Ces travaux de thèse, effectués dans le cadre des études sur le retraitement des combustibles nucléaires usés par extraction-liquide-liquide, concernent l’étude cinétique d’extraction de l’uranium(VI) et du plutonium(IV) en milieu acide nitrique par un mélange d’extractants de type monoamide. Trois techniques ont été utilisées pour étudier les cinétiques d’extraction et identifier les régimes de transfert : la méthode de la goutte unique, la cellule d’extraction de Nitsch et la cellule à membrane tournante (RMC). Les résultats obtenus avec la goutte unique indiquent que les cinétiques de transfert d’U(VI) et Pu(IV) sont proches. Une étude hydrodynamique des gouttes et la modélisation du transfert de l’U(VI) ont montré que la présence d’une circulation interne à la goutte diminue les résistances diffusionnelles pour des tailles de goutte croissantes. L’influence de la concentration d’U(VI) dans le solvant sur la cinétique d’extraction a montré que le transfert d’U(VI) et Pu(IV) ralentit lorsque la concentration d’U(VI) en phase organique et la viscosité augmentent. L’ensemble de ces résultats couplé à l’étude du transfert de l’U(VI) par la cellule de Nitsch conduisent à supposer une cinétique gouvernée par la diffusion principalement localisée dans la phase organique. Les études réalisées avec la RMC ont permis de déterminer la constante chimique d’extraction de l’U(VI), du même ordre de grandeur que les cinétiques obtenues par la goutte unique et de confirmer la présence d’une réaction chimique interfaciale. Enfin, le transfert d’U(VI) et Pu(IV) s’avère 3 fois plus lent que celui par le TBP mais reste adapté pour un procédé d’extraction à l’échelle industrielle. / This thesis was conducted in the framework of the reprocessing of spent nuclear fuels. The kinetics extraction of uranium(VI) and plutonium(IV) by N,N-dialkylamides or monoamides in an aliphatic diluent were studied from an aqueous nitric solution using 3 different techniques: “single drop technique”, Nitsch cell and Rotating Membrane Cell (RMC). All these experiments were useful to attempt the identification of the transfer process between the phases which can be controlled by kinetic or diffusional regime. The kinetics of extraction of U(VI) by monoamides solvent seems to be similar to that of the Pu(IV). In general, molecular diffusion in organic phase slows down the extraction process and the limiting thickness of organic phase increases with solvent viscosity. The process in “single drop technique” seems to be controlled not only by diffusion but also by the chemical reaction. Then the extraction kinetics of U(VI) has been carried out by Nitsch cell and the RMC. Diffusionnal regime is the limiting step in Nitsch cell and the results confirm that molecular diffusion in organic phase should mainly control the kinetics transfer. Then experiments performed by the RMC indicate that the kinetics is in the same order as transfer coefficient obtained by “single drop” and the chemical reaction occurs at the interface. Finally, these results were compared with data obtained with a TBP solvent (tributyl phosphate) currently used in the PUREX process in order to estimate the interest of such a new solvent. The kinetics of extraction of U(VI) by this monoamide-based solvent is three times lower that of the TBP 30 % but remains fast and suitable for a future industrial process.

Esta dissertação foca um processo de criação de imagens fotográficas a partir de manipulações químicas feitas sobre superfícies fotossensíveis, diretamente sobre o papel ou negativo, sem utilizar para isso o aparato tecnológico – a câmera. A partir dessa abordagem, a dissertação analisa os limites daquilo que conhecemos como fotografia. A pesquisa convergiu para a investigação das possibilidades fotográficas inerentes às superfícies sensíveis e fotossensíveis, às soluções fotoquímicas e para o cruzamento dessas possibilidades fotográficas com outras linguagens artísticas. O trabalho confrontou a fotografia com suas especificidades e buscou desvendar os domínios da fotograficidade, que se configurou como um lugar apropriado para vasculhar as certezas e incertezas do que é a fotografia. As reflexões teóricas foram instituídas pelo trabalho plástico. / This dissertation aims at a photographic images creation process from chemistry manipulations made over light sensitive surfaces, directly on the paper or negative, without the camera. From this point of view the dissertation analyses the limits of which we know as photograph. The research converged to an investigation of the inherent photographic possibilities to sensitive and light sensitive surfaces, to photochemical solutions and to an approaching of these photographic possibilities to other kinds of artistic languages. The creation process faced photograph and its unique features and, by dealing with them, tried to reveal the photographs unique characteristics, which showed a specific place to search the certainties and uncertainties of what photography is. The theoretic reflections were established by the artistic work.

