Este trabalho apresenta os detalhes da construção de um espectrômetro para a realização de experimentos de transferência de polarização elétron-núcleo (Efeito Overhauser). São também mostrados: as implementações e modificações feitas no espectrômetro de RPE existente no Laboratório de Ressonância Magnética do DFCM, os circuitos para a construção de um equipamento de RMN para operar de forma pulsado na freqüência fixa de 14 MHz, os desenhos da cavidade de RPE construída para a banda-X (~ 9,2 GHz), os \"softwares\" modificados e desenvolvidos para aquisição de dados, tratamento e reconstrução de imagens. São apresentados os resultados do aumento do sinal de RMN e as imagens obtidas através da Tomografia de Ressonância Magnética, usando amostras menores do que 1 mm de diâmetro (volume ~10 ul), a uma concentração de 2,2 mM de TEMPOL dissolvido em água destilada. / This work describes in details the arrangements that must be accomplished for development of a spectrometer for Dynamic Nuclear Polarization - DNP (Overhauser Effect). Also, the construction project of a 14 MHz pulsed NMR spectrometer and drawings of a homemade EPR cavity for X-band (~ 9,2 GHz) are shown. The DNP probe built for the experiments and modifications done on the EPR spectrometer existing at Laboratory of Magnetic Resonance are discussed in detail. Results on the enhancement of the NMR signal due electron-proton dynamic interactions are presented. NMR imaging of very small objects, 1 mm diameter glass tube filled with 5 ~10 ul of 2,2 mM of free radicals (TEMPOL) solution, obtained through back projection reconstruction NMR tomography method, are presented

La maitrise de l'information représente un avantage concurrentiel de nos jours. Malgré une intensification des moyens développés pour protéger les flux de données, il n'est actuellement pas possible d'échanger à distance et de façon complètement sure, une information entre deux interlocuteurs. Néanmoins, des travaux menés par Bennett et Brassard ont montré qu'il est possible d'atteindre un niveau de sécurité maximum en utilisant un protocole quantique de transmission de l'information. Ce protocole se base sur l'utilisation de réseaux télécom utilisant des répéteurs quantiques à la place des répéteurs classiques. La voie étudiée dans cette thèse, réalisée en partie dans le cadre du projet européen QuRep, a pour but l'amélioration des connaissances sur les monocristaux dopés aux ions de terre rare qui sont des candidats de choix pour la mise au point de répéteurs quantiques. Deux grands axes ont émergés : dans un premier temps nous avons essayé de comprendre quels sont les facteurs de succès et limitatifs dans l'utilisation du cristal de Nd : YSO en tant qu'hôte pour les mémoires quantiques avec pour objectif le transfert de la cohérence électronique vers des niveaux hyperfins. Dans un second temps, nous avons étudié un cristal présentant une structure hyperfine directement accessible en optique, Er : YLF afin de vérifier sa potentielle utilisation pour les mémoires quantiques. Ces travaux ont permis, entre autre, de réaliser un transfert de cohérence d'un niveau Zeeman électronique vers un niveau hyperfin avec un temps de stockage de plus de 300 µs, ce qui permet d'envisager une mémoire quantique dans Nd : YSO permettant de réémettre un photon à la demande.

L'utilisation de nanoparticules semi-conductrices colloïdales, appelées quantum dots, est nouvelle pour des applications en photocatalyse redox. Ces nano-objets possèdent une forte absorption dans le visible modulable avec leur taille. De plus, la présence d'une couche de ligands peut rendre ces nanostructures éco-compatibles. Dans ce manuscrit de thèse, une étude de la photocatalyse redox avec des nanoparticules cœur CdSe et cœur-coquille CdSe/ZnS a été réalisée dans le visible en solvant aqueux. Les quantum-dots hydrosolubles sont tout d'abord fonctionnalisés par des ligands de type acide aminé. Trois systèmes photochimiques ont ensuite été testés avec les substrats suivants : dérivés d'acides aminés, vanilline et 8oxodG. L'activité photochimique des nanoparticules semi-conductrices a pu être démontrée et évaluée par la détection de produits de réaction. Le mécanisme de transfert de charge photoinduit a par ailleurs été élucidé par des techniques spectroscopiques durant l'irradiation. / The use of colloidal semiconductor nanoparticles, called quantum dots, is recent in redox photocatalysis. Those nanostructures exhibit strong and size depending visible absorption properties. The ligand shell on the nanoparticle surface can be furthermore managed to realize green applications. Thus the study of redox photocatalysis with core CdSe nanoparticles and core-shell CdSe/ZnS was carried out with visible light in aqueous solvent. Firstly, we synthetized hydrophilic quantum-dots stabilized by amino acids ligands. Secondly, we perform three different studies of photocatalytic systems with the following substrates : modified amino acids, vanillin and 8oxodG. The nanoparticle's photochemical activity was first demonstrated and evaluated by the detection of photochemical products. The photoinduced charge transfer mechanism was elucidated during irradiation by spectroscopic techniques.

