Syftet med denna studie är att undersöka hur förskolebarn i en ålder av tre till fem år utforskar hur enkel teknik fungerar och hur pedagogerna kan hjälpa barnen med detta. En ökad kunskap om barns lärande inom ämnet teknik kan vara till nytta för den vardagliga verksamheten. För att få insikt i barnens utforskande användes ostrukturerade kvalitativa observationer som metod. Även användes kvalitativa semi-strukturerade intervjuer för att få kännedom om hur pedagogerna ser på teknik och hur de anser att barn upptäcker teknik. Resultatet visar att barnens utforskande påverkas hur pedagogerna ställer frågor. Dessutom visar observationerna att barn lär sig genom  att göra och att de utforskar med hela sin kropp. Det kan även konkluderas att pedagogerna känner sig okunniga inom ämnet. / The purpose of this study is to examine how preschool children at the age of three to five years explore how simple technology works and how teachers can help them with this. A better understanding of children's learning in the subject can be beneficial for daily activities. To gain insight into children's exploratorations, unstructured qualitative observation method was used. Qualitative semi-structured interviews were also used to ascertain how teachers look at technology and how they think children discover technology. The results show that children's exploration is influenced by the way the teachers ask their questions. Moreover, the observations show that children learn by doing, and that they explore with their entire body. It can even be concluded that the teachers feel ignorant on this subject.

Le motif fluoropyridine est très fréquent dans les molécules d’intérêt thérapeutique et diagnostique pour l’imagerie par tomographie par émission de positons. Bien que les 3/5-[18F]fluoropyridines soient a priori plus stables in vivo que leurs analogues radiofluorés en position 2, 4 ou 6, leur radiosynthèse par voie nucléophile à partir du [18F]fluorure reste difficile et peu documentée. Dans ce contexte, les travaux ont porté sur l’étude du radiomarquage au fluor-18 de 3/5-fluoropyridines par radiofluoration de précurseurs iodoniums. Dans une première partie, la préparation d’une vingtaine de sels de iodonium possédant une structure pyridine ou pyridinium différemment substituée, a été mise au point. Une seconde partie a été dédiée à l’optimisation de la réaction de radiofluoration des sels de iodoniums. Cette étude a conduit à la mise en évidence du rôle important du TEMPO et de la base K2CO3 dans cette réaction. Enfin, la dernière partie a été consacrée à la radiosynthèse de 3/5-[18F]fluoropyridines portant des groupements carboxamides ou aminés par une approche multi-étape. L’ensemble des résultats ont montré que la radiofluoration de triflates de iodonium en présence de TEMPO était une méthode efficace, générale et robuste de radiosynthèse de 3/5-[18F]fluoropyridines. Cette méthode a été transposée avec succès sur deux automates de synthèse. / The fluoropyridinyl moieties have become of increasing importance in the development of drug candidates as well as of radiotracers for positron emission tomography (PET) imaging after radiolabeling with fluorine-18. Although 3/5-[18F]fluoropyridines are more stable in vivo than their 2,4 or 6 radiofluorinated analogues, their radiosynthesis by nucleophilic pathway from cyclotron produced [18F]fluoride remains difficult and poorly documented. In this context, the work focused on the development of a robust and general route to 3/5-[18F]fluoropyridines based on the radiofluorination of iodonium precursors. In a first part, the preparation of about twenty iodonium salts containing a pyridine or pyridinium scaffold, has been developed. A second part was devoted to the optimization of the radiofluorination reaction of iodonium salts. This study highlighted the important role of TEMPO and K2CO3 in this reaction. Finally, the last part was dedicated to the radiosynthesis of 3/5-[18F]fluoropyridines bearing carboxamide or amine groups according to a multistep approach. All the results showed that radiofluorination of iodonium triflates in the presence of TEMPO was an efficient, general and robust method for the radiosynthesis of 3/5-[18F]fluoropyridines. This method was also successfully transposed on two automated systems.

