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Development of Methods for Protein Delivery and the Directed Evolution of RecombinasesThompson, David Brandon 01 January 2015 (has links)
As a class, protein-based therapeutics offer tremendous advantages over traditional small molecule drugs. Due to their sizes and folding energies, proteins are ideal for catalyzing chemical reactions, and can bind tightly and selectively to extended target surfaces. However, due to their large size, virtually all proteins are unable to spontaneously enter cells, and as a result protein therapeutics are restricted to extracellular targets. We developed a platform for delivery of proteins to intracellular target sites by engineering the surface chemistry of a model protein, green fluorescent protein (GFP). We found that 'supercharged' cationic GFP variants (scGFPs) bind to anionic cell surface molecules and initiate endocytosis, resulting in the efficient delivery of translationally fused cargo to intracellular targets. We discovered that scGFPs, and cationic delivery reagents in general, alter endosomal trafficking in a manner proportional to both their charge and their delivery efficiency, suggesting that avoidance of endosomal maturation is a key step in the endosomal escape of delivered protein cargos. We also developed a method for encapsulation of recombinant proteins by cationic lipid delivery reagents using negatively supercharged GFP.
Genetic modification technologies have matured rapidly following the discovery of protein classes with programmable DNA-binding specificities. While site-directed genetic knockout technologies are highly effective, targeted integration and repair remain comparatively inefficient. Site-specific recombinases directly catalyze strand exchange and ligation between DNA molecules, offering an approach to efficient genomic integration. However, most site-specific recombinases are not easily reprogrammable. To address this problem, we developed a genetic selection technique based on the Phage-Assisted Continuous Evolution (PACE) system, to enable the rapid evolution of recombinase proteins towards targets of interest. Using Cre recombinase as a model, the PACE system was optimized, validated, and used to evolve Cre variants with higher activity on their native loxP target site, as well as altered specificity towards a human genomic sequence within the hROSA26 locus.
Finally, we developed a method for enhancing the specificity of RNA-guided nucleases by restricting activity to sites of obligate dimeric nuclease assembly. We engineered a FokI nuclease fusion to a catalytically inactivated Cas9 protein that mediates efficient modification with significantly reduced off-target activity.
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Development of molecular diagnostic system for detection of hepatitis B virus in blood donationsFun, Sze-tat., 范思達. January 2003 (has links)
published_or_final_version / Medical Sciences / Master / Master of Medical Sciences
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När barn talar om politik : En studie om barns ord om politik och samhälleJohansen, Suzie, Jurcevic, Kristina January 2015 (has links)
Abstrakt Syftet med den här uppsatsen är att lyfta fram barns tankar om parlamentarisk politik och samhällsfrågor utifrån deras perspektiv. Uppsatsen är av kvalitativ art och vi har använt oss både av fokusgrupp och individuella intervjuer. Vi har inspirerats av tidigare forskning där barn som medborgare och FN:s barnkonvention artikel 12 är i fokus för att öka legitimiteten i vår studie. Uppsatsen har utgått ifrån politisk teori, socialisationsprocessen, genus och gruppstyrda. Vi har fått resultat ur både grupp och individperspektiv som stärker barn som tänkande individer utan rättslig handlingsförmåga. Trots att barn inte talar vuxenspråk har de åsikter om och påverkas av den omvärld de lever i. Den grupp med barn vi har lyssnat på kunde inte mycket om politiska partier, däremot kunde de mycket om fattigdom, ebola och välgörenhet. De hade problem med partiledarnas namn, däremot berättade de för oss hur de upplever skolmiljön och gav förslag till förbättringar i skolan. Ur barnens perspektiv har vi försökt fånga essensen i deras ord. / Abstract The purpose of this paper is to highlight children’s thoughts regarding issues of society and politics. Our paper is qualitative and we´ve used both a focus group and several individual interviews. We were inspired by previous research in which children, as citizens, are in focus. We also used UNCRC article 12 in order to increase the legitimacy of our study. The thesis has been based on political theory, socialization, gender and other-directed. We have received results from groups and individuals perspective that strengthen children as thinking individuals without legal capacity. Although children do not use adult speech, they have opinions and are influenced by the society they live in. The group of children we listened to didn´t know much about political parties, they did, however, know about poverty, Ebola and charity. They had problems naming the leaders of political parties, but could tell us how they perceived the school-environment and suggested improvements while we interviewed them. We´ve tried to capture the essence of the childrens words in their perspective.