Este trabalho apresenta os detalhes da construção de um espectrômetro para a realização de experimentos de transferência de polarização elétron-núcleo (Efeito Overhauser). São também mostrados: as implementações e modificações feitas no espectrômetro de RPE existente no Laboratório de Ressonância Magnética do DFCM, os circuitos para a construção de um equipamento de RMN para operar de forma pulsado na freqüência fixa de 14 MHz, os desenhos da cavidade de RPE construída para a banda-X (~ 9,2 GHz), os \"softwares\" modificados e desenvolvidos para aquisição de dados, tratamento e reconstrução de imagens. São apresentados os resultados do aumento do sinal de RMN e as imagens obtidas através da Tomografia de Ressonância Magnética, usando amostras menores do que 1 mm de diâmetro (volume ~10 ul), a uma concentração de 2,2 mM de TEMPOL dissolvido em água destilada. / This work describes in details the arrangements that must be accomplished for development of a spectrometer for Dynamic Nuclear Polarization - DNP (Overhauser Effect). Also, the construction project of a 14 MHz pulsed NMR spectrometer and drawings of a homemade EPR cavity for X-band (~ 9,2 GHz) are shown. The DNP probe built for the experiments and modifications done on the EPR spectrometer existing at Laboratory of Magnetic Resonance are discussed in detail. Results on the enhancement of the NMR signal due electron-proton dynamic interactions are presented. NMR imaging of very small objects, 1 mm diameter glass tube filled with 5 ~10 ul of 2,2 mM of free radicals (TEMPOL) solution, obtained through back projection reconstruction NMR tomography method, are presented

Neste trabalho, usamos a técnica de Ressonância Paramagnética Eletrônica em seu modo convencional para o estudo da enzima clorocatecol 1,2-dioxigenase e em seu modo pulsado para o estudo da estrutura dinâmica de biomembranas. Clorocatecol 1,2-dioxigenase é uma enzima que catalisa a clivagem de estruturas aromáticas, como a do clorocatecol, via a a ativação e incorporação de uma molécula de oxigênio e que possui em seu sítio ativo um íon Fe(III). Nossos resultados de CD e atividade enzimática mostram o intervalo de temperaturas entre 20-25 ºC como aquele no qual a enzima apresenta atividade máxima. A maior contribuição para sua estrutura secundária vem de folhas β anti-paralela. A quantificação do número de spin feita por EPR indicou a presença de um íon Fe(III) por molécula de CCD, estando esse íon em estado de spin alto e simetria rômbica, como se depreende da linha estreita em g=4,3. Os parâmetros de desdobramentos de campo zero foram determinados pela medida em função da temperatura da linha em g=4,3, fornecendo λ=E/D=1/3 e D=(1,3±0,2) cm -1. O gráfico de Scatchard construído a partir dos espectros de EPR quando da titulação da enzima com o substrato catecol indicou a presença de um único sítio ligante de catecol, em acordo com a quantificação anterior do metal, e cuja constante de afinidade enzima-substrato é k=(2,7±0,1)x10-6 M. No que tange a EPR pulsado, foram utilizadas as chamadas técnicas modernas de EPR, com ênfase em 2D-ELDOR, para a investigação de diversos aspectos em membranas de interesse biológico. Primeiramente, investigamos a diferenciação entre a fase de líquido ordenado (Lo) e a de líquido cristalino (Lc) em membranas modelo de lipídio puro e lipídio/colesterol (1/1) contendo diferentes marcadores de spin (16-PC, CSL e DPPTC). Aí mostramos que é possível distinguir-se diferentes fases lipídicas apenas por simples inspeção visual dos espectros de 2D-ELDOR de um determinado marcador de spin incorporado à membrana. Além disso, realizamos simulações através do pacote de programas NLSPMC e determinamos as diferenças quantitativas entre as fases lipídicas: o estado de líquido ordenado apresenta maior fluidez e ordenamento na região das cadeias acílicas, ao passo que, na região da cabeça polar, mostra menor ordenamento. Em seguida, estudamos o efeito da presença de colesterol em alta concentração sobre o comportamento da