A persistência bacteriana correlacionada à formação de biofilmes bacterianos é, há algum tempo, fonte de grande preocupação médica em virtude de sua ampla associação com a dificuldade de tratamento de infecções crônicas. Por outro lado, as perspectivas de utilização de biofilmes bacterianos em novas aplicações biotecnológicas e até mesmo para fins terapêuticos são promissoras. Há, portanto, grande interesse em compreender os mecanismos que levam as células bacterianas a deixar o estado planctônico, de vida livre, e associarem-se nesses conglomerados celulares altamente complexos. Ao longo das últimas décadas, o segundo mensageiro c-di-GMP – em conjunto com as moléculas que catalisam sua síntese (diguanilato ciclases) e sua degradação (fosfodiesterases) e seus receptores – estabeleceu-se como um elemento central de regulação de uma série de respostas celulares que determinam a formação ou a dispersão de biofilmes. Curiosamente, as proteínas que participam do metabolismo deste segundo mensageiro estão, frequentemente, codificadas múltiplas vezes em um mesmo genoma bacteriano. Em vista dessa observação, estudos mais recentes apontam que, para reger paralelamente uma variedade tão ampla de fenótipos, este sistema opera em modo de alta especificidade de sinalização e que, portanto, o sinal metabolizado por determinados conjuntos de diguanilato ciclases e fosfodiesterases tem alvos celulares específicos. Evidências robustas, porém isoladas até o momento, apontaram que um dos meios pelo qual ocorre a segregação entre sinal produzido e alvo específico é a interação direta entre as proteínas componentes das vias de sinalização. Mais, demonstrou-se que, em algumas vias, a transmissão de sinal ocorre exclusivamente via interação proteica, dispensando a intermediação do sinalizador em si. Para avaliar a validade e relevância global deste mecanismo, propôs-se, neste estudo, a investigação da rede total de interações entre as proteínas tipicamente associadas às vias de sinalização por c-di-GMP em Pseudomonas aeruginosa, utilizando ensaios de duplo-hibrido bacteriano. Para tanto, foram construídas duas bibliotecas de DNA direcionadas e foram feitos testes de interação de forma estratégica para possibilitar o esgotamento e averiguação de todas as possíveis interações entre as proteínas alvo identificadas. O resultado obtido, um mapa inicial, porém abrangente, da rede de interações proteicas em P. aeruginosa, indica uma grande probabilidade de que os mecanismos previamente descritos sejam realmente recorrentes e relevantes para o intermédio da sinalização nesse organismo. Algumas das interações mais robustas encontradas são bastante interessantes e serão, em estudos futuros, mais extensivamente estudadas. / Persister bacteria are correlated to biofilm formation and have been a source of great medical concern due to its close association with the impairment of traditional treatment in combating chronic infections. On the other hand, using bacterial biofilms to create original biotechnological applications or even as a means of therapeutic treatment in medical settings constitutes a promising prospect. There is, therefore, a great interest in understanding the mechanisms that allow bacteria to leave the free-living planktonic lifestyle and associate in these highly complex cellular aggregates. Over the last decades, the second messenger c-di-GMP – and also the molecules involved in its synthesis (diguanylate ciclases) and degradation (phosphodiesterases) along with its receptors – has been established as a key element implicated in regulation of a series of cellular responses that determine biofilm formation or dispersion. Curiously, the proteins that play a part in the metabolism of this second messenger are frequently coded multiple times in single bacterial genomes. Taking this into account, recent studies indicate that, in order to control such a wide range of phenotypes, this system operates via high specificity of signaling – which means that the signal metabolized by a certain set of diguanylate ciclases and phosphodiesterases has specific cellular targets. Robust but yet isolated evidence indicate that a means by which a signal is segregated with its correlated phenotypic response is through direct protein-protein interaction involving the components of these signaling pathways. Even more, there has been strikingly evidence that, in some of these pathways, signal transduction occurs exclusively through protein-protein interaction, entirely dismissing any mediation by the signal molecule. In order to validate and evaluate the global relevance of this type of mechanism, this study proposed the investigation of the entire network of interactions between proteins typically associated with c-di-GMP signaling pathways of Pseudomonas aeruginosa by employing bacterial two-hybrid system assays. To make that possible, two DNA libraries were constructed and interaction essays were performed in a strategic way so that all possibilities of interaction between target proteins were explored. The results obtained from these experiments allowed the construction of a broad map of interactions that, although still primitive, indicates that, chances are, the mechanisms previously described are both recurrent and relevant to signaling regulation in this organism. Some of the interaction partners found are particularly interesting and will be further investigated in future studies.