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The Effects of Cultural and Economic Capital on both Formal and Informal Learning for the WorkplaceStowe, Susan Lynn 31 August 2012 (has links)
The aim of the thesis was to explore the magnitude of inequity in accessibility to initial formal education, continuing adult education, and work-related informal learning for the workplace. The two main issues that the thesis attempted to determine is whether social background characteristics that affect initial educational attainment continue to influence participation in adult education and work-related informal learning. More specifically, this research focused on three main questions: First, to what extent does parents’ social background influence educational attainment levels for Canadians from different generations? Second, to what extent does parents’ social background influencing participation in adult education for their offspring beyond the effects of an individual’s own social background? And, third, to what extent does parents' social background have on their offspring’s participation in informal learning for the workplace beyond the effects of an individual's own social class background? A secondary data quantitative analysis was carried out on the data collected in the 2004 Work and Lifelong Learning (WALL) survey. Both crosstab analysis and structural equation analysis were used to obtain an overview of inequities in participation in formal education and informal learning and to test the applicability of Bourdieu’s social reproduction theory. Overall, the findings of this thesis indicate that social reproduction occurs not only through the formal education system, but also through the adult education system. More specifically, parents’ education continues to be a good predictor of the level of education attained by offspring. Moreover, one’s level of education continued to be a predictor of participation in adult education. Social reproduction was not present for work-related informal learning. In fact, those from low incomes were more likely to engage in informal learning than those from high incomes. These findings indicate that despite level of cultural and economic capital, the majority of Canadians engage in a learning activity. It is apparent that structures that are present in our formal education system continue to advantage students with high cultural and economic capital; however, work-related informal learning is accessible to all.
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A Convenient Synthesis of Pyrrolnitrin and Related Halogenated PhenylpyrrolesMORRISON, MATTHEW 07 October 2009 (has links)
This thesis details a straightforward synthetic route to the antifungal compound pyrrolnitrin 1.2, along with several analogous halogenated phenylpyrroles. The proposed synthetic protocol involved the Suzuki-Miyaura cross-coupling of appropriately halogenated pyrrole pinacolboronate esters and aryl compounds.
In the efforts towards preparing the cross-coupling partners, we report a regiospecific and high yielding synthesis of a 3-chloro pyrrole compound 2.14, its brominated analog 2.16, an iodinated analog 2.17, and the corresponding pinacolboronate ester 2.18. We also report a generalized reaction sequence (lithiation/carboxylation/Schmidt reaction/oxidation) for the preparation of halogenated benzoic acids, anilines and nitrobenzenes. In particular, we synthesized the desired halogenated nitrobenzene coupling partner 3.27 in excellent yield. We were also able to show that the conditions employed in this sequence were mild enough to allow preparation of the 2-bromo-6-iodo compound 3.33.
Once the coupling partners were prepared, we developed the optimal conditions for our Suzuki-Miyaura cross-coupling reactions. In doing so, we were able to prepare our target compound 1.2 and several halogenated analogs in good yields. We also prepared brominated and deuterated arylpyrroles 4.27 and 4.28, respectively, for future use in mechanistic studies of the pyrrolnitrin biosynthetic enzymes, PrnB, Prn C and PrnD. This required preparation of the corresponding brominated and deuterated pyrrole pinacolboronate esters 4.24 and 4.26. / Thesis (Master, Chemistry) -- Queen's University, 2009-09-29 13:58:35.186
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UNDERSTANDING THE CHEMICAL GYMNASTICS OF ENZYME-CATALYZED 1’-1 AND 1’-3 TRITERPENE LINKAGESBell, Stephen A 01 January 2014 (has links)
Squalene synthase (SS) is an essential enzyme in eukaryotic systems responsible for an important branch point in isoprenoid metabolism that leads to sterol formation. The mechanistic complexity of SS has made it a difficult enzyme to study. The green alga Botryococcus braunii race B possesses several squalene synthase-like (SSL) enzymes that afford a unique opportunity to study the complex mechanism of triterpene biosynthesis. SSL-1 catalyzes presqualene diphosphate (PSPP) formation, which can either be converted to squalene by SSL-2 or botryococcene by SSL-3. A rationally designed mutant study of B. braunii squalene synthase (BbSS) and SSL-3 was conducted to understand structure-function relations among these enzymes. These studies revealed two amino acid positions in SSL-3 (N171, G207) that appeared to control 1’-3 versus 1’-1 linkages. The reciprocal mutations in the corresponding positions of BbSS did not convert this enzyme into a botryococcene synthase.
Next, a genetic selection was developed to evolve SSL enzymes towards a fully functional SS. Previous studies have shown that Saccharomyces cerevisiae squalene synthase (ScSS) can be knocked out and although lethal, growth can be restored by providing an exogenous source of ergosterol. Additional studies have shown that successful complementation of the ScSS knockout with a non-fungal SS is possible but requires a fungal SS carboxy- terminus region. Given these observations, proof-of-principle experiments were conducted to demonstrate that SSL-SSL fusion enzymes could complement the ScSS knockout followed by construction of a mutant SSL-SSL fusion enzyme library that was screened in the ScSS knockout yeast line. From this library, mutant SSL-SSL fusion enzymes were identified that were able to complement, which demonstrated the feasibility of this approach as a genetic selection for mutant SSL enzymes.
