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Micelle-Catalyzed Domain Swapping in the Cytoplasmic Domain of E. coli Rhomboid ProteaseKwok, Jason Ka-Cheong January 2014 (has links)
Domain swapping is a mechanism of protein oligomerization whereby subunits exchange identical structural elements with each other. This type of interaction is important in a number of biologically important processes, including the regulation of enzyme activity, the modulation of molecular recognition and in the development of some protein deposition diseases. Domain swapping can be promoted by exposure to chemical denaturants, high temperatures, or redox reagents, although these factors can deviate greatly from conditions encountered in vivo. Moreover, in some cases these conditions can alter the monomer-oligomer equilibrium or even promote the formation of alternate domain-swapped oligomers. In contrast, we have found that it is possible for detergent micelles to be used as a catalyst in the domain-swapping interaction involving the N-terminal cytoplasmic domain of E. coli GlpG rhomboid protease. Our results show that hexadecylphosphocholine micelles can catalyze domain swapping of NGlpG by lowering the Gibbs free energy of the kinetic barrier by 12 kcal/mol, while preserving the equilibrium populations of monomer and dimer. Micelle charge and size were found to be important for this catalysis, which involved the formation of a partially unfolded micelle-bound intermediate containing significant secondary structure. Overall, the results from this work reveal how detergent micelles can affect the energy landscape of domain swapping for NGlpG, and provide insight into the potential ability of local irregularities in lipid membrane environments to play a role in domain swapping in vivo.
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Predictive Modeling of Uniform Differential Item Functioning Preservation Likelihoods After Applying Disclosure Avoidance Techniques to Protect PrivacyLemons, Marlow Q. 04 April 2014 (has links)
The need to publish and disseminate data continues to grow. Administrators of large-scale educational assessment should provide examinee microdata in addition to publishing assessment reports. Disclosure avoidance methods are applied to the data to protect examinee privacy before doing so, while attempting to preserve as many item statistical properties as possible. When important properties like differential item functioning are lost due to these disclosure avoidance methods, the microdata can give off misleading messages of effectiveness in measuring the test construct. In this research study, I investigated the preservation of differential item functioning in a large-scale assessment after disclosure avoidance methods have been applied to the data. After applying data swapping to protect the data, I attempted to empirically model and explain the likelihood of preserving various levels of differential item functioning as a function of several factors including the data swapping rate, the reference-to-focal group ratio, the type of item scoring, and the level of DIF prior to data swapping. / Ph. D.
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Caracterização funcional e estrutural da primeira arabinofuranosidase da família 62 de Thielavia terrestris dimerizada através de domain swapping / Functional and structural characterization of the first arabinofuranosidase of the Thielavia terrestris from family 62 dimerized through the domain swappingCamargo, Suelen 10 August 2018 (has links)
As enzimas hidrolíticas são consideradas, por plena definição, aquelas com a capacidade de realizar a reação de hidrólise. Devido à sua capacidade de degradar substâncias naturais, as enzimas hidrolíticas são de grande uso industrial e podem ser aplicadas nas indústrias têxtil, alimentar, farmacêutica, biocombustíveis- agrícola, assim como na produção de derivados biotecnológicos de maior valor agregado. Conhecidas como enzimas que liberam arabinose e arabinofuranose, as arabinofuranosidases da família 62 são capazes de quebrar as conexões de arabinoxilanos. A importância biotecnológica de tais proteínas é desde o uso na agricultura à indústria. Elas podem ser utilizadas, por exemplo, em coquetéis na digestão de alimentos para animais, vinhos e para melhorar a eficiência de clarificação de sucos. Além disso, sua ação de deslignificação é promissora na degradação da biomassa para a produção de bicombustíveis. Este projeto procura realizar a clonagem, expressão heteróloga em bactérias e caracterização estrutural de uma α-L-arabinofuranosidase do fungo termofílico Thielavia terrestris. Esta enzima pertence à família 62 das hidrolases de glicosídeos, que é conhecida por melhorar a ação das celulases e, portanto, de interesse na degradação da biomassa lignocelulósica, como o bagaço de cana-de-açúcar. Estudos biofísicos, bioquímicos e estruturais apresentados neste estudo buscam promover a compreensão dos mecanismos de ação desta enzima e sua aplicação na hidrólise do bagaço de cana-de-açúcar. O trabalho destaca uma estrutura tridimensional inédita dentro da família 62 portadora de um fenômeno conhecido como domain swapping, possibilitando maiores investigações dentro do grupo das arabinofuranosidases. / Hydrolytic enzymes are considered, for the full definition, those with the ability to perform the hydrolysis reaction. Because of their ability of degrading natural substances, hydrolytic enzymes are of wide industrial use and can be applied in the textile, food, pharmaceutical, and agricultural industries, in addition to being used in the production of