membrana como função da temperatura. Nesse caso, o colesterol atua de maneira a manter a membrana em uma fase altamente ordenada e fluída dentro do intervalo de temperaturas medido, abolindo a transição gel-Lc do lipídio DPPC. Uma tentativa de aplicação de nossa metodologia ao estudo de membranas biológicas foi feita ao estudarmos membranas bleb que são ricas em colesterol. Estas mostram comportamento semelhante àquele observado para as membranas modelo com alta concentração de colesterol, um indicativo da existência de domínios Lo naquelas membranas biológicas. Por fim, estudamos o efeito da presença do peptídeo Gramicidina A\' (GA) sobre a estrutura da membrana de DPPC. Os resultados de 2DELDOR mostram claramente a presença de duas populações de lipídios (\"boundary\" e \"bulk\"). As simulações desses espectros foram as primeiras feitas com espectros contendo duas componentes e com os dados nas formas Sc- e Secsy. Essas mostram que os lipídios \"bulk\" são pouco afetados pela presença de moléculas de GA, ao passo que os lipídios \"boundary\" têm suas cadeias acílicas dobradas na porção terminal em torno das moléculas de GA, mecanismo que suporta a formação de domínios em fase HII na membrana. / In this work, EPR-CW and 2D-FT-EPR are used to study the enzyme chlorocatechol 1,2-dioxygenase and the dynamic structure of biological relevant membranes, respectively. Chlorocatechol 1,2-dioxygenase (CCD) is a non-heme Fe(lIl) enzyme that catalyses the ring cleavage of aromatic compounds like chlorocatechol. The structure stability as a function of the temperature was determined via circular dichroism and catalytic activity assays. The enzyme has its maximum activity at 20-25 ºC. The main contribution to its secondary structure comes from antiparallel Β sheets. The iron content was determined by EPR measurements, indicating the presence of one Fe(llI) per molecule. The Fe(III) ion shows a very narrow line at g=4.3 indicating a high spin state in a rhombic symmetry with λ=E/D=1/3 and D=(1,3±0,2) cm -1. The Scatchard plot based on EPR spectra suggests the existence of one site for substrate binding with a binding constant k=(2,7±0,1)x10-6 M. As for 2D-FT-EPR, the so-called modem EPR techniques, like 2D-ELDOR, were used to investigate several aspects of biologically relevant membranes. Firstly, we determined the differences between the liquid-ordered (Lo) and the liquid-crystalline (Lc) phases in model membranes of pure lipid and of lipid/cholesterol (1/1) mixtures containing different spin labels (16-PC, CSL, and DPPTC). In this case, we show how 2D-ELDOR makes possible the differentiation between Lo and Lc phases just by a pattern recognition scheme. We also performed simulations of those spectra and the results are: the Lo phase shows higher fluidity and ordering in the acyl chain region, whereas it shows lower ordering in the headgroup polar region. After that the membrane behaviour as s function of temperature was studied. We showed that cholesterol maintains the membrane in a highly ordered and fluid structure over the entire range of temperatures, abolishing the gel-Lc phase transition of the lipid DPPC. The biological membrane bleb was the first attempt to apply our methodology to real membranes. The 2D-ELDOR results for bleb membranes indicate the existence of Lo domains in the structure of those membranes. Finally, we studied the effects of the peptide Gramicidin A\' (GA) on the lipid organization of DPPC membranes. The 2D-ELDOR results show very clear two-component spectra (assigned to bulk and ,boundary lipids), which were simulated by the NLSPMC programs. This is the first time that 2D-FT-EPR multi-component spectra are simulated using both the Sc- and Secsy formal. The simulations indicate that the GA molecules do not significantly affect the bulk lipid, whereas the boundary lipids present the end portion of their acyl chain bent towards the GA molecule. This mechanism is probably responsible for the local formation of the HII phase in the membranes.