La transcriptase inverse (RT) est un hétéro-dimère p66/p51 avec des activités ADN polymérase et ribonucléase H qui jouent un rôle critique dans le cycle viral du VIH-1. La RT convertit l’ARN génomique viral simple brin en ADN proviral double brin dans le cytoplasme de la cellule infectée. L’efficacité de la RT est augmentée par la protéine de la nucléocapside (NC) grâce à son activité chaperonne vis-à-vis des acides nucléiques et/ou une coopération entre les deux protéines. Dans ce travail, nous avons étudié par la technique de smFRET (single molecule Fluorescence Resonance Energy Transfer) les effets de la NC sur l’interaction entre la RT et un substrat d’ADN au niveau de deux sites de pause de la synthèse de l’ADN(+). Nous avons d’abord réalisé et validé le montage de smFRET. Lors de la validation du montage avec des fluorophores Cy3 encapsulés dans des vésicules lipidiques, nous avons mis en évidence deux mécanismes différents entrainant le photoblanchiment du Cy3. Ensuite, après avoir déterminé les propriétés de liaison à l’équilibre de la RT et la NC sur différents substrats amorce/matrice à l’aide de mesures d’ensemble en solution, nous avons confirmé par smFRET que la RT adopte plusieurs conformations sur son substrat d’ADN, incluant celle qui conduit à la polymérisation de l’ADN. En présence de la NC, nous n’avons observé qu’une réorganisation modérée des différentes conformations du complexe RT/substrat. Par contre, une réorganisation beaucoup plus importante est induite par la NC en présence du dNTP, avec une très forte exaltation des conformations compétentes pour la polymérisation. Nous avons également montré que la NC augmente l’efficacité de synthèse de l’ADN au niveau de sites de pause en diminuant ou en augmentant le temps de dissociation du complexe RT/substrat/dNTP selon le type de site de pause. L’ensemble de ces données permet de mieux comprendre les mécanismes polyvalents par lesquels NC facilite l’activité de la RT. / The reverse transcriptase (RT) is a p66/p51 hetero-dimer with DNA polymerase and ribonuclease H activities which plays a critical role in the viral cycle of HIV-1. RT converts the viral genomic RNA to proviral DNA in the cytoplasm of infected cells. The efficiency of RT is increased by the nucleocapsid protein (NC) through its nucleic acids chaperone properties and/or via direct interaction with RT. In the present work, we investigated the effects of NC on the interaction between RT and its DNA substrate attwo pause sites during the synthesis of (+)DNA by using the smFRET (single molecule Fluorescence Resonance Energy Transfer) technique. In a first step, we implemented and validated the smFRET set-up. Within the validation step, using Cy3 fluorophores encapsulated, in lipid vesicles, we monitored the photobleaching of Cy3 dyes and found out that it was governed by two parallel mechanisms. In a second step, we determined the affinity of RT and NC to different primer/template substrates by using steady-state fluorescence. Then, we confirmed by smFRET that RT adopts different conformations on its DNA substrate, including the one that leads to DNA polymerization. In the presence of NC, we observed only a moderate reorganization of the different conformations of RT/substrate complex. However, NC was found to induce a more important reorganization in the presence of dNTP, with a very strong promotion of the polymerization-competent conformations. We also showed that NC increases the efficiency of DNA synthesis at pause sites by either decreasing or increasing the dissociation time of the RT/substrate/dNTP complex, depending on the type of pause site. Together, these data allow us to further elucidate the mechanisms by which NC facilitates the RT.

O presente trabalho tem por objetivo investigar o pensamento de Antonio Gramsci (1891- 1937) acerca da educação, com ênfase em suas elaborações teóricas e conceitos políticos relacionados a sua perspectiva pedagógica. Tendo por propósito a identificação do discurso gramsciano sobre educação e sua atualidade teórica, esta pesquisa analisará, principalmente, os escritos feitos no cárcere (Quaderni del carcere), em que o teórico teria se debruçado sobre o temário educacional e formativo. Procurando mapear a perspectiva pedagógica de Gramsci, esta pesquisa procurou efetuar também uma revisão bibliográfica da literatura já produzida sobre o tema. Gramsci ansiava pela constituição de um plano de ação capaz de alterar a estrutura político-econômica da sociedade. Nesse sentido, a dimensão cultural e educativa ocupa uma posição destacada na sua reflexão sobre a transformação social. Em sua acepção, uma reforma intelectual e moral seria imprescindível para a edificação da hegemonia popular, o que justifica a importância que a educação assume em suas reflexões e em seu trabalho teórico. Todos os conceitos e formulações que Gramsci desenvolve ao longo de sua vida apresentam-se correlacionados com tal perspectiva de elaboração de estratégias de atuação em prol da modificação da ordem vigente. Por essa razão, foi necessário examinar também conceitos e escritos de Gramsci acerca do debate político, especialmente sobre os temas seguintes: Estado, sociedade civil, sociedade política, bloco histórico, partido, estrutura, superestrutura, Ocidente, Oriente, guerra de posição, guerra de movimento, hegemonia, ideologia e filosofia. Acreditamos ser primordial compreender o encadeamento entre a esfera política e essa proposição de reforma intelectual e moral; sendo que para Gramsci - a transformação social passaria necessariamente pela questão cultural. Esta, por sua vez, teria uma inegável dimensão educativa. A educação e a cultura são, na ótica gramsciana, as peças fundamentais para a constituição de um novo homem: autônomo, consciente, crítico, capaz de criar novas relações e as demais condições necessárias para a superação da estratificação social em prol da edificação de uma