biofuels. Known as enzymes that release arabinose, the arabinofuranosidases from family 62 are able to break arabinoxylans connections. The biotechnological importance of these proteins are used in the agricultural and industry. These enzymes are employed to process as cocktails for animal feed digestion, wines and to improve the efficiency of juices clarification. Moreover, its delignification action is promising in the biomass degradation for biofuel production. This project seeks to carry out cloning, heterologous expression in bacteria and characterization of an α-L-arabinofuranosidase from thermophilic fungus Thielavia terrestris. This enzyme belonging to the glycoside hydrolase family 62, which is known to enhance the action of cellulases, and thus of interest in the lignocellulosic biomass degradation such as sugar cane bagasse. This study will characterize the protein structure and biochemistry in order to understand the mechanism and optimal conditions of action. The biophysical, biochemical and structural tests seek to promote an understanding of the mechanisms of action of this enzyme and its application in the hydrolysis of sugarcane bagasse. The work shows a new three- dimensional structure within the family 62 caused by a phenomenon known as domain swapping. Thus, it is possible to predict further investigations on the arabinofuranosidases group.
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Potential for N pollution swapping from riparian buffer strips and an instream wetlandBoukelia, Willena Esther January 2012 (has links)
Diffuse agricultural pollution is a major contributor to poor water quality in many parts of the world. Consequently agri-environment policy promotes the use of riparian buffer strips and/or denitrifying wetlands to intercept and remove diffuse NO3--N pollution. However, these methods have the potential to cause ‘pollution swapping’: the exchange of one form of pollution as a result of measures implemented to reduce another. Thus the benefits of intercepting NO3--N could be offset by enhanced emissions of the potent greenhouse gases, nitrous oxide (N2O) and methane (CH4), from buffer strips and wetlands. This research aimed to: (1) quantify the direct N2O emissions from an irrigated buffer strip (IBS), using nitrate-rich agricultural drainage water, compared to a non-irrigated control (CP); (2) improve the understanding of N2O production and consumption within soils using controlled soil monolith experiments; (3) assess the effectiveness of a small (60 m2) instream wetland at intercepting and removing diffuse NO3--N pollution, and quantify pollution swapping in the form of CH4 and N2O emissions; (4) assess the production of CH4 and N2O within the sediment, and their emissions as well as inorganic-N concentrations in the overlying water column in response to temperature and turbulence, using intact wetland sediment and membrane inlet mass spectrometry (MIMS). The research focused on mitigating diffuse NO3--N pollution from grazed pasture at a farm in north-east England. Annual N2O-N emissions from the IBS and CP were not statistically different (P > 0.05): 509 and 263 g N2O-N ha-1, respectively, in 2007 and 375 and 500 g N2O-N ha- 1 year-1, respectively, in 2008. Irrigation of the IBS increased spatial variability in flux and generated hotspots of denitrification compared to the CP. However, these changes were short-lived. Direct N2O emission factors (EF1) calculated using the available NO3--loading data (September 2007 - December 2008) for the IBS were lower (c.0.1%) than those calculated for the CP assuming N input from biological N fixation only (<1.9%). Soil monolith experiments under a variety of irrigation and NO3--N loading regimes confirmed low direct and indirect (of dissolved N2O-N in leachate) emissions (<3.1 and <2.3% of applied NO3--N emitted as N2O-N, respectively), similar to the IPCC default emission factors. However, N loss in leachate was high, up to 82% of added NO3--N with concentrations reaching 24 mg NO3--N L-1. Therefore even though no pollution swapping occurred the high leachate losses indicate irrigation of buffer strips are not effective mitigation methods. Monitoring for 2 years of the instream wetland that received median NO3--N concentrations of c. 6 mg N L-1, but up to c. 20 mg N L-1, showed it to be ineffective at intercepting diffuse NO3- pollution: likely a result of the relatively high discharge and short water residence time, as well as the direct input of NO3--N to the wetland from secondary sources: field drains and/or overland flow. The wetland was a net source of NH4+-N in both 2007 and 2008, and a net sink of NO3--N in 2007 only. Annual wetland CH4 and N2O emissions were 713 and 237 mg CH4 m-2 year-1, and 3.5 and 1.9 mg N2O-N m-2 year-1, for 2007 and 2008, respectively and were highly variable between seasons. N pollution swapping was minimal from either direct or indirect emissions, but CH4 emissions were found to be of greater importance at a net cost of ~ £600 ha-1 over the study period (2007 to 2008), compared to N2O emissions (~ £60 ha-1) and low NO3--N interception savings (~ £24 ha-1). Incubation experiments suggest that spatially variable microsites of nitrifying, denitrifying or methanogenic activity and CH4 oxidation occur within the wetland sediment. Therefore off-line, larger wetland systems offer the best prospects of enhanced NO3--N interception and potentially reduced CH4 emissions by maintaining shallow water depths (increased CH4 oxidation) and long residence times (increased opportunity for denitrification), within the wetland or wetland cells.