A persistência bacteriana correlacionada à formação de biofilmes bacterianos é, há algum tempo, fonte de grande preocupação médica em virtude de sua ampla associação com a dificuldade de tratamento de infecções crônicas. Por outro lado, as perspectivas de utilização de biofilmes bacterianos em novas aplicações biotecnológicas e até mesmo para fins terapêuticos são promissoras. Há, portanto, grande interesse em compreender os mecanismos que levam as células bacterianas a deixar o estado planctônico, de vida livre, e associarem-se nesses conglomerados celulares altamente complexos. Ao longo das últimas décadas, o segundo mensageiro c-di-GMP – em conjunto com as moléculas que catalisam sua síntese (diguanilato ciclases) e sua degradação (fosfodiesterases) e seus receptores – estabeleceu-se como um elemento central de regulação de uma série de respostas celulares que determinam a formação ou a dispersão de biofilmes. Curiosamente, as proteínas que participam do metabolismo deste segundo mensageiro estão, frequentemente, codificadas múltiplas vezes em um mesmo genoma bacteriano. Em vista dessa observação, estudos mais recentes apontam que, para reger paralelamente uma variedade tão ampla de fenótipos, este sistema opera em modo de alta especificidade de sinalização e que, portanto, o sinal metabolizado por determinados conjuntos de diguanilato ciclases e fosfodiesterases tem alvos celulares específicos. Evidências robustas, porém isoladas até o momento, apontaram que um dos meios pelo qual ocorre a segregação entre sinal produzido e alvo específico é a interação direta entre as proteínas componentes das vias de sinalização. Mais, demonstrou-se que, em algumas vias, a transmissão de sinal ocorre exclusivamente via interação proteica, dispensando a intermediação do sinalizador em si. Para avaliar a validade e relevância global deste mecanismo, propôs-se, neste estudo, a investigação da rede total de interações entre as proteínas tipicamente associadas às vias de sinalização por c-di-GMP em Pseudomonas aeruginosa, utilizando ensaios de duplo-hibrido bacteriano. Para tanto, foram construídas duas bibliotecas de DNA direcionadas e foram feitos testes de interação de forma estratégica para possibilitar o esgotamento e averiguação de todas as possíveis interações entre as proteínas alvo identificadas. O resultado obtido, um mapa inicial, porém abrangente, da rede de interações proteicas em P. aeruginosa, indica uma grande probabilidade de que os mecanismos previamente descritos sejam realmente recorrentes e relevantes para o intermédio da sinalização nesse organismo. Algumas das interações mais robustas encontradas são bastante interessantes e serão, em estudos futuros, mais extensivamente estudadas. / Persister bacteria are correlated to biofilm formation and have been a source of great medical concern due to its close association with the impairment of traditional treatment in combating chronic infections. On the other hand, using bacterial biofilms to create original biotechnological applications or even as a means of therapeutic treatment in medical settings constitutes a promising prospect. There is, therefore, a great interest in understanding the mechanisms that allow bacteria to leave the free-living planktonic lifestyle and associate in these highly complex cellular aggregates. Over the last decades, the second messenger c-di-GMP – and also the molecules involved in its synthesis (diguanylate ciclases) and degradation (phosphodiesterases) along with its receptors – has been established as a key element implicated in regulation of a series of cellular responses that determine biofilm formation or dispersion. Curiously, the proteins that play a part in the metabolism of this second messenger are frequently coded multiple times in single bacterial genomes. Taking this into account, recent studies indicate that, in order to control such a wide range of phenotypes, this system operates via high specificity of signaling – which means that the signal metabolized by a certain set of diguanylate ciclases and phosphodiesterases has specific cellular targets. Robust but yet isolated evidence indicate that a means by which a signal is segregated with its correlated phenotypic response is through direct protein-protein interaction involving the components of these signaling pathways. Even more, there has been strikingly evidence that, in some of these pathways, signal transduction occurs exclusively through protein-protein interaction, entirely dismissing any mediation by the signal molecule. In order to validate and evaluate the global relevance of this type of mechanism, this study proposed the investigation of the entire network of interactions between proteins typically associated with c-di-GMP signaling pathways of Pseudomonas aeruginosa by employing bacterial two-hybrid system assays. To make that possible, two DNA libraries were constructed and interaction essays were performed in a strategic way so that all possibilities of interaction between target proteins were explored. The results obtained from these experiments allowed the construction of a broad map of interactions that, although still primitive, indicates that, chances are, the mechanisms previously described are both recurrent and relevant to signaling regulation in this organism. Some of the interaction partners found are particularly interesting and will be further investigated in future studies.