outra ordem econômica e política, não mais pautada pela desigualdade social. Para isso, o alcance da hegemonia seria o primeiro passo, só atingido quando a concepção de mundo compatível com tal plano de transformação fosse majoritária na sociedade. Eis nos escritos gramscianos - o grande papel ocupado pela figura do intelectual - ator público disseminador de ideologias - e da educação no processo de luta política pela emancipação do homem. Esse processo requereria, contudo, uma ampla formação humanista, integral, crítica, criativa, capaz de agregar trabalho intelectual e trabalho manual, sendo que sua maior expressão estaria no ideal da escola única. / This study aims at investigating the thought of Antonio Gramsci (1891-1937) about education, with emphasis on his theoretical elaborations and political concepts related to his educational perspective. Having as a goal the identification of the Gramscian discourse on education and its theoretic relevance, this research will examine mainly the writings made in prison (Quaderni del carcere), in which the theorist developed educational and formative themes. Seeking to map the pedagogical perspective of Gramsci, this research has also aimed at carrying out a bibliographic review of the literature produced on the subject. Gramsci longed for the establishment of a plan of action capable of altering the political-economic structure of society. In this sense, the cultural and educational dimension has a prominent position in his discussions on social transformation. In his meaning, an \"intellectual and moral reform\" would be essential for the construction of popular hegemony, which explains the importance given to education in his thoughts and his theoretical work. All concepts and formulations developed by Gramsci throughout his life are correlated with such perspective of developing strategies of action focusing on modifying the existing order. For this reason, it was also necessary to examine Gramscis concepts and writings about the political debate, especially on the following topics: State, civil society, political society, historical bloc, party, structure, superstructure, West, East, war of position, motion war, hegemony, ideology and philosophy. We believe it is essential to understand the linkage between politics and this proposition of \"intellectual and moral reform\", remembering that - for Gramsci - the social transformation would necessarily involve the cultural issue. This, in turn, would have an undeniable educational dimension. Education and culture are, in Gramscis perspective, the key pieces to form a new man: autonomous, conscious, critical, able to create new relationships and other necessary conditions for overcoming the social stratification in favor of building another economic and political order, no longer dominated by social inequality. To do so, the scope of hegemony would be the first step, only achieved when the world conception compatible with such a transformation is predominant in society. It is stated in the writings of Gramsci the great role played by the intellectual figure - public actor in charge of disseminating ideologies - and education in the process of political struggle for the emancipation of man. This process would demand, however, a broad humanistic, integral, critical, creative education, capable of putting together intellectual and manual labor, having as its greatest expression the ideal of unique school.

Objetivo: caracterizar a dinâmica da produção física e financeira de internações hospitalares de um hospital de ensino, do interior do Estado de São Paulo, por meio do Sistema de Informações Hospitalares do Sistema Único de Saúde, segundo especialidade e ano. Metodologia: pesquisa avaliativa, utilizando dados quantitativos e qualitativos. Para a coleta de dados quantitativos foi realizada pesquisa documental em banco de dados oficiais, do total de internações hospitalares nas especialidades de clínica médica, clínica cirúrgica, obstetrícia e pediatria, no período de 1996 a 2011, utilizando a estatística descritiva. Para os dados qualitativos foram realizadas entrevistas com membros da Administração Superior do hospital. Os dados foram agrupados por similaridade de conteúdo e articulados aos dados quantitativos. Resultados: a população de estudo constituiu- se de 504.529 internações; apresentam-se os dados dos anos correspondentes ao menor e ao maior valor das internações hospitalares, no que diz respeito a aspectos físicos e financeiros no período. O percentual de internações seguiu certa linearidade, variando de 5,52 a 7,09%, no ano de menor e o de maior produção de internações, respectivamente. O montante financeiro teve expressivo aumento, sendo que, em 1996, correspondia a 2,83% dos recursos despendidos e, em 2011, esse valor representou 11,58% dos gastos com internações. Analisando as especialidades, a clínica cirúrgica, em 1997, correspondia a 4,92% das internações e, em 2009, houve aumento para 7,49%; quanto aos recursos financeiros, em 1996, o percentual de internações pagas era de 2,73%; em 2010, esse número era de 10,90%. Em clínica médica, as internações, em 2007, representavam 5,25%, em 2011, o percentual era de 8,11%; quanto aos valores pagos, houve aumento entre 1997 e 2011, de 2,75% para 14,03%, respectivamente. Em pediatria, em 1998, as internações representavam 8,69% e, em 2008, esse número caiu para 4,64%; quanto ao montante financeiro, em 1997, as internações pagas representavam 2,76% e, em 2010, aumentaram para 12,88%. Em obstetrícia, as internações, em 1996, correspondiam a 9,33% e, em 2010, eram de 4,58%; em relação à produção financeira, em 1997 houve um percentual de 4,11% e, em 2009, aumentou para 9,73%. Das entrevistas, emergiram aspectos relevantes que podem explicar as variações físicas e financeiras das internações hospitalares, a saber: incorporação tecnológica (a inclusão de recursos tecnológicos implica