Information and communication technology has for long been integrated into learning and teachers utilize all forms of digital technology for communication as well as to simplify learning. To adapt, teachers need to personally or through informal learning process, learn about new technologies and how to utilize them to improve learning. To personally educate themselves, the teachers need to dedicate time and resources to identify ICT competence areas where is needed and sort for resources to solve it. This thesis investigates the process of self-directed learning with a group of teachers in a planned competence development program within a local municipality’s educational department, on the use of digital technology to integrate into classrooms. With action research that integrates instructional learning from the organizations perspective and inquiry learning from teacher’s perspective, self-directed learning process was tested as a simple and structured process for self/collaborative learning, for participants. The result was a series of events that summarized why teachers could not follow the learning process, with a conclusion that in order for teachers to be self-directed in learning new ICT, the organizational need to allocate time not only for instructional learning, but also for inquiry learning.

Chloroperoxidase (CPO) is a heme-containing glycoprotein secreted by the marine fungus Caldariomyces fumago. Chloroperoxidase contains one ferriprotoporphyrin IX prosthetic group per molecule and catalyzes a variety of reactions, such as halogenation, peroxidation and epoxidation. The versatile catalytic activities of CPO coupled with the increasing demands for chiral synthesis have attracted an escalating interest in understanding the mechanistic and structural properties of this enzyme. In order to better understand the mechanisms of CPO-catalyzed enantioselective reactions and to fine-tune the catalytic properties of chloroperoxidase, asparagine 74 (N74) located in the narrow substrate access channel of CPO was replaced by a bulky, nonpolar valine and a polar glutamine using site-directed mutagenesis. The CPO N74 mutants displayed significantly enhanced activity toward nonpolar substrates compared to wild-type CPO as a result of changes in space and polarity of the heme distal environment. More interestingly, N74 mutants showed dramatically decreased chlorination and catalase activity but significantly enhanced epoxidation activity as a consequence of improved kinetic perfection introduced by the mutation as reflected by the favorable changes in kcat and kcat/KM of these reactions. It is also noted that the N74V mutant is capable of decomposing cyanide, the most notorious poison for many hemoproteins, as judged by the unique binding behavior of N74V with potassium cyanide. Histidine 105 (H105) was replaced by a nonpolar amino acid alanine using site-directed mutagenesis. The CPO H105 mutant (H105A) displayed dramatically decreased chlorination and catalase activity possibly because of the decreased polarity in the heme distal environment and loss of the hydrogen bonds between histidine 105 and glutamic acid 183. However, significantly increased enantioselectivity was observed for the epoxidation of bulky styrene derivatives. Furthermore, my study provides strong evidence for the proposed histidine/cysteine ligand switch in chloroperoxidase, providing experimental support for the structure of the 420-nm absorption maximum for a number of carbon monoxide complexes of heme-thiolate proteins. For the NMR study, [dCPO(heme)] was produced using 90% deuterated growth medium with excess heme precursors and [dCPO(Phe)] was grown in the same highly deuterated medium that had been supplemented with excess natural phenylalanine. To make complete heme proton assignments, NMR spectroscopy has been performed for high-resolution structural characterization of [dCPO(heme)] and [dCPO(Phe)] to achieve unambiguous and complete heme proton assignments, which also allows important amino acids close to the heme active center to be determined.