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Role of the amino acid sequences in domain swapping of the B1 domain of protein G by computation analysisMaurer-Stroh (née Sirota Leite), Fernanda 12 October 2007 (has links)
Domain swapping is a wide spread phenomenon which involves the association between two or more protein subunits such that intra-molecular interactions between domains in each subunit are replaced by equivalent inter-molecular interactions between the same domains in different subunits. This thesis is devoted to the analysis of the factors that drive proteins to undergo such association modes. The specific system analyzed is the monomer to swapped dimer formation of the B1 domain of the immunoglobulin G binding protein (GB1). The formation of this dimer was shown to be fostered by 4 amino acid substitutions (L5V, F30V, Y33F, A34F) (Byeon et al., 2003). In this work, computational protein design and molecular dynamics simulations, both with detailed atomic models, were used to gain insight into how these 4 mutations may promote the domain swapping reaction.
The stability of the wt and quadruple mutant GB1 monomers was assessed using the software DESIGNER, a fully automatic procedure that selects amino acid sequences likely to stabilize a given backbone structure (Wernisch et al., 2000). Results suggest that 3 of the mutations (L5V, F30V, A34F) have a destabilizing effect. The first mutation (L5V) forms destabilizing interactions with surrounding residues, while the second (F30V) is engaged in unfavorable interactions with the protein backbone, consequently causing local strain. Although the A34F substitution itself is found to contribute favorably to the stability of the monomer, this is achieved only at the expense of forcing the wild type W43 into a highly strained conformation concomitant with the formation of unfavorable interactions with both W43 and V54.
Finally, we also provide evidence that A34F mutation stabilizes the swapped dimer structure. Although we were unable to perform detailed protein design calculations on the dimer, due to the lower accuracy of the model, inspection of its 3D structure reveals that the 34F side chains pack against one another in the core of the swapped structure, thereby forming extensive non-native interactions that have no counterparts in the individual monomers. Their replacement by the much smaller Ala residue is suggested to be significantly destabilizing by creating a large internal cavity, a phenomenon, well known to be destabilizing in other proteins. Our analysis hence proposes that the A34F mutation plays a dual role, that of destabilizing the GB1 monomer structure while stabilizing the swapped dimer conformation.
In addition to the above study, molecular dynamics simulations of the wild type and modeled quadruple mutant GB1 structures were carried out at room and elevated temperatures (450 K) in order to sample the conformational landscape of the protein near its native monomeric state, and to characterize the deformations that occur during early unfolding. This part of the study was aimed at investigating the influence of the amino acid sequence on the conformational properties of the GB1 monomer and the possible link between these properties and the swapping process. Analysis of the room temperature simulations indicates that the mutant GB1 monomer fluctuates more than its wild type counter part. In addition, we find that the C-terminal beta-hairpin is pushed away from the remainder of the structure, in agreement with the fact that this hairpin is the structural element that is exchanged upon domain swapping. The simulations at 450 K reveal that the mutant protein unfolds more readily than the wt, in agreement with its decreased stability. Also, among the regions that unfold early is the alpha-helix C-terminus, where 2 out of the 4 mutations reside. NMR experiments by our collaborators have shown this region to display increased flexibility in the monomeric state of the quadruple mutant.
Our atomic scale investigation has thus provided insights into how sequence modifications can foster domain swapping of GB1. Our findings indicate that the role of the amino acid substitutions is to decrease the stability of individual monomers while at the same time increase the stability of the swapped dimer, through the formation of non-native interactions. Both roles cooperate to foster swapping.