em maiores investimentos financeiros, justificando os gastos com internações hospitalares); organização da rede de atenção à saúde (a ordenação dos serviços de saúde, com ênfase na atenção básica pode justificar a diminuição das internações em pediatria e obstetrícia, cujos atendimentos podem ser realizados em serviços de menor densidade tecnológica, assim como o crescimento de internações que requerem maior densidade tecnológica) e perfil demográfico (o envelhecimento progressivo da população, seguido das comorbidades relacionadas, também têm influência sobre o perfil de internações em clínica cirúrgica e clínica médica). Conclusões: o acompanhamento das internações possibilita ao gestor estruturar a atenção à saúde e o financiamento do hospital, constituindo-se em importante ferramenta gerencial. A organização das redes de atenção, o perfil etário da população e a incorporação de tecnologias repercutem na atenção hospitalar, particularmente nas internações / Objective: characterize the dynamics of financial and physical production of hospitalizations at a school hospital in a city of São Paulo State through the Hospital Information System of the Unique Health System, according to the speciality and year. Methodology: evaluative research using quantitative and qualitative data. To collect quantitative data, it was used the documental research in official database, the total of clinical, surgical, obstetrics and pediatrics hospitalizations, during 1996 to 2011, using descriptive statistics. To the qualitative data, members of the Upper Management of the hospital were interviewed. The data were grouped by its similarities and articulated to the quantitative data. Results: the population under study were 504.529 hospitalizations, presenting data from the relative years, as of the lowest and to the highest number of hospitalizations, about physics and financial during that period of time. The hospitalizations percentage followed such linearity, ranging between 5,52% to 7,09% during the years of lowest and highest number of hospitalizations, respectively. The financial total had an important raise, and in 1996 it corresponded to 2,83% of the spent means and, in 2011 this number corresponded to 11,58% of the hospitalizations spent. Analysing the specialities, the surgical clinic in 1997 corresponded to 4,92% of hospitalizations and, in 2009 there was a raise to 7,49%, regarding the financial means, in 1996 the hospitalizations percentual spent were 2,73%, in 2010 these number were 10,90%. In internal medicine, in 2007 the hospitalizations represented 5,25%, in 2011 the percentual was 8,11%, regarding the spent values, there was a percentual raise between 1997 and 2011, as of 2,75% to 14,03%, respectively. In 1998, pediatrics hospitalizations represented 8,69% and in 2008, this number dropped to 4,64%, regarding the financial amount, in 1997, paid admissions represented 2,76% and, in 2010, increased to 12,88%. In obstetrics, during 1996 the hospital admissions corresponded to 9,33% and, in 2010, they were 4,58%, regarding the financial production, in 1997 there was a 4,11% percentual and, in 2009, it increased to 9,73%. During the interviews, relevants aspects emerged that may explain the physical and financial hospitalization variations, namely: technological incorporation (adding the technologics means implies in biggest financial investiments, justifying the hospitalizations outlay); organization of the health care network (the health services assortment, with emphasis on basic attention may justify the pediatrics and obstetrics admission dicrease, whose treatment may be performed in smaller technological services, as well as the increase on admissions that request superior technological density) and demographic profile (the progressive population aging followed by the related comorbidities, also influences on the surgical and clinical admissions profiles). Conclusions: following the admissions enables the manager to organize the health attetion and the hospital financial constituting an important management tool. The attetion network organization, the population age profile and the technology incorporation affects the hospitalar attention, specially on admissions

Esta dissertação foca um processo de criação de imagens fotográficas a partir de manipulações químicas feitas sobre superfícies fotossensíveis, diretamente sobre o papel ou negativo, sem utilizar para isso o aparato tecnológico – a câmera. A partir dessa abordagem, a dissertação analisa os limites daquilo que conhecemos como fotografia. A pesquisa convergiu para a investigação das possibilidades fotográficas inerentes às superfícies sensíveis e fotossensíveis, às soluções fotoquímicas e para o cruzamento dessas possibilidades fotográficas com outras linguagens artísticas. O trabalho confrontou a fotografia com suas especificidades e buscou desvendar os domínios da fotograficidade, que se configurou como um lugar apropriado para vasculhar as certezas e incertezas do que é a fotografia. As reflexões teóricas foram instituídas pelo trabalho plástico. / This dissertation aims at a photographic images creation process from chemistry manipulations made over light sensitive surfaces, directly on the paper or negative, without the camera. From this point of view the dissertation analyses the limits of which we know as photograph. The research converged to an investigation of the inherent photographic possibilities to sensitive and light sensitive surfaces, to photochemical solutions and to an approaching of these photographic possibilities to other kinds of artistic languages. The creation process faced photograph and its unique features and, by dealing with them, tried to reveal the photographs unique characteristics, which showed a specific place to search the certainties and uncertainties of what photography is. The theoretic reflections were established by the artistic work.