Squalene and botryococcene have valuable industrial applications in vaccine adjuvant formations, cosmetic products, and renewable energy feedstock material. Limitations in natural sources of these molecules have made heterologous production of them an important research target. Algae represent a desirable group of organisms that could be engineered to produce these metabolites because they are photosynthetic and capable of using non-arable farmland. The feasibility, approach, and progress for engineering green algae to produce squalene and botryococcene are discussed.
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Mémorisation de séquences dans des réseaux de neurones binaires avec efficacité élevéeJIANG, Xiaoran 08 January 2014 (has links) (PDF)
Sequential structure imposed by the forward linear progression of time is omnipresent in all cognitive behaviors. This thesis proposes a novel model to store sequences of any length, scalar or vectorial, in binary neural networks. Particularly, the model that we introduce allows resolving some well known problems in sequential learning, such as error intolerance, catastrophic forgetting and the interference issue while storing complex sequences, etc. The quantity of the total sequential information that the network is able to store grows quadratically with the number of nodes. And the efficiency - the ratio between the capacity and the total amount of information consumed by the storage device - can reach around 30%. This work could be considered as an extension of the non oriented clique-based neural networks previously proposed and studied within our team. Those networks composed of binary neurons and binary connections utilize the concept of graph redundancy and sparsity in order to acquire a quadratic learning diversity. To obtain the ability to store sequences, connections are provided with orientation to form a tournament-based neural network. This is more natural biologically speak- ing, since communication between neurons is unidirectional, from axons to synapses. Any component of the network, a cluster or a node, can be revisited several times within a sequence or by multiple sequences. This allows the network to store se- quences of any length, independent of the number of clusters and only limited by the total available resource of the network. Moreover, in order to allow error correction and provide robustness to the net- work, both spatial assembly redundancy and sequential redundancy, with or without anticipation, may be combined to offer a large amount of redundancy in the activation of a node. Subsequently, a double layered structure is introduced with the purpose of accurate retrieval. The lower layer of tournament-based hetero-associative network stores sequential oriented associations between patterns. An upper auto-associative layer of mirror nodes is superposed to emphasize the co-occurrence of the elements belonging to the same pattern, in the form of a clique. This model is then extended to a hierarchical structure, which helps resolve the interference issue while storing complex sequences. This thesis also contributes in proposing and assessing new decoding rules with respect to sparse messages in order to fully benefit from the theoretical quadratic law of the learning diversity. Besides the performance aspect, the biological plausibility is also a constant concern during this thesis work.
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The potential of a learning management system to enhance self-directed learning / Chantelle TredouxTredoux, Chantelle January 2012 (has links)
The use of Learning Management Systems (LMSs) in higher education institutions is not a new tendency. Although this is an innovative way of implementing e-learning in the educational process, there are a few problems concerning these systems. Educators tend to apply traditional classroom ideas and pedagogy in computer-supported e-learning environments, assuming that because these environments allow the interaction that we see in the classroom, traditional pedagogy can be used. Although most of the pedagogical principles that apply to the traditional classroom-delivery method also apply to e-learning, the e-environment supports such interactions in a different manner. Traditional pedagogical principles should be adapted to accommodate the e-learning environment and should form the very basis for inclusion of features in LMSs. These principles should be integrated into the LMS where every feature included is accompanied by explicit guidelines on how to use the feature in such a way that it will effect pedagogically sound instruction.
The aim of this study is to determine how an LMS could be used in order to enhance self-directed learning. In order to reach this aim a brief history of SDL was given and a number of SDL models were discussed. These models were analyzed in order to compile a list of guidelines to foster SDL. The first set of guidelines didn’t focus on any specific learning environment and it was necessary to refine these guidelines for an online environment. To be able to refine the guidelines for an online environment, LMSs in general were discussed and a few models for SDL in an online environment were reviewed. The SDL guidelines were further refined for implementation in eFundiTM. eFundiTM is the LMS used at the North-West University, Potchefstroom campus, South Africa. The nature of the AGLE 121 module (a literacy module for all first year students) and the specific functionalities of eFundiTM were discussed and taken in consideration when the final set of guidelines was compiled.