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Physicochemical Studies of the Grb2-Sos1 InteractionMcDonald, Caleb Benton 16 June 2009 (has links)
Grb2, a modular protein comprised of a central SH2 domain flanked between a N-terminal SH3 (nSH3) domain and a C-terminal SH3 (cSH3) domain, is a component of cell signaling networks involved in the transmission of extracellular information in the form of growth factors and cytokines to downstream targets such as transcription factors within the nucleus. The Grb2-Sos1 interaction is mediated through the combinatorial binding of nSH3 and cSH3 domains of Grb2 to various sites - designated S1, S2, S3, and S4 - containing PXpsiPXR motifs within Sos1. Here, using a diverse array of biophysical techniques, including in particular isothermal titration calorimetry coupled with molecular modeling and semi-empirical analysis, I provide new insights into the Grb2-Sos1 interaction in thermodynamic and structural terms. My data show that Grb2 exists in monomer-dimer equilibrium in solution and that the dissociation of dimer into monomers is entropically-driven. The heat capacity change observed was much smaller than that expected from the rather large molecular surfaces becoming solvent-occluded upon dimerization, implying that monomers undergo conformational rearrangement upon dimerization. 3D structural models suggest strongly that such conformational rearrangement may arise from domain swapping. I further show that the nSH3 domain of Grb2 binds to the S1 site containing the proline-rich consensus motif PXpsiPXR with an affinity that is nearly three-fold greater than that observed for the binding of the cSH3 domain. It is also demonstrated that such differential binding of the nSH3 domain relative to the cSH3 domain is largely due to the requirement of a specific acidic residue, in the RT loop, to engage in the formation of a salt bridge with the arginine residue in the consensus motif PXpsiPXR. The data further reveal that, while binding of both SH3 domains to Sos1 is under enthalpic control, the nSH3 binding suffers from entropic penalty in contrast to entropic gain accompanying the binding of cSH3, implying that the two domains employ differential thermodynamic mechanisms for Sos1 recognition. Additionally, my data reveal that while the nSH3 domain of Grb2 binds with affinities in the physiological range to all four sites S1-S4, the cSH3 domain can only do so at the S1 site. Further scrutiny of these sites yields rationale for the recognition of various PXpsiPXR motifs by the SH3 domains in a discriminate manner. Unlike the PXpsiPXR motifs at S2, S3 and S4 sites, the PXpsiPXR motif at S1 site is flanked at its C-terminus with two additional arginine residues that are absolutely required for high-affinity binding of the cSH3 domain. In contrast, these two additional arginine residues augment the binding of the nSH3 domain to the S1 site but their role is not critical for the recognition of S2, S3 and S4 sites. Molecular modeling is employed to rationalize my new findings in structural terms. Taken together, this thesis provides novel insights into the physicochemical basis of a key protein-protein interaction pertinent to cellular signaling and cancer. My studies bear the potential for the development of novel therapies with less toxicity but more effectiveness for the treatment of disease.
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Componentization of IP and Netfilter Architecture in Linux KernelLin, Jiun-nan 25 July 2007 (has links)
In this thesis, we exercised the componentization technique to componentize the Netfilter architecture in Linux network system. Netfilter is a software architecture for filtering packets. System administrator can register packet-matching rules and target handling function into the system. Netfilter matches packets according to the rules and processes them by the corresponding target functions. By componentizing the architecture, we can improve the elasticity and the reusability of Netfilter. Hot-swapping is an important procedure in componentized software system. In this study, we implemented hot-swapping based on the work developed by Fan[1]. It stores the relocation information of exporting symbols into the module symbol table. With this information, we are able to dynamically change the caller-callee relationship of modular components at run time. In addition, we extend their work to allow the same modular component to be loaded into Linux kernel for more than once so that the same component can be replicated in the system.
We started with decomposing all the ¡§hook¡¨ functions into smaller and simpler components and then for each component, we added in-ports and out-ports and registered its own iptables, and we fixed the limitation of only one instance of a module allowed in kernel and broke the hard rule in iptables. As a result, after Netfilter componentization, we are able to illustrate new configurations that cannot be done in the original architecture, and the system becomes further compact with only necessary components loaded in the system. This reflects in slight performance improvement in our experiments, which is not usually seen in other frameworks due to componentization overhead.