Aim: The aim of this study is to analyse potential determinants of cycle commuting speed, with the following research questions: 1. How does cycle commuting speed relate to amount of gears, access to shower facilities, and rating of perceived exertion (RPE), when sex, age, weight, body mass index (BMI), duration, last digit in self-reported duration, and cycling area are controlled for? 2. How are the earlier found relationships between speed and duration, and speed and sex, affected when amount of gears, access to shower facilities, and RPE are added to the control variables age, weight, BMI, last digit in self-reported duration, and cycling area?   Method: In this cross-sectional study, 1526 adult cycle commuters (67% females) in Stockholm County, Sweden, were recruited through advertisements. In a self-administered questionnaire, the respondents reported their sex, age, height, weight, duration of their typical cycle commuting journey, amount of gears, access to shower facilities at the destination, and rating of perceived exertion (measured by the Borg RPE Scale) while cycling. They also drew their cycling route on an individually adjusted map. Multiple linear regression was used to facilitate the analyses.   Results: In a regression model with the independent variables sex, age, weight, body mass index, duration, last digit in self-reported duration (1–4 or 6–9 compared to 0 or 5), cycling area (inner urban compared to suburban and suburban – inner urban), gears, shower facilities, and RPE, the dependent variable cycle commuting speed was positively related to using a bicycle with five or more (as compared to four or fewer) gears, having convenient (as opposed to inconvenient or non-existent) access to shower facilities, and perceiving a higher degree of exertion while cycling. Gears, showers, and RPE were also found to substantially affect the earlier found relationships between speed and duration, and speed and sex. The respondents showed a clear tendency of giving odd-numbered, compared to even-numbered, ratings of perceived exertion.   Conclusions: This study highlights the importance of gears, showers, and RPE in activities such as estimating, planning or taking measures in relation to cycle commuting. It also shows that adding new factors to control for might change earlier established relationships within this field. When the Borg RPE Scale is used, correct instructions strengthens the validity. / Syfte och frågeställningar: Syftet med denna studie är att analysera potentiella påverkansfaktorer gällande cykelpendlingshastighet, med följande frågeställningar: 1. Hur relaterar hastighet till antal växlar, tillgång till duschfaciliteter och skattning av egenupplevd fysisk ansträngningsgrad (RPE), när kön, ålder, vikt, BMI, duration, slutsiffra i självrapporterad duration samt cykelområde kontrolleras? 2. Hur påverkas de tidigare funna förhållandena mellan hastighet och duration, samt hastighet och kön, när antal växlar, tillgång till duschfaciliteter och RPE adderas till kontrollvariablerna ålder, vikt, BMI, slutsiffra i självrapporterad duration, och cykelområde?   Metod: I denna tvärsnittsstudie rekryterades 1526 vuxna cykelpendlare (67% kvinnor) i Stockholms län genom annonser. I ett själv-administrerat formulär rapporterade de kön, ålder, längd, vikt, duration vid deras typiska cykelpendlingsresa, antal växlar, tillgång till duschfaciliteter vid destinationen, och skattning av egenupplevd fysisk ansträngningsgrad (enligt Borgskalan) under cyklingen. De ritade även in sin cykelväg på en individuellt anpassad karta. Multipel linjär regression användes för att möjliggöra analyserna.   Resultat: I en regressionsmodell med de oberoende variablerna kön, ålder, vikt, BMI, duration, slutsiffra i självrapporterad duration (1­–4 eller 6–9 jämfört med 0 eller 5), cykelområde (innerstad i jämförelse med ytterstad samt ytterstad – innerstad), växlar, duschmöjligheter och RPE, var den beroende variabeln cykelhastighet positivt relaterad till användandet av en cykel med fem växlar eller fler (i jämförelse med fyra eller färre), att ha bekväm tillgång till dusch (i motsats till obekväm eller ingen tillgång alls) samt att uppleva en högre grad av fysisk ansträngning under cykling. Växlar, duschmöjligheter och RPE visade sig även substantiellt påverka de tidigare funna förhållandena mellan hastighet och duration, samt hastighet och kön. Respondenterna visade en tydlig tendens att ge ojämna, i jämförelse med jämna, RPE-skattningar.   Slutsats: Denna studie belyser vikten av växlar, duschmöjligheter och egenupplevd ansträngningsgrad vid exempelvis bedömning, planering eller vidtagande av åtgärder i relation till cykelpendling. Den visar även att nya kontrollfaktorer kan förändra tidigare etablerade förhållanden inom detta fält. Korrekta instruktioner stärker validiteten vid användandet av Borgskalan.