O Brasil está passando por uma transformação demográfica, com o grupo etário dos idosos crescendo rapidamente no país, e a demanda deste grupo por recursos de saúde é intensa, tanto no que se refere à utilização de serviços de saúde quanto no que diz respeito ao uso de medicamentos. Os medicamentos em idosos se comportam de maneira diferente devido às alterações na farmacocinética e na farmacodinâmica, portanto as reações adversas dos medicamentos (RAM) nesses pacientes podem ocorrer de maneira mais proeminente. A discussão sobre a qualidade da farmacoterapia nesses indivíduos é um tema importante relacionado com a atenção, tendo em vista que o medicamento é considerado um instrumento de recuperação e manutenção da saúde dos indivíduos. Diante disso este projeto de pesquisa tem por objetivo estudar a utilização de medicamentos por usuários idosos do SUS. Para coleta de dados utilizou-se um formulário, previamente padronizado e aprovado pelo Comitê de Ética em Pesquisa do Centro de Saúde Escola da Faculdade de Medicina de Ribeirão Preto, Universidade de São Paulo (CEP-CSE-FMRP-USP). Com este instrumento entrevistou-se 1000 idosos no período de novembro de 2008 a maio de 2009, os dados relativos a esses pacientes foram lançados no programa estatístico, Epi Info® versão 3.4.3. A média de idade foi de 69,8 anos, sendo 66,1% do sexo feminino, com renda per capita média de R$ 581,00, com predomínio de brancos (56,2%), casados (51%), com ensino fundamental incompleto (65%). A morbidade de maior prevalência entre os entrevistados foi a Hipertensão Arterial Sistêmica (72,8%); em se tratando dos cuidados com a saúde 58,8% são sedentários, 93,2% ingerem bebida cafeinada, 18% utilizam bebida alcoólica, 8,5% são tabagistas. Considerando o acesso aos medicamentos e serviços de saúde 46,8% dos idosos retiram todos os medicamentos na farmácia dos SUS; 15,8% possuem planos de saúde privado; a média de consultas agendadas pelo SUS foi de quatro consultas/paciente/ano; 16,3% não recebem orientação sobre o uso correto dos medicamentos de nenhum profissional de saúde; 12,6% recebem orientações do farmacêutico no momento da dispensação dos medicamentos. Com relação ao perfil farmacoepidemiológico encontrou-se um intervalo de um a vinte e um fármacos utilizados por paciente, sendo a média de sete fármacos/paciente; a maior prevalência foram dos medicamentos do aparelho cardiovascular (83,4%); 30,9% realizam automedicação. A polifarmácia (uso de seis ou mais princípios ativos) esteve presente em 60,1% dos idosos, sendo que 74% são mulheres; 80,2% utilizam MIPs (medicamentos isentos de prescrição); 46,2% relataram pelo menos uma RAM; 36% utiliza medicamentos controlados pela portaria 344/98; 44,2% utilizam medicamentos considerados inapropriados ao idoso; e encontrou-se 282 interações medicamentosas. As variáveis com maior correlação com o uso de mais seis princípios ativos são: sexo feminino, uso de medicamentos considerados inapropriados aos indivíduos idosos, automedicação, quantidade maior de problemas de saúde, número de consultas médicas agendadas, presença de RAM, uso de MIPs, falta de exercício físico, uso de adoçante e uso de medicamentos controlados pela portaria 344/98. Diante de tais evidências verifica-se a necessidade de se adotar estratégias para melhoria da farmacoterapia e a assistência prestada à saúde do paciente idoso. / Brazil is undergoing a demographic transformation which means elderly group is growing rapidly in the country and the demand of health resources for them is intense, referring to the use of health services and to the use of drugs. The drugs in elderly behave differently due to changes in pharmacokinetics and pharmacodynamics, thus adverse drugs reactions (ADR) in these patients may occur more prominent. The discussion about the quality of pharmacotherapy in these individuals is an important issue related to attention, as the drug is considered an instrument of rehabilitation and maintenance of peoples health. Therefore, this research has the purpose to study the use of drugs by elderly users of SUS. To collect the data, a form standardized and approved by Ethics Committee in Research of Health School Center of the College of Medicine of Ribeirão Preto, University of São Paulo (CEP-CSE-FMRP-USP) was used. With this instrument, 1000 elderly was interviewed in the period of November 2008 to May 2009, the data of these patients were inserted in statistic program, Epi Info ® version 3.4.3.The average age was 69.8 years, 66.1% were female, the average income per capita was R$ 581.00, with predominance of whites (56.2%), married (51%), with elementary school incomplete (65%). The morbidity most prevalent among the elderly was the Hypertension (72.8%), in the case of health care, 58.8% are sedentary, 93.2% ingest caffeinated drink, 18% use alcohol, 8,5% are smokers. Considering the access to medicines and health services, 46.8% of elderly took out all drugs at the pharmacy of SUS; 15.8% have private health insurance; and the average medical visits scheduled by SUS was four visits/patient/year; 16.3% did not receive guidance about correct form of use of drugs from any health professional, 12.6% receive guidance from