Nesse projeto trabalhamos com uma β-glicosidase digestiva da larva da lagarta Spodoptera frugiperda (Sfβgli50, 50 kD - AF052729), expressa na forma de proteína recombinante em E.colli. O nosso objetivo foi estudar o papel de dois resíduos de aminoácidos envolvidos na atividade catalítica da Sfβgli50. O primeiro resíduo estudado foi o Y456, envolvido na afinidade pela porção redutora do substrato (aglicone), o segundo resíduo foi o N329 envolvido na modulação do pH ótimo. Estudo do papel do resíduo Y456 na afinidade pelo aglicone do substrato. O sítio-ativo da Sfβgli50 é formado por quatros subsítios (-1, +1, +2, e +3). O subsítio que acomoda a porção não-redutora do substrato (glicone) recebe numeração negativa (-1), já os subsítios que acomodam a porção redutora do substrato (aglicone) recebem números positivos (+1, +2 e +3). Trabalhando com duas β-glicosidases de plantas (milho e sorgo), Cicek et al. (2000) demonstraram que uma pequena porção da extremidade C-terminal destas β-glicosidases (462SSGYTERF469 - numeração da enzima do sorgo) está envolvida na especificidade pelo aglicone do substrato, sendo que muitos desses aminoácidos são conservados em outras β-glicosidases da família 1. O alinhamento das sequências destas duas enzimas com a Sfβgli50 sugere que Y456 pode fazer parte do sítio de ligação do aglicone nesta β-glicosidase de inseto. Utilizando experimentos de mutação sítio-dirigida, o Y456 foi substituído por uma alanina (mutante Y456A) sendo que este foi expresso na forma de proteína recombinante em bactérias BL21 DE3 utilizando o vetor pT7-7. O mutante Y456A foi parcialmente purificado através de uma cromatografia hidrofóbica em sistema de FPLC, e caracterizado utilizando diversos inibidores competitivos (glucono δ-lactona, celobiose, celotriose, pentilbglicosídeo e octilbtioglicosídeo). Comparando os Kis obtidos para a Sfβgli50 selvagem e mutante Y456A com os inibidores glucono δ-lactona, celobiose e celotriose, foi proposto que Y456 encontra-se no subsítio +1 do sítio ativo da Sfβgli50. Já através da comparação entre os inibidores octilβtioglicosídeo e pentilβglicosídeos constatou-se que Y456 interage com uma porção polar do aglicone do substrato, talvez através de uma ligação de hidrogênio. Baseando-se nestes Kis foi calculada a energia de associação de resíduos de glicose e grupos alquila nos subsítios +1 e +2, indicando que o subsítio +1 do mutante Y456A tem uma especificidade mais ampla frente à ligantes polares (glicose) e apolares (grupos butil) do que a enzima selvagem. Sabendo que este resultado foi obtido removendo-se um resíduo com um grupo polar na cadeia lateral (Y456), estes dados estão de acordo com a hipótese de que a especificidade dos subsítios da região de ligação do aglicone é determinada por um balanço entre resíduos polares e apolares (Marana et al., 2001). Estudo do papel do resíduo N329 na modulação do pH ótimo. O mecanismo de catálise da Sfβgli50 é dependente de dois resíduos de ácido glutâmico: um doador de prótons (E187 - pKa= 7,5) e um nucleófilo (E399 - pKa = 5,0). Sendo o pH ótimo da Sfβgli50 (6,2) uma média aritmética dos pKas destes dois resíduos catalíticos. Uma análise estrutural do sítio ativo da Sfβgli50 mostra que o resíduo N329 forma ligações de hidrogênio com o resíduo E187 (doador de prótons), talvez atuando na modulação do seu pKa. Para estudar o papel do resíduo N329 na atividade da Sfβgli50 foram construídos 3 mutantes, nos quais tal resíduo foi substituído por alanina (N329A), ácido aspártico (N329D) e uma glutamina (N329Q). Os mutantes foram expressos na forma de proteína recombinante em bactérias BL21 DE3 utilizando os vetores pT7-7 e pCal-n-Flag. Entretanto, tentativas de purificação das SfΒgli50 mutantes através de cromatografia hidrofóbica foram infrutíferas, sugerindo uma possível inativação destas enzimas. Esta hipótese foi reforçada pela purificação das Sfβgli50 mutantes e selvagem contendo o peptídeo de fusão CBP (calmodulin binding peptide) através de cromatografia de afinidade. Este experimento demonstrou que as enzimas mutantes eram de fato inativas. Frente à estes resultados não foi possível concluir a caracterização do efeito do pH na atividade catalítica das Sfβgli50 mutantes N329A, N329D e N329Q. Por fim, foi proposto que a inativação da Sfβgli50 devido à mutações na posição N329 pode resultar de uma desnaturação das enzimas mutantes