Finally, a study of the D-NPI classification tree algorithm with noisy data is presented. Noisy data are data that contain incorrect values for the attribute variables or class variable. The performance of the D-NPI classification tree algorithm with noisy data is studied and compared to other classification tree algorithms when different levels of random noise are added to the class variable or to attribute variables. The results indicate that the D-NPI classification algorithm performs well with class noise and slightly outperforms other classification algorithms, while there is no single classification algorithm that acts as the best performing algorithm with attribute noise
Metal binding to the surface lipoprotein AdcAI from Streptococcus pyogenes and to the salivary antimicrobial peptide Histatin-5
Get PDFThe family of human salivary His-rich antimicrobial peptide, histatins, as exemplified by Histatin-5 (Hst5), bind zinc (Zn) and copper (Cu), but the role of metal binding in histatins have not been fully elucidated. Whether Hst5 is involved in nutritional immunity by limiting Zn availability to cause bacterial Zn starvation or by increasing Cu availability to cause Cu toxicity was examined in this study. Streptococcus pyogenes or Group A Streptococcus (GAS) was used as a model organism as it represents the oral streptococci that make up approximately 60 % of the oral microbiome. The metal binding affinity of Hst5 was measured by equilibrium competition assays using colourimetric probes and was compared against the high-affinity Zn uptake protein AdcAI from GAS. These experiments confirmed that Hst5 binds Zn weakly, and it does not strongly influence Zn availability. In addition, Hst5 cannot compete for Zn binding with AdcAI, which has an affinity that is 8 orders of magnitude higher than that of Hst5. In contrast, Hst5 has a high affinity for Cu and therefore influences the Cu availability. However, it does not promote Cu toxicity in GAS by delivering excessive Cu into the cytoplasm. It rather protects from the accumulation of intracellular Cu in ΔcopA mutant strain that is hypersensitive to increased Cu availability due to the loss of ability to export intracellular Cu. Whether Cu induces Cu starvation in Cu-requiring microbes remains unsolved, but this study has opened the possibility of Hst5 providing a buffering effect from metal toxicity to maintain a healthy oral microbiome
Control and Collisions of 87Rb and 133Cs Atoms in Optical Tweezers
Get PDFThis thesis reports the first steps towards the production of an optical tweezer array of ultracold 87Rb133Cs molecules. Such a system represents a longstanding goal: the full quantum control of individual polar molecules. Harnessing the long-range dipolar interactions and the rich rovibrational structure of ultracold molecules will unlock new applications in quantum computation, quantum simulation and quantum chemistry.
We demonstrate loading of single Rb and Cs atoms into species-selective optical tweezers of wavelengths 814 nm and 938 nm from magneto-optical traps of each species. We characterise the tweezers, detailing measurements of the trap frequency, light shift, trap waist and mean atom temperature. These measurements are performed using a newly-built apparatus, the design and construction of which are described in depth.
Next, we demonstrate control of the internal state of the atoms through optical pumping and microwave transfer. Using an acousto-optic deflector, we demonstrate control of the tweezer position, which is used to produce and rearrange a 1D array of five Cs atoms. Furthermore, we use this positional control to merge species-selective tweezers, which is used to prepare exactly one Rb atom and one Cs atom in the same optical tweezer.
We then use our control of individual atoms to study interatomic collisions. We demonstrate transfer of two or three Cs atoms into the same optical tweezer without heating, and discuss a method of separating tweezers to image multiple homonuclear atoms. We perform Feshbach resonance spectroscopy using pairs of Cs atoms prepared in the same optical tweezer. We observe nine loss features corresponding to Cs Feshbach resonances, and one loss feature corresponding to a zero in the scattering length. Finally, we study interspecies collisions of one Rb atom and one Cs atom.
The next steps towards an array of ground-state polar molecules, namely magnetoassociation and stimulated Raman adiabatic passage, will build on the work presented here
Modelling Mine Energy Systems: A Feasibility Study of the Implementation of a Minewater Heating System on the University Estate
No full textMinewater heating is a form of shallow geothermal energy provision that exploits abandoned subsurface mines that have since flooded after mining activity ceased. Groundwater that occupies the void space left from material extraction is warmed to ambient temperatures by heat transfer exchanges with surrounding rock. Using ground source heat pumps, the temperatures of these waters can be lifted to those suitable for domestic heating with efficient electrical input.
This thesis assesses the feasibility of implementing a minewater heating system on the Durham University campus, specifically Van Mildert College. Assuming abstraction and reinjection of the minewater, the available subsurface heat resource was investigated using numerical modelling. The model calculated minewater flow through the void space of the mines and thermal interaction with the surrounding rock. A new methodology was developed for the digitisation of historic mine plans provided by the U.K.'s Coal Authority, which have then been modelled to provide 3D visualisation of technical factors critical to the success of such a project.
Preliminary results indicate over 11 million kWh of heat in place resource available at temperatures of 13.5°C. However, modelling suggests that Van Mildert College would not be a suitable candidate for minewater heat. Mine plans indicate a shaft connection between the Hutton and Busty Seams close to the case study site, resulting in thermal breakthrough when modelled. This occurred in all modelled configurations, with extracted water being up to 2°C cooler after one year of operation.
After reviewing these model results, a different site was assessed. The Teaching and Learning Centre is a recently constructed educational facility approximately 420 m North-East from Van Mildert College. Due to the layout of the mine workings beneath the site, a sustainable, secure heat supply was identified, with no temperature reduction over time.
Calculations of the project's economic feasibility were performed, considering current energy market prices and potential CO₂ emissions reductions. A social acceptance study was also undertaken with members of the University's senior leadership and staff, identifying stakeholder motivations and concerns regarding minewater heat on the University estate
Soviet Snapshooters: Amateur Photography and Self-Fashioning in Interwar Russia
No full textThis thesis examines the practice of leisure-time snapshot photography in 1930s Soviet Russia. It explores how photography and leisure constituted interlinked processes of self-fashioning. For some citizens, the snapshot camera was a very important tool with which to mediate, represent, and make sense of their experience of life in Stalin-era Russia. Their photographs therefore make visible how ordinary citizens lived through a period of rapid and extensive societal transformation at a time of sweeping state censorship. This study is the first to use snapshot photographs as a source for the examination of the experience of Soviet life in the interwar years. Contributing to a body of literature before now largely focused on the verbal, the findings of this thesis nuance and deepen our understanding of how citizens encountered Stalin-era society. By approaching the snapshot photograph album as a mode of self-representation through which people communicate information about themselves and attribute value to particular spaces, objects, practices, interactions, and relationships, the analysis highlights the rich complexity of processes of self-fashioning in interwar Soviet Russia. This thesis shows how snapshot photographers and their subjects engaged with representational and bodily practices championed by the regime as well as pre-revolutionary customs and forms of behaviour criticised by Soviet organs. Significantly, the thesis demonstrates the active role that citizens performed in the shaping of Soviet culture through their participation. By engaging with a diverse range of models of identity, citizens reimagined certain ideas and practices, at once constructing their own identities and remaking the Soviet world they inhabited
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