Mon projet a porté sur la caractérisation du mécanisme moléculaire des enzymes d'arenavirus (une 3'-5' exoribonucléase et une endonuclease) impliquées dans l'inhibition de la réponse innée IFN de type I et dans le vole de coiffe respectivement, et le développement d'une stratégie thérapeutique basée sur leur structures. Premièrement, j'ai résolu deux structures cristallographiques à haute résolution du domaine exoribonucléases du virus Mopeia (NP-exo MOPV) -un homologue du virus Lassa pathogène- en complexe avec deux ions différents. Ensuite, j'ai effectué une caractérisation fonctionnelle de l’activité exoribonucléase 3'-5' codée par ce domaine. Une corrélation entre la structure et la fonction de NP-exo MOPV démontre que; L’activité exoribonucléase 3'-5' est conservée chez les arenavirus pathogènes ainsi que chez les non-pathogènes. J'ai démontré pour la première fois que l'exoribonucléase est capable d'exciser un ARN misapparié, suggérant ainsi une potentielle activité de correction d'erreur par cette enzyme. Avec la structure de NP-exo MOPV, j'ai développé une stratégie in silico pour identifier des inhibiteurs potentiels spécifiques contre son activité et un inhibiteur a était identifié.En parallèle, nous avons résolu deux structures cristallographiques du domaine de l'endonuclease du virus de la LCMV en complexe avec deux ions catalytiques et deux composés appartenant a la famille des diketo. En résumé, ce travail éclaircit le rôle des exoribonucléases de la famille d'Arenaviridae allant de l’évasion de l'immunité innée à son implication directe dans la réplication. Il ouvre également la voie au développement des inhibiteurs contre ces nucléases. / My PhD work focused on the characterization of the molecular mechanism of two arenavirus enzymes - a 3'-5' exoribonuclease and an endonuclease - implicated in type I IFN suppression and mRNA cap-snatching respectively and the design of a structure based-drug strategy against them. First I solved two high resolution crystal structures of the exoribonuclease domain of Mopeia virus (NP-exo MOPV) -a non pathogenic homologue of the highly pathogenic Lassa virus- in complex with different metal ions. Next I performed an in depth functional characterization of the 3'-5' exoribonuclease activity encoded by this domain. By correlating the structure and function of NP-exo MOPV, I showed that; the 3'-5' exoribonuclease activity is conserved in pathogenic as well as in non-pathogenic arenaviruses. Also, I showed for the first time that this enzyme is able to excise a mismatched RNA suggesting that, arenaviruses might posses a mechanism to limit error incorporation by the RdR polymerase during replication. Using the crystal structure of NP-exo MOPV I designed a structure-based strategy to identify potential inhibitors specific for the 3'-5' exoribonuclease activity and have identified a potential inhibitor.Alongside, we solved two crystal structures of the endonuclease domain of LCMV in complex with two catalytic ions and two compounds belonging to the diketo family.In conclusion, this work has a deep implication extending the role of the Arenaviridae exoribonuclease from innate immunity evasion to direct implication in replication. It also paves the way for the development of inhibitors against these arenavirus nucleases.