ou do reposicionamento do ácido catalítico devido à perda ou alteração da interação com o resíduo 329. / In this project it was studied the role of two residues (N329 and Y456) in the catalytic activity of a digestive β-glycosidase from Spodoptera frugiperda (SfΒgli50 - AF052729). N329 is believed to modulate the enzyme pH optimum, whereas Y456 may participate in the binding of the substrate aglycone. Role of Y456 The peptide 462SSGYTERF469 of the sorghum β-glycosidase is proposed to be part of the aglycone binding site in that enzyme. Some of those residues are conserved in Sfβgli50, among them Y456. Using site-directed mutagenesis Y456 was replaced by A and this mutant (Y456A) expressed in bacteria. Following that, this mutant enzyme was partially purified using hydrophobic chromatography. Inhibition experiments showed that binding of δ-gluconolactone, which occupies subsite -1, is not affected by that mutation. In contrast, Ki values for cellobiose (that binds to subsites -1 and +1) and cellotriose (that binds to subsites -1, +1 and +2) are two-fold higher than those of wild-type enzyme, indicating that mutation Y456A decrease the interaction with these oligocellodextrins. Moreover, binding of pentyl and octylβglucosides is not affected by mutation Y456A, suggesting that Y456 interacts with aglycone polar groups. Finally, evaluation of glucose and butyl binding energies in subsite +1 revealed that mutant Y456A specificity is broader than that of wild-type enzyme. Role of N329 A structural model of Sfβgli50 active site revealed that catalytic proton donor (E187) may interact with N329. In order to study the role of this interaction in the activity of Sfβgli50, N329 was replaced by A, D and Q (mutants N329A, N329D and N329Q, respectively). These mutants were expressed as recombinant proteins in bacteria and purified through affinity chromatography, revealing that Sfβgli50 was inactivated by those mutations. It was proposed that this inactivation may be due to protein desnaturation or a wrong positioning of the catalytic proton donor.

Les virus adéno-associés (AAVs) font partie des vecteurs les plus efficaces pour le transfert de gène, en particulier dans la rétine. Ils sont utilisés aussi bien pour des études biologiques que pour la thérapie génique. Malgré cela, il reste encore des barrières qui limitent leur utilisation. Nous proposons ici d’utiliser une technique d’évolution dirigée pour surmonter ces barrières et améliorer l’efficacité des AAVs en tant que vecteurs de gènes. Dans un premier temps, nous avons créé trois librairies virales hautement diversifiées basées sur l’AAV2. Ces librairies étaient constituées de capsides modifiées aléatoirement pour leur donner de nouvelles propriétés. Nous avons ensuite réalisé deux types de sélections. D’une part, nous avons sélectionné nos librairies virales dans le système visuel de la souris pour obtenir une capside capable de transport axonal antérograde trans-synaptique afin de pouvoir étudier simultanément l’activité et la connectivité de réseaux neuronaux. Cette sélection a fortement convergée vers une capside nommée AAV2-7mD, dont la capacité de transport axonal antérograde trans-synaptique est plus efficace que les AAVs 1 et 2. D’autre part, nous avons sélectionné nos librairies virales directement sur des explants de maculas de rétine humaine afin découvrir une capside capable de traverser la membrane limitante interne de la macula humaine. Ceci a pour but d’avoir un vecteur efficace pour des traitements de thérapie génique par voie intra-vitréenne. Cette librairie a commencé à converger mais nous sommes toujours en attente du dernier cycle de sélection. Nous traitons donc dans cette thèse des résultats de deux évolutions dirigées sur l’AAV2 afin de créer des vecteurs de gènes plus performants dans le système visuel. / Adeno-associated viruses (AAVs) are among the most efficient vectors for gene transfer, particularly in the retina. They are used for asking biological questions as well as for gene therapy. Nonetheless, some barriers are still restraining their use. Here, we used a directed evolution method to overcome those barriers and improve the efficiency of AAVs for gene transfer. First, we created three highly diversified viral libraries based on AAV2. Those libraries were based on randomly modified capsids displaying new properties. Then, we