The researcher did an empirical study to gather valid and reliable data. A mixed methods inquiry approach was used to obtain reliable evidence. The population consisted of all the students that were enrolled for the AGLE modules over 2 years. These students were divided into 2 groups, the AGLE 121 in 2010 (237 students) and the AGLE 121 (287 students) in 2011. The questionnaire that was used for the quantitative research in this study was based on the Fisher, King and Taque (2001) SDL readiness scale for nursing education. Semi-structured interviews were conducted with seven participants from each group in order get a better understanding of the data collected from the quantitative research, and to elaborate further on the students’ development of SDL. Findings indicated that the students from the second year of the study did not necessarily improve their SDL-skills. Most of the results from the quantitative data showed small practical significant differences. However, the qualitative data indicated that the SDL skills of the students improved in two of the three factors after they used the newly developed eFundiTM site, in the second year of the study and therefore the researcher is of opinion that the intervention had a positive impact on the students’ SDL skills. / Thesis (MEd (Computer Science Education))--North-West University, Potchefstroom Campus, 2012
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The potential of a learning management system to enhance self-directed learning / Chantelle TredouxTredoux, Chantelle January 2012 (has links)
The use of Learning Management Systems (LMSs) in higher education institutions is not a new tendency. Although this is an innovative way of implementing e-learning in the educational process, there are a few problems concerning these systems. Educators tend to apply traditional classroom ideas and pedagogy in computer-supported e-learning environments, assuming that because these environments allow the interaction that we see in the classroom, traditional pedagogy can be used. Although most of the pedagogical principles that apply to the traditional classroom-delivery method also apply to e-learning, the e-environment supports such interactions in a different manner. Traditional pedagogical principles should be adapted to accommodate the e-learning environment and should form the very basis for inclusion of features in LMSs. These principles should be integrated into the LMS where every feature included is accompanied by explicit guidelines on how to use the feature in such a way that it will effect pedagogically sound instruction.
The aim of this study is to determine how an LMS could be used in order to enhance self-directed learning. In order to reach this aim a brief history of SDL was given and a number of SDL models were discussed. These models were analyzed in order to compile a list of guidelines to foster SDL. The first set of guidelines didn’t focus on any specific learning environment and it was necessary to refine these guidelines for an online environment. To be able to refine the guidelines for an online environment, LMSs in general were discussed and a few models for SDL in an online environment were reviewed. The SDL guidelines were further refined for implementation in eFundiTM. eFundiTM is the LMS used at the North-West University, Potchefstroom campus, South Africa. The nature of the AGLE 121 module (a literacy module for all first year students) and the specific functionalities of eFundiTM were discussed and taken in consideration when the final set of guidelines was compiled.
The researcher did an empirical study to gather valid and reliable data. A mixed methods inquiry approach was used to obtain reliable evidence. The population consisted of all the students that were enrolled for the AGLE modules over 2 years. These students were divided into 2 groups, the AGLE 121 in 2010 (237 students) and the AGLE 121 (287 students) in 2011. The questionnaire that was used for the quantitative research in this study was based on the Fisher, King and Taque (2001) SDL readiness scale for nursing education. Semi-structured interviews were conducted with seven participants from each group in order get a better understanding of the data collected from the quantitative research, and to elaborate further on the students’ development of SDL. Findings indicated that the students from the second year of the study did not necessarily improve their SDL-skills. Most of the results from the quantitative data showed small practical significant differences. However, the qualitative data indicated that the SDL skills of the students improved in two of the three factors after they used the newly developed eFundiTM site, in the second year of the study and therefore the researcher is of opinion that the intervention had a positive impact on the students’ SDL skills. / Thesis (MEd (Computer Science Education))--North-West University, Potchefstroom Campus, 2012
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The Effects of Cultural and Economic Capital on both Formal and Informal Learning for the WorkplaceStowe, Susan Lynn 31 August 2012 (has links)
The aim of the thesis was to explore the magnitude of inequity in accessibility to initial formal education, continuing adult education, and work-related informal learning for the workplace. The two main issues that the thesis attempted to determine is whether social background characteristics that affect initial educational attainment continue to influence participation in adult education and work-related informal learning. More specifically, this research focused on three main questions: First, to what extent does parents’ social background influence educational attainment levels for Canadians from different generations? Second, to what extent does parents’ social background influencing participation in adult education for their offspring beyond the effects of an individual’s own social background? And, third, to what extent does parents' social background have on their offspring’s participation in informal learning for the workplace beyond the effects of an individual's own social class background? A secondary data quantitative analysis was carried out on the data collected in the 2004 Work and Lifelong Learning (WALL) survey. Both crosstab analysis and structural equation analysis were used to obtain an overview of inequities in participation in formal education and informal learning and to test the applicability of Bourdieu’s social reproduction theory. Overall, the findings of this thesis indicate that social reproduction occurs not only through the formal education system, but also through the adult education system. More specifically, parents’ education continues to be a good predictor of the level of education attained by offspring. Moreover, one’s level of education continued to be a predictor of participation in adult education. Social reproduction was not present for work-related informal learning. In fact, those from low incomes were more likely to engage in informal learning than those from high incomes. These findings indicate that despite level of cultural and economic capital, the majority of Canadians engage in a learning activity. It is apparent that structures that are present in our formal education system continue to advantage students with high cultural and economic capital; however, work-related informal learning is accessible to all.
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