pharmacist when the drug was dispensed. Regarding the Pharmacoepidemiological profile, a range of one to twenty-one drugs used was found per patient, with an average of seven drugs/patient, the greater prevalence was cardiovascular drugs (83.4%) 30.9% perform self-medication. Polypharmacy (use of six or more drugs) was present in 60.1% of the elderly, and 74% are women, 80.2% use OTC\'s (over-the- counter), 46.2% reported at least one ADR, 36% use controlled drugs by decree 344/98, 44.2% used drugs considered inappropriate for the elderly, and we found 282 drug interactions. The variables most strongly correlated with the use of six drugs are: female gender, use of inappropriate drugs by elderly, self-medication, increased amount of health problems, number of medical visits scheduled, the presence of ADR, use of OTC\'s , lack of physical exercise, use of artificial sweetener, and use of controlled drugs by decree 344/98. Facing these evidences, it is perceptible the need to adopt strategies to improve pharmacotherapy and health assistance offered to the elderly.

La relation entre le repliement du génome et les processus cellulaires majeurs, notamment la transcription, la réparation de l’ADN et la réplication est une question biologique centrale. De nouvelles méthodes de la génomique ont révélé un niveau inconnu de l’architecture tridimensionnelle de la chromatine. A l’échelle en dessous de la mégabase, certaines séquences génomiques se trouvent préférentiellement à proximité les unes des autres formant ainsi des domaines topologiques associés (TAD). Les gènes situés dans le même TAD ont des propriétés épigénétiques similaires, et leur expression au cours de la différentiation semble corrélée, ce qui suggère un lien fort entre la structure de la chromatine et la transcription. Les TADs sont à leur tour séparés par des régions avec peu de contacts, appelées frontières, qui sont généralement occupées par des protéines dites isolatrices. Les déterminants de cette organisation chromatinienne particulière et ses implications fonctionnelles sont largement méconnus. Selon une hypothèse récente, les TADs seraient formés par des contacts entre les séquences des frontières, stabilisés par la formation de boucles via les protéines isolatrices. Les travaux présentés ici ont pour but d’étudier le rôle des protéines isolatrices dans le mécanisme de formation des TADs chez la drosophile. La microscopie super-résolue a été implémentée et une série de développements ont été réalisés en microscopie à illumination structurée (SIM) et la microscopie à localisation de molécules uniques (SMLM), avec une attention particulière sur le marquage fluorescent. Ces développements ont directement été appliqués à l’étude de l’organisation nucléaire de la protéine associée aux éléments frontières (BEAF), une des 11 protéines isolatrices identifiées à ce jour chez la drosophile. Le fort enrichissement aux frontières des TADs, ainsi que son activité dans la formation de boucles d’ADN font de BEAF un candidat intéressant pour tester l’hypothèse de regroupement de frontières comme mécanisme général de repliement de la chromatine. La technique SMLM multi-couleurs a systématiquement localisé BEAF à la périphérie des larges domaines portant la marque épigénétique H3K27me3. L’analyse quantitative des images SMLM a révélé que BEAF forme des centaines de foyers d’une taille de 45 nm, composés en moyenne de 5 molécules, ce qui est en désaccord avec la présence de boucles de chromatine à large échelle. Afin de tester le regroupement de gènes directement au niveau de l’ADN, des frontières ont été marquées par des oligonucléotides fluorescents. Le nombre de foyers détectés par SIM s’est à nouveau révélé incompatible avec le modèle de contacts entre les frontières tout le long du génome. Par ailleurs, les distances entre paires de frontières au niveau de deux régions génomiques ont montré <5% de contacts. Ensemble, ces résultats sont en désaccord avec l’établissement d’interactions entre barrières chez la drosophile. Enfin, ces travaux de thèse ont contribué au développement méthodologique de la microscopie super-résolue, ce qui a permis d’apporter des preuves expérimentales invalidant le modèle de regroupement des frontières comme mécanisme général du repliement chromatinien. / The interplay between genome folding and key cellular functions such as transcription, DNA repair and replication is a fundamental question in chromatin biology. Recent genome-wide developments unveiled a new level of three-dimensional chromatin architecture. At the sub-megabase scale, some genome sequences are preferentially found in proximity with one another forming Topologically Associating Domains (TADs). Genes located within the same TAD display common epigenetic properties and tend to have coordinated dynamics of expression during differentiation, suggesting a strong link between chromatin structure and transcription. TADs are in turn separated by regions of low contact, termed TAD borders, which are generally occupied by factors called chromatin insulators. What are the determinants of this particular type of chromatin organization and what are the functional implications is still largely