[pt] O planejamento de estruturas push-pull tem levado a compostos com propriedades fotofísicas interessantes. Isto inclui a Emissão Induzida por Agregação (AIE) e a Emissão Melhorada Induzida por Agregação (AIEE), que são importantes para emissão em estado sólido e, portanto, para aplicações em dispositivos optoeletrônicos. Visando AIEE, novos compostos fluorescentes com características D-Pi-A e D-A-D foram planejados. Usando ferramentas computacionais, foram feitas simulações para calcular as propriedades teóricas dos compostos desenhados e validar se eram apropriadas para o desenvolvimento sucessivo das rotas sintéticas. No presente trabalho, duas novas séries de derivados de piridina3,5-dicarbonitrilo difenil-substituído (PPC) e derivados de 2,1,3- benzothiadiazol fluorenil-substituído (FL-BTD) foram sintetizados usando estruturas D-Pi-A e D-A-D, respectivamente. As estratégias sintéticas usadas para a sínteses dos novos compostos incluem reações multicomponente de Hantzsch (na produção do principal intermediário na série PPC), assim como reações de acoplamento cruzado C-C catalisadas por paládio, incluindo Heck, Sonogashira e Suzuki e acoplamentos N-C via reação Buchwald-Hartwig para a obtenção dos produtos. Um total de seis novos derivados de PPC, foram obtidos e caracterizados, com rendimentos de 35 a 73 por cento, e quatro novos derivados de BTD com rendimentos de reação de 39 a 95 por cento são reportados. Experimentos de espectroscopia foram realizados para a avaliação das propriedades fotofísicas dos novos derivados de PPC e BTD, abrangendo medidas de absorção, emissão, e de rendimento quântico fluorescente (Rendimento quântico de fluorescência). Os compostos apresentaram altos Rendimentos quânticos de fluorescência em solução, até 0,71 na série PPC e até 0,67 na série FL-BTD, exibindo band gaps adequados para sua aplicação como camadas emissoras em OLEDs. O comportamento no estado agregado dos derivados de PPC e BTD foi avaliado, quatro compostos exibiram aumento de luminescência em soluções THF contendo fração de água (fw) de 70 por cento até maior que 90 por cento, indicando as propriedades AIEE. Um dos compostos da série PPC, MT-PPC, apresentou AIE com indução da florescência na mistura com fw 80 por cento. Na série BTD, destacou-se o derivado FL-BTD-OAM com AIEE e Rendimento quântico de fluorescência significativo, assim, um OLED foi construído utilizando-o como camada emissora, obtendo resultados promissores com um dispositivo operando com alto brilho (luminância de 6450 cd m-2), boa irradiação (503 mW cm-2) e eficiência de corrente razoável (0,84 cd/A). / [en] The design of push-pull structures has led to compounds with interesting photophysical properties. This includes Aggregation-Induced Emission (AIE) and Aggregation-induced Enhanced Emission (AIEE), which are important for emission in solid state and therefore for applications in optoelectronic devices. Aiming AIEE, new fluorescent compounds with D-Pi-A and D-A-D characteristics were designed. Using computational tools, simulations were made to calculate their theoretical properties and validate if were appropriate for the successive development of the synthetic routes. The present work reports two new series of diphenyl-substituted pyridine3,5-dicarbonitrile (PPC) derivatives and fluorenyl-substituted 2,1,3-benzothiadiazole (FL-BTD) derivatives, synthetized using D-Pi-A and D-AD structures, respectively. The compounds presented high fluorescence quantum yields in solution, up to 0.71 in the PPC series and up to 0.67 in the FL-BTD series, displaying suitable band gaps for application as emitting layers in OLEDs. Five compounds displayed enhancement of luminescence in THF solutions containing water fraction (fw) from 70 percent up to bigger than 90 percent, indicating AIEE properties. Among these, the derivative FL-BTD-OAM stood out, and an OLED device was built using it as an emitting layer, obtaining promising results with a device operating with high bright (luminance of 6450 cd m-2), good irradiance (503 mW cm-2) and reasonable current efficiency (0.84 cd/A).