did two types of selections. On one hand, we selected our libraries in the retinofugal pathway in order to obtain a capsid with enhanced axonal anterograde trans-synaptic transport capacities, so we could study simultaneously the activity and the connectivity of neuronal networks between the retina and the brain. This selection converged strongly toward a new capsid, named AAV2-7mD, with enhanced axonal anterograde trans-synaptic transport capacities compared to AAV1 and AAV2. On the other hand, we directly selected our viral libraries on human macular explants, to select capsids capable of crossing the human macular inner limiting membrane. Such a capsid would be very useful for retinal gene therapy via intravitreal injections. This library started to converge but we are still waiting to complete the last cycle of selection. In this thesis we discuss the results of these two directed evolution studies on AAV2 to create enhanced gene delivery vectors in the visual system.

Les organophosphorés (OPs) sont des composés neurotoxiques qui sont largement utilisés comme pesticides. Cette utilisation intensive a conduit à une importante pollution des sols et des effluents agricoles et sont retrouvés jusque dans les aliments. Ces pesticides sont responsables de 300 000 morts à travers le monde. Les OPs ont également été développés comme agents neurotoxiques de guerre tel que le sarin. Actuellement, il n’existe pas de méthode de décontamination externe satisfaisante pour dégrader les OPs, c’est pourquoi l’utilisation d’enzymes est une stratégie attractive. Parmi les enzymes capables de dégrader les OPs, les phosphotriestérases (PTEs) sont les plus actives mais sont peu stables ce qui limite leurs applications. Les enzymes hyperthermostables ont donc été considérées. Ainsi l’enzyme SsoPox, isolée de l’archée Sulfolobus solfataricus ayant une activité lactonase et une activité de promiscuité phosphotriestérase, a été plus particulièrement étudiée. SsoPox est extrêmement robuste mais son activité phosphotriestérase est en revanche plus faible. Une stratégie d’ingénierie protéique a été réalisée afin d’obtenir un compromis entre l’activité d'une PTE et la stabilité de SsoPox. En utilisant les similarités structurales entre ces deux enzymes, une base de données mutationnelle a été réalisée pour transférer le site actif hautement performant d’une PTE dans la structure hyperstable de SsoPox. Cette stratégie a permis d’obtenir des variants de SsoPox améliorés jusqu’à 2000 fois. L'efficacité de ces variants a été démontrée in vivo chez un modèle animal, la planaire, permettant d'améliorer la survie ainsi que la mobilité et la capacité de régénération. / Organophosphates (OPs) are neurotoxic compounds widely used as pesticides. Over the years, utilization of OP led to a considerable environmental contamination of soils and agricultural wastewaters, this pollution is furthermore a major health issue as these insecticides can be found in food. OP are highly toxic and are responsible for 300,000 deaths in the world every year. OPs were also developed as chemical warfare nerve agents such as sarin. Currently, no satisfying method for external decontamination is available, therefore bioremediation with enzymes is highly appealing. Among OP degrading enzymes, phosphotriesterases (PTEs) are the most active biocatalysts but are poorly stable what hinders their potential for bioremediation. Hyperthermostable enzymes from extreme environments were thus considered to circumvent this limitation. In particular, SsoPox isolated from the archaeon Sulfolobus solfataricus, displaying a lactonase activity and a promiscuous phosphotriesterase activity was deeply investigated. SsoPox is extremely robust but its activity for OP degradation is from far lower. A protein engineering strategy was started in order to reach a compromise between PTE activity and SsoPox robustness. Using structural similarities between PTEs and SsoPox, a mutational database was designed in order to transfer the highly performant active site of PTE into the hyperstable scaffold of SsoPox. This strategy led to variants displaying up to 2,000-fold increase against OPs as compared to wild-type enzyme. The variants efficiency was demonstrated in vivo using an original animal model planarian, allowed to enhance survival rate as well as mobility and regeneration capacity.