unknown. In an emerging hypothesis, TADs could be formed through contacts between TAD border sequences stabilized by the looping activity of insulator proteins. This thesis investigates the roles of insulator proteins in the TAD formation mechanism using Drosophila melanogaster as model system. Superresolution imaging was implemented and a series of developments were performed in Structured Illumination Microscopy (SIM) and Single-molecule Localization Microscopy (SMLM), with particular attention on fluorescent labeling for single-molecule detection. These developments were directly applied to study the nuclear organization of the Boundary Element Associated Factor (BEAF), one of the 11 insulator proteins discovered to date in Drosophila. The strong enrichment on TAD borders and its demonstrated looping activity make BEAF a potent candidate to test for the clustering of TAD borders as a general mechanism of chromatin folding. Multicolor SMLM systematically located BEAF foci at the periphery of large H3K27me3 chromatin domains. Quantitative analysis of SMLM images indicated BEAF forms hundreds of 45 nm foci, containing a mean of 5 molecules, which argues against a large-scale looping of BEAF-bound chromatin. To directly probe for gene clustering at the DNA level, TAD borders were labeled using fluorescent oligonucleotide probes. The number of foci detected by SIM was once more incompatible with a model of chromosome-wide contacting of multiple TAD borders. Furthermore, TAD border pairs distances were measured in two genomic regions, resulting in <5% of paired contacts among the measured barriers. Taken together, these results are inconsistent with constitutive interactions between consecutive or non-consecutive barriers in Drosophila.In conclusion, this study contributed to the methodological development of super-resolution microscopy which was applied to provide experimental evidence invalidating the TAD border clustering model as a general mechanism of chromatin folding.

Lean Startup lacks a method specifically designed to evaluate whether a HCI-design is fulfilling the promises delivered by the unique value proposition (UVP). At the time of writing, concurrent think aloud protocols during usability testing and interviews are used to that end. There are, however, a couple of issues bundled with the use of such methods. Using a case study conducted on two iterations of high-fidelity prototypes of a meal planning website currently in development, a method called Directed Product Reaction (DPR) is proposed. DPR is a result of the adaptation of Product Reaction Cards, and the addressing of issues uncovered during case study iterations. DPR aspires to be that specifically designed method needed for assessment of user experience in relation to UVP in Lean Startup, and while the resulting method proposed has far from achieved that goal, it serves as a foundation moving forward.

Au sein des synapses comme dans les régions extra synaptiques, la diffusion latérale joue un rôle critique dans la densité membranaire des récepteurs. En face des zones actives, l’accumulation de récepteurs détermine en particulier l’efficacité de la transmission synaptique. Il est important de comprendre les paramètres cellulaires qui jouent sur l’accès au compartiment synaptique, qu’ils soient d’origine moléculaires ou morphologiques. Dans les synapses excitatrices, la tige de l’épine dendritique se comporte comme une barrière à la diffusion. Cette barrière pourrait être fonction de la longueur et du diamètre de la tige (paramètre géométrique), ou résider dans la présence d’éléments spécifiques constituant des obstacles à la diffusion. Une sous-population d’épines contient dans sa tige une forme spécialisée de réticulum endoplasmique, appelé appareil épineux et constituée d’un empilement des accules de réticulum. Une protéine liant l’actine, nommée synaptopodine, est associée de façon étroite à l’appareil épineux et participe aux mécanismes de plasticité synaptique. La question centrale de ce travail de thèse était de définir si la présence de synaptopodine influait sur les caractéristiques de la diffusion dans la tige de l’épine, et d’identifier les mécanismes sous-jacents. Afin d’étudier la diffusion membranaire, j’ai utilisé trois protéines recombinantes différentes: une protéine associée au feuillet extérieur de la membrane plasmique (GFP-GPI), une protéine avec un domaine transmembranaire et une courte séquence intracellulaire (TMD-pHluorin), et la sous-unitéGluR5 du récepteur métabotropique (mGluR5) contenant 7 domaines transmembranaires et une séquence intracellulaire volumineuse. Les trois constructions portent une étiquette (GFP ou pHluorin) du côté extracellulaire. Les propriétés diffusives de ces molécules ont été mesurées par un suivi de particules uniques, à base de quantum dots. Ces expériences ont révélé que la diffusion des protéines membranaires est fonction du diamètre de la structure cylindrique considérée, et par conséquent moins rapide dans la tige de l’épine que dans le tronc du dendrite. Mais les propritétés diffusives dépendent aussi de la taille et delà complexité des molécules membranaires considérées. En effet, la diffusion