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Peer-to-Peer Bartering: Swapping Amongst Self-interested AgentsCabanillas, David 01 April 2009 (has links)
Large--scale distributed environments can be seen as a conflict between the selfish aims of the participants and the group welfare of the population as a whole. In order to regulate the behavior of the participants it is often necessary to introduce mechanisms that provide incentives and stimulate cooperative behavior in order to mitigate for the resultant potentially undesirable availability outcomes which could arise from individual actions.The history of economics contains a wide variety of incentive patterns for cooperation. In this thesis, we adopt bartering incentive pattern as an attractive foundation for a simple and robust form of exchange to re-allocate resources. While bartering is arguably the world's oldest form of trade, there are still many instances where it surprises us. The success and survivability of the barter mechanisms adds to its attractiveness as a model to study.In this thesis we have derived three relevant scenarios where a bartering approach is applied. Starting from a common model of bartering: - We show the price to be paid for dealing with selfish agents in a bartering environment, as well as the impact on performance parameters such as topology and disclosed information.- We show how agents, by means of bartering, can achieve gains in goods without altruistic agents needing to be present.- We apply a bartering--based approach to a real application, the directory services.The core of this research is the analysis of bartering in the Internet Age. In previous times, usually economies dominated by bartering have suffered from high transaction costs (i.e. the improbability of the wants, needs that cause a transaction occurring at the same time and place). Nowadays, the world has a global system of interconnected computer networks called Internet. This interconnected world has the ability to overcome many challenges of the previous times. This thesis analysis the oldest system of trade within the context of this new paradigm. In this thesis we aim is to show thatbartering has a great potential, but there are many challenges that can affect the realistic application of bartering that should be studied.The purpose of this thesis has been to investigate resource allocation using bartering mechanism, with particular emphasis on applications in largescale distributed systems without the presence of altruistic participants in the environment.Throughout the research presented in this thesis we have contributed evidence that supports the leitmotif that best summarizes our work: investigation interactions amongst selfish, rational, and autonomous agents with incomplete information, each seeking to maximize its expected utility by means of bartering. We concentrate on three scenarios: one theoretical, a case of use, and finally a real application. All of these scenarios are used for evaluating bartering. Each scenario starts from a common origin, but each of them have their own unique features.The final conclusion is that bartering is still relevant in the modern world.
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Entanglement Swapping in the Strong Coupling Interaction between the Atoms and the Photonic Crystal MicrocavitiesLay, Chun-feng 06 June 2005 (has links)
The cavity quantum electrodynamics has been applied to investigate the strong coupling interaction dynamics process between the microcavity field and the atom. The high quality cavity is a key to the realization of cavity quantum electrodynamics. Photonic crystal nanocavities are with small mode volumes and large quality factors. Lights are confined within the nanocavity. They can be used for cavity QED experiments of Fabry-Perot cavity. We have provided a realization of a quantum entanglement method for quantum information processing.
In this paper, we discuss the entanglement swapping in the strong coupling process between two level atoms interacting with the photonic crystal microcavities fields of coherent states. We investigate the atomic level population and the entanglement degree of the system. We have found that the atomic maximal entangled state can be transformed into the photonic crystal microcavity maximal coherent entangled state cavity field, whereas the photonic crystal microcavity maximal coherent entangled state cavity field can be transformed into the atomic maximal entangled state.
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Playfulness as a motivational tool : Using play to develop for an enhanced reception of swapping reusable goods / Lekfullhet som ett motiverande verktyg : Att använda lek i utvecklingen för en förbättrad upplevelse av att byta återanvändbara varorFaraj, Zarah January 2015 (has links)
This study examines the field of playful design as a tool to affect and enhance the reception of a mundane activity. The management of swapping reusable household goods between neighbors is explored by designing playful components to enhance the experience. A pilot field study was carried out to observe recycling areas, in conjunction to 20 interviews being conducted regarding their point of views on recycling habits and their perception on their own local recycling area. The results show a clear indifferent attitude towards recycling and the environment surrounding the recycling area. However the interviewees were predominantly optimistic to the idea of swapping reusable goods with neighbors for its well-known advantages. Concerns were expressed on the practical efficiency and organization of such an activity, as well as on the shameful label perceived by using pre-used goods. A hybrid mobile application prototype was developed as one suggestion on how to incorporate playful design in a way that takes into consideration the needs and concerns of the users for swapping goods. Future research includes carrying out case studies on such playful applications for the current intended uses to gather insight on how they are utilized by potential users. Relevant are also empirical studies on how playful design as a tool may be used and its impact on users to get rid of perceived negative labels.
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