This study aims to bring clarity to a discussion of whether the Chinese relationship with Africa can be regarded as an act of Neo-colonialism or if it contains Neo-colonial elements. As China has increasingly engaged with the continent giving extensive amounts of foreign aid and loans as well as intensifying their trade relations, the question arises whether or not this can be connected to Neo-colonial dynamics. This study will use a newly constructed framework with the help of the Neo-colonial theory, different definitions by scholars will be used to create the framework, and the basis for this analysis. The research approach is a qualitative design and the research design is a case study with a focus on China's engagement in Africa. This study finds that there are Neo-colonial elements in the processes of engagement exercised by China on the African continent in some of the variables used in the constructed theoretical framework. Lastly, this study argues that African states must be increasingly cautious when exporting raw materials and in letting private Chinese companies invest and buy shares in important African domestic sectors.

This study focused on investigating students’ complex L2 motivational systems in an equally complex educational environment. It analyzed students’ motivation while learning Spanish in a Language for Specific Purposes (LSP) course taught in a student-centered technology-enhanced classroom at a university in the Midwest. The innovative curriculum for the course emphasized student interaction, and revolved around the development of a collaborative entrepreneurial wiki project. This study addressed the expanding call for considering motivation as multidimensional, changing and contextualized (Crookes & Schmidt, 2006; Dörnyei, MacIntyre, & Henry, 2015) by steering away from simplistic cause–effect quantitative paradigms. It addressed the topic through the lens of Complex Dynamic Systems Theory (CDST) and utilized two contemporary L2 motivation frameworks for making sense of the data: the L2 Motivational Self System (L2MSS) (Dörnyei, 2005, 2009), and Directed Motivational Currents (DMCs) (Muir & Dörnyei, 2013; Dörnyei, Ibrahim, & Muir 2015). It adopted in-depth qualitative case study methodology to answer the following research questions: 1. How can students' L2 motivations be described while learning Business Spanish through an innovative curriculum? 2. What are the factors affecting students’ L2 motivations throughout the course? Four students enrolled in this class during the Fall 2015 were randomly selected as the participants for this study. Data were collected throughout the academic semester and included: 1) four in-depth interviews with each student; 2) the work students developed collaboratively on the wiki; 3) course evaluations submitted to the instructor of the course; 4) students’ academic records and 5) classroom observations of the times students worked on the wiki. Findings revealed that the self system interacted with the motivational system of students in this class, and was determinant in guiding their motivational trajectories throughout the semester. The self system was also instrumental in shaping experiences students had related to the elements of the immediate L2 learning context. Moreover, factors stemming from the immediate L2 context that fulfilled students’ self-concordant goals were also instrumental in keeping students engaged with the process of learning; and completing the wiki project became a shared goal for students in each group. These factors led students to experience a group motivational wave — with characteristics of group DMCs — as they became more and more involved with the wiki project for the course. In terms of the work completed, students’ motivations translated into detailed wiki projects that incorporated more content than specified by the project’s guidelines and requirements. Finally, the study also generated insights into areas in which the L2MSS and DMCs could be expanded or refined in order to better account for students’ complex motivational trajectories.