L’étude des organismes vivants, la biologie, s’étend sur de nombreux domaines et notamment s’applique à comprendre le fonctionnement des êtres vivants. Les organismes les plus complexes comme les êtres Humains possèdent plusieurs niveaux d’organisation : ils sont constitués successivement d’organes, de tissus, de cellules, de biomolécules. On trouve plusieurs types de biomolécules dont les protéines, qui sont comme des minuscules outils qui permettent aux cellules de vivre et d’interagir avec leur environnement. Pour cela, les protéines doivent entrer en contact les unes avec les autres de manière très précise et déterminée. Cette thèse teste l’existence de forces électrodynamiques de longue portée qui leur permettraient d’interagir de manière rapide et guidée, via l’étude de l’absorption ou l’émission de ce type d’onde par des protéines, puis la diffusion de ces protéines en solution pour observer leur comportement. / The study of living organisms, biology, extends over many fields and in particular, applies to understanding the functioning of living beings. The most complex organisms, such as human beings, have several levels of organization: they are made up successively of organs, tissues, cells, and biomolecules. There are several types of biomolecules including proteins, which are like tiny tools that allow cells to live and interact with their environment. To do this, proteins must come into contact with each other in a very precise and determined way. This thesis tests the existence of long-range electrodynamic forces which would allow them to interact in a rapid and guided way, by studying the absorption or emission of this type of wave by proteins, then the diffusion of these proteins in solution to observe their behavior.

Les émulsions fluorées de Pickering sont étudiées et mises au point dans la technologie demicrofluidique en gouttes pour des applications d’études sur des cellules adhérentes isolées.Les principaux résultats de ce projet sont : l’établissement d’un lien entre la couverture desurface des nanoparticules et la fluidité de l’émulsion de Pickering ; l’établissement deslignes directrices pour la stabilisation des gouttes avec un débit de production élevé et unminimum de déchets de particules ; et la mise en oeuvre d’une plateforme technologiquecomplète pour l’étude des cellules RPE, pour mesurer leur hétérogénéité phénotypique auniveau de la cellule individuelle. / Fluorinated Pickering emulsions are studied and engineered within droplet-based microfluidicstechnology for adherent-cell studies applications. The main findings of this projectinclude: linking the nanoparticles surface coverage to the bulk flowability of the Pickeringemulsion; deriving guidelines for droplet stabilization with high production throughput andminimal particle waste; and implementing the full technological platform for the study ofRPE cells, while unraveling their phenotypic heterogeneity at the single cell level.