de molécules comportant des domaines transmembranaires est particulièrement faible dans les tiges contenant de la synaptopodine. Cet aspect a été approfondi par l’utilisation de traitements pharmacologiques, qui ont permis de modifier la structure interne de la tige dendritique. Les variations des tailles des domainesoccupés par l’actine-F, et par lesaggrégats de synaptopodine, ont été observées à l’échelle nanoscopique en utilisant l’imagerie PALM/STORM. En conditions contrôle, la synaptopodine occupe la partie centrale de la tige. La dépolymérisation indirecte de l’actine-F par le 4-Aminopyridineentraîne une diminution des zones occupées par ces deux composants, corrélée à une augmentation de la vitesse de diffusion de mGluR5. En revanche, la dépolymérisation par la latrunculin-A (effet direct sur l’actine) induit une augmentation de la taille des clusters de synaptopodine et donc de la surface occupée par ceux-ci dans la tige. Les mesures de la diffusion de la sous-unité mGluR5 réalisées dans ces conditions montrent une accélération de la vitesse de diffusion, indiquant que la mobilité de mGluR5 n’est pas régulée par une interaction directe avec la synaptopodine. En conclusion, je propose un rôle de stabilisation mutuel pourl’actine-F et la synaptopodinedans la tige des épines dendritiques de neurones d’hippocampe en culture. Les épines contenant de la synaptopodine dans leur tige auraient une organisation unique du cytosquelette qui agirait comme une barrière additionnelle pour la diffusion de récepteurs aux neurotransmetteurs. / Lateral diffusion in and outside synapses plays a key role in the accumulation of receptors at synapses, which critically determines the efficacy of synaptic neurotransmission. Therefore, to better understand the trapping of neurotransmitter receptors in synapses, it is important to investigate the mechanisms that may affect receptors diffusion and their capacity to reach synapses. The neck of dendritic spine imposes a diffusional barrier that is considered to depend on the length and diameter of the spine neck. The origin of this barrier could be purely geometrical or could be induced by the presence of specific barriers/obstacles for diffusion. A subpopulation of spines contains a specialized form of endoplasmic reticulum in the spine neck called spine apparatus. The actin-binding protein synaptopodin (SP) is tightly associated with the spine apparatus and participates in synaptic plasticity mechanisms. The central question of my research was to assess whether the presence of the SP affects the diffusion of receptors in the spine neck and to characterize the underlying molecular mechanisms. To study membrane diffusion, I have developed three different probes: a construct associated with the outer leaflet of the plasma membrane (GFP-GPI), a construct with one transmembrane domain and a short intracellular sequence (TMD-pHluorin), and a recombinant metabotropic mGluR5 receptor construct containing an extracellular domain tagged with pHluorin, seven transmembrane domains, as well as a large intracellular region. The diffusion properties of these molecules were measured by single particle tracking using quantum dots. My experiments revealed that the diffusion of membrane proteins was slower in the spine neck than in the dendrite as a result of the different diameter of the two compartments. Furthermore, the diffusion properties depended on the molecular size and complexity of the membrane proteins. Interestingly, the diffusion of membrane proteins with transmembrane domains was particular slow in spine necks containing SP. This could be the result of direct molecular interactions between the membrane proteins and SP or due to spatial constraints that are related to the structural organization of spine necks expressing SP. To address these questions further I used pharmacological treatments to change the internal organization of the spine neck, and measured their effect on the diffusion properties of mGluR5. The distribution of SP and F-actin in the spine neck was determined on the nanoscopic scale using PALM/STORM imaging. This showed that under control condition SP occupies only the central region of the spine neck. Activity-dependent depolymerization of F-actin by 4-Aminopyridine led to a simultaneous decrease of the amount of F-actin and SP and enhanced the diffusion of mGluR5 in all analyzed neck regions. Disruption of F-actin by latrunculin A induced the re-distribution of SP and the formation of larger SP clusters, occupying an increased region within the spine neck. The recruitment of SP was accompanied by an acceleration of mGluR5 diffusion in SP-positive spines, demonstrating that the mobility of mGluR5 is not controlled by direct interactions with SP. Instead, the diffusion of mGluR5 is dependent on the organization of the spine cytoskeleton. In conclusion, I propose that SP and the polymerization of actin filaments have a reciprocal effect on the stability of each other in the spine neck of cultured hippocampal neurons. Spine necks bearing SP have a unique F-actin cytoskeletal organization that acts as an additional diffusion barrier for neurotransmitter receptors such as mGluR5.