La neuroglobine humaine (Ngb) est une globine découverte en 2000 dont la fonction principale demeure encore inconnue. Par comparaison avec l’hémoglobine (Hb) et la myoglobine (Mb), les globines les plus étudiées, la Ngb possède une séquence en acides aminés particulière. Il en résulte des caractéristiques structurales propres à la Ngb. L’hème, qui constitue le site actif de la Ngb, est hexacoordiné par l’histidine distale 64 et existe sous deux formes isomères A et B. La Ngb comprend également un pont disulfure Cys46-Cys55 intramoléculaire.La relation entre ces spécificités et d’éventuelles fonctions de la Ngb demeure cependant assez mal explorée. Notre objectif durant la thèse, était de mettre en évidence in vitro l’influence de différents éléments structuraux sur les propriétés et la réactivité de la Ngb. Pour ce faire, les mutations H64V, F106L, A90P et C46G ont été réalisées. Des études expérimentales à l’aide de spectrophotométrie UV-visible, de dichroisme circulaire et de RMN, ont été effectuées pour caractériser les mutants synthétisés, tester leur stabilité en fonction du pH et évaluer leur réactivité vis-à-vis de la fixation du ligand CN.Nous avons ainsi montré que la structure de la Ngb était influencée par la présence de l’histidine distale, du pont disulfure et de l’environnement de l’hème. L’étude, pour la première fois, des coefficients d’extinction molaire des protéines mutées a permis de souligner l’impact des acides aminés au voisinage de l’hème mais aussi du pont disulfure sur l’environnement électronique de l’hème. Nous avons aussi mis en évidence que le pont disulfure et les acides aminés mutés influaient sur la capacité de la forme isomère A de la Ngb à fixer le cyanure. La forme isomère B est en revanche peu impactée par ces deux paramètres. Cela soulève la question de l’existence et de la fonction des deux formes isomères de l’hème in vivo. / The physiological function of Human Neuroglobin (Ngb), discovered in 2000, is still unknown. Compared to other classical globins Haemoglobin and Myoglobin, Ngb has some structural specificities. Its haem, which is its reactive centre, is hexacoordinated by distal histidine 64 and exists under two isomer forms A and B. Moreover, Ngb possesses an intramolecular disulfide bridge between two cysteines 46 and 55.The relationship between its structural characteristics and its functions in vivo does not remain well-understood. The goal of this thesis was to underline the impact of some structural features on the Ngb properties and reactivity in vitro. Thus Ngb variants H64V, F106L, A90P and C46G were produced. Experimental studies were performed by UV-Visible spectrophotometry, circular dichroism and NMR. Variants were characterized : their stability as a function of pH were tested and their reactivity trough the CN binding reaction were evaluated.We have shown that the Ngb structure was strongly dependant on the presence of the distal histidine, the disulfide bridge and the haem environment. The first and unique determination of variants’ molar absorption coefficients underlined the influence of the haem vicinity and disulfide bridge on the electronic haem environment. We have brought some evidence that the disulfide bridge and the mutated amino acids have an impact on the isomer A Ngb ability to bind the cyanide whereas isomer B is poorly affected by those two parameters. This phenomenon raises the issue of the existence and function of the two isomer forms in vivo.

The purpose of this study was to determine whether the teams that received interpersonal training would function more effectively as a team than the teams that did not receive training. Individuals from a large division of a major defense contractor in the southern part of the United States served as subjects. Data were collected using the Team Effectiveness Profile designed to measure team effectiveness. This survey measures the overall score as well as five sub-scores. It was hypothesized that the teams that received training would function more effectively than the teams that did not receive training. The hypotheses were not supported. Results were explained, among other things, by the internal and external changes that hampered the transition towards self-managed work teams.

This study investigates the effects of a participative-type management approach termed self-managed work teams (SMWTs) and work spillover into the family environment. The perspective of isomorphism by Aldous (1969), and Rapoport and Rapoport (1965), was used as a paradigm to examine both positive and negative effects of the work-family relationship. A total of 76 employees from the Department of Defense's Quality Division was used in the regression analysis, due to recent transitions into SMWTs. Results reported overwhelming support for the perspective of isomorphism: over 40% of the variation in positive work spillover was explained by SWMT characteristics. Communication with other teams was the single most important factor found to have a significant effect on positive work spillover.