TREX2, a 3’-5’ exonuclease, plays a pivotal role in cleaving nucleoside monophosphates at the 3’ terminus of ssDNA. Despite previous insights into its biochemical functions—homodimerisation, DNA binding, and enzymatic activity—the precise biological significance of TREX2 remains elusive. The broad objective of this thesis was to elucidate the physiological function of the 3’-5’ DNA exonuclease TREX2, aiming to deepen our understanding of the cellular mechanisms governed by TREX2. Considering the implications of DNA exonuclease dysregulation in autoimmune disorders, we aimed to explore the potential implications of TREX2 deficiency on immune function. Due to the lack of anti-TREX2 antibodies recognising endogenous TREX2, we worked on overexpression systems. During interphase, GFP-tagged TREX2 predominantly localised to the cytoplasm; however, throughout all stages of mitosis, it accumulated within the nucleus, showing significant colocalisation with chromatin. This suggests a multifaceted role for TREX2 in maintaining chromatin integrity during cell division. Utilising the CRISPR-Cas9 knockout technique, we targeted the TREX2 gene in wildtype HaCaT cells. Our unexpected findings revealed that TREX2 deficiency led to reduced basal type I IFN activity, contrary to the anticipated effect observed with TREX1 deficiency. This suggests that TREX2 plays a crucial role in maintaining the homeostasis of the type I IFN pathway. Moreover, upon stimulation with cGAS- specific agonists, TREX2 knockout HaCaT cells exhibited diminished responsiveness, supporting a positive regulatory function of TREX2 in the cGAS-sensing pathway. Live imaging further demonstrated colocalisation of TREX2 and cGAS with mitotic chromatin, underscoring their collaborative role in cellular dynamics. In conclusion, TREX2 emerges as a crucial player in regulating the type I IFN pathway, influencing immune homeostasis in association with cGAS. These findings pave the way for comprehending the intricate interplay of TREX2 in cellular processes and its implications for immune modulation.

No description available.

Escherichia coli is the best characterized bacterium; it grows rapidly, and it is easy to manipulate genetically. An increased knowledge about the physiology of this model organism will facilitate the development of engineered E.coli strains for applications such as production of biofuels and biofilm control. The aims of this work were the application of protein engineering to increase E. coli hydrogen production, the identification of the proteins regulating extracellular DNA production (eDNA), and the evaluation of the effect of the proteins synthesizing the signal 3'-5'-cyclic diguanylic acid (c-di-GMP) on biofilm formation. The Escherichia coli hydrogen production rate was increased 9 fold through random mutagenesis of fhlA. Variant FhlA133 (Q11H, L14V, Y177F, K245R, M288K, and I342F) enhances hydrogen production by increasing transcription of the four transcriptional units regulated by FhlA. The amino acid replacements E363G and L14G in FhlA increased hydrogen production 6 fold and 4 fold, respectively. The complete E. coli genome was screened to identify proteins that affect eDNA production. The nlpI, yfeC, and rna mutants increased eDNA production and the hns and rfaD mutants decreased eDNA production. Deletion of nlpI increases eDNA 3 fold while overexpression of nlpI decreases eDNA 16 fold. Global regulator H-NS is required for eDNA with E. coli since deletion of hns abolished eDNA production while overexpression of hns restored eDNA to 70 percent of the wild-type levels. Our results suggest that eDNA production in E. coli is related to direct secretion. Deletions of the genes encoding the diguanylate cyclases YeaI, YedQ, and YfiN increased swimming motility and eDNA as expected for low c-di-GMP levels. However, contrary to the current paradigm, early biofilm formation increased dramatically for the yeaI (30 fold), yedQ (12 fold), and yfiN (18 fold) mutants. Hence, our results suggest that c-di-GMP levels should be reduced for initial biofilm formation because motility is important for initial attachment to a surface.

Thesis (Ph. D.)--University of Washington, 2007. / Vita. Includes bibliographical references (leaves 102-118).