Chez les vertébrés, la perception de la lumière est possible grâce à la transformation de l'énergie des photons en un signal électrochimique dans les photorécepteurs. La photo-isomérisation du chromophore RAL-11-cis en RAL-tout-trans déclenche une cascade d'événements de transduction du signal et conduit à la perte de la sensibilité à la lumière des photorécepteurs, un processus de recyclage du chromophore visuel entre alors en jeu afin de rétablir cette sensibilité, c'est le cycle visuel de rétinoïdes. Le cycle visuel s'effectue dans les photorécepteurs et l'épithélium pigmentaire rétinien et fait intervenir plusieurs protéines. L'étape limitante de ce cycle est catalysée par la protéine spécifique de l'EPR RPE65. Mieux comprendre la régulation de cette étape permettra d'ouvrir des pistes thérapeutiques pour différentes pathologies liées l'activité du cycle visuel. Notre laboratoire a montré que FATP1, une protéine du métabolisme lipidique, inhibe l'activité de RPE65 in vitro. Dans ce travail, nous avons évalué l'effet de l'absence de FATP1 sur l'activité du cycle visuel et la fonction visuelle dans un modèle murin. La souris ko FATP1 est caractérisée par une baisse des amplitudes des ondes a et b de l'ERG, un retard de régénérescence de l'ERG et d'accumulation de rétinylesters après éblouissement. La formation du RAL-11-cis reste comparable à celle des souris sauvages. Les souris FATP-/- montrent une plus grande susceptibilité aux effets du vieillissement : des anomalies de structure ont été observées au niveau des photorécepteurs, de la membrane de Brüch et de la choroïde, sans accumulation excessive de lipides ou de lipofuscine ni de dégénérescence rétinienne. La perturbation de la fonction visuelle observé en l'absence de FATP1 chez la souris pourrait avoir un effet plus accru chez l'homme qui vie plus longtemps et dont la rétine reçoit plus de lumière. En conclusion, FATP1 pourrait être une composante importante pour le vieillissement de la rétine chez l'homme. / In vertebrates, perception of light is made possible through the conversion of photon energy into an electrochemical signal in photoreceptors. The photo-isomerization of the chromophore 11-cis-RAL into all-trans-RAL triggers a cascade of signal transduction and leads to loss of light sensitivity of photoreceptors, thus, a recycling process of chromophore called the rétinal visual cycle is involved to restore this sensitivity. The visual cycle takes place in the photoreceptors and retinal pigment epithelium and involves several proteins. The limiting step of this cycle is catalyzed by RPE-specific protein RPE65. Better understanding the regulation of this step might open up new ways for treatment of various pathologies related to the visual cycle activity. Our laboratory has shown that FATP1, a protein involved in lipid metabolism, inhibits the activity of RPE65 in vitro. In this study, we evaluated the effect of the lack of FATP1 on the activity of the visual cycle and visual function in a mouse model. FATP1 ko mice are characterized by lower a and b amplitudes of the elctroretinogram, delayed ERG and retinylesters accumulation recovery after bleache. 11-cis-RAL synthesis rates are similar to those of wt mice. FATP1 ko mice show a greater susceptibility to the aging effects: structural abnormalities were observed in the photoreceptors, Bruch's membrane and choroid without excessive accumulation of lipid or lipofuscin or retinal degeneration. The disturbance of visual function which accopagne the lack of FATP1 in mice could have a more increased effect in human who live longer and wich the retina receives more light. In conclusion, FATP1 might be an important component for the aging of the retina in humans.

Mitf and Otx transcription factors have been identified as essential to the development of the retinal pigmented epithelium (RPE), but the relationship between these factors and their specific role in the RPE developmental pathway have not been clearly defined. The role of the two Mitf transcription factors (Mitfa and Mitfb) and two Otx transcription factors (Otx1a and Otx2) in zebrafish RPE development was explored in these experiments. The loss of Mitf activity in mitfa, mitfb, or double mitf null mutant fish lines had no effect on RPE pigmentation or development. The loss of Otx2 activity through morpholino knockdown produced a RPE deficient phenotype in a small percentage of embryos, while the additional knockdown of Otx1a caused widespread and severe RPE developmental abnormalities. Analysis of ocular sections revealed that the retinal layers remain unaffected in mitf mutants, as well as in most RPE-deficient otx morphants. Mitf and Otx combined loss of function experiments suggest that Mitfa and Mitfb may still play a role in zebrafish RPE development. Expression analysis through in situ hybridization has demonstrated that Otx transcription factors are necessary for the proper expression of mitfa and mitfb while Mitf transcriptions factors are not required for the expression of otx genes. The transcriptional regulation of Mitf by Otx transcription factors may explain why only Otx transcription factors are necessary for zebrafish RPE development despite the somewhat overlapping functions of Mitf and Otx transcription factors.