With the introduction of nanoparticles in health care and daily products, the interaction between cells and nanoparticles is of great interest. There are many ways to examine these interactions, one of them being soft X-ray microscopy. It is a technique utilizing the water window to image biological samples, its greatest benefit being the non-invasive sample preparation, keeping the cell in a near-native state. The Stockholm Laboratory Soft X-ray Microscopy is a compact soft X-ray microscope that is currently being used to examine cell interactions. However, the sample preparations have been inconsistent and have yielded few useful and reproducible results. The purpose of this project is to optimize the cell preparation for better imaging using the Stockholm Laboratory Soft X-ray Microscope.  To reach the project goal to enable imaging of the nucleus and organelles in macrophages, this thesis will present a sample preparation protocol for macrophages where the concentration, blotting and cryo-fixation of the samples have been improved. The macrophage preparation was also tried on two other types of cells, amoeba and HEK 293 cells, to see if this preparation is universal for all cell lines. The results indicated that the most difficult and crucial step is the blotting, as too dry samples destroy the cells and too wet samples will yield almost no transmission in the microscope, making it impossible to image. / Med introduktionen av nanopartiklar i vården och dagliga produkter är samspelet mellan celler och nanopartiklar av stort intresse. Det finns många sätt att undersöka dessa interaktioner, ett av dem är mjuk röntgenmikroskopi. Det är en teknik som använder vattenfönstret för att avbilda biologiska prover, vars största fördel är den icke-invasiva provberedningen, som håller cellen i ett nästan naturligt tillstånd. Stockholm laborativa mjukröntgenmikroskop är ett kompakt mjuktröntgenmikroskop som för närvarande används för att undersöka cellinteraktioner. Provberedningarna har dock varit inkonsekventa och har gett få användbara och reproducerbara resultat. Syftet med detta projekt är att optimera cellförberedelsen för bättre avbildning med hjälp av Stockholm laborativa mjukröntgenmikroskop. För att uppnå projektmålet att möjliggöra avbildning av cellkärnor och organeller i makrofager, så kommer denna avhandling att presentera ett provberedningsprotokoll för makrofager där koncentrationen, blotting och kryofixering av proverna har förbättrats. Makrofag-preparatet testades även på två andra typer av celler, amobea- och HEK 293-celler, för att se om detta preparat är universellt för alla cellinjer. Resultaten visade att det svåraste och mest avgörande steget är blotting, eftersom för torra prover förstör cellerna och för våta prover kommer att ge nästan ingen överföring i mikroskopet, vilket gör det omöjligt att avbilda.

Cette thèse vise à étudier les nanoparticules d'or (NPOs) comme radio sensibilisateur et nano catalyseur afin d'améliorer la production d'espèces réactives de l'oxygène (EROS) et à fournir un aperçu de l'effet protecteur potentiel des acides aminés de base contre les effets des rayonnements ionisants afin d'étudier le mécanisme de dommages de l'ADN. En outre, il vise à améliorer notre connaissance des effets directs et indirects sur l'ADN plasmatique qui est une cible importante et sensible lors de l'étude des effets des rayonnements ionisants. Cette étude nécessite à la fois Ia bonne distribution et le contrôle de l'ADN déposé sur graphite HOPG. Nous avons procédé à la préparation de films (couches nanométriques à l’échelle) faite d'un complexe d'ADN avec des amines, ainsi que les acides aminés basiques. Nous avons caractérisé le déposé sec par microscopie a force atomique et par XPS. Nous avons montré que les couches contenant des acides aminés basiques sont très denses et que la protection contre les dommages de l'ADN après re-dissolution dans l'eau est très efficace. Ensuite, nous avons déposé l'ADN plasmatique à sec soul sur des feuilles minces de mylar et d'exposé ces films à un faisceau de protons à différentes énergies, Nous avons montré que, dans le value de pic de Bragg d'énergie a protons énergies inferieure a 500 keV il y a un rendement Cleve de protons a endommagé l'ADN. L'effet indirect a donc été étudié en exposant l'ADN plasmatique en solution aqueuse par rayons X ultra-mous. Nous avons procédé a de telles expériences en utilisant 100 lit de solution d'ADN plasmatique avec et sans différentes concentrations de 2-amino-2-hydroxymethyl-propane-1,3-diol, (Tris) et l'arginine (Arg) à température ambiante et sous agitation. Une très bonne procédure expérimentale. Nous avons montré que les rendements pour les pauses ADN simples brins ont permis au ratio indirect des effets directs à déterminer à 96% en bon accord avec la valeur de 97% découlant d’une étude basée sur l’irradiation aux rayons gamma de solutions congelées de l’ADN plasmatique. Nous analysons nos résultats et nous suggérons que les produits secondaires de Arginine réagissent avec les radicaux OH pour produire des effets secondaires; notamment en raison du fait que, contrairement au Tris, Arg est capable de liaison à l'ADN à l'extérieur et également à l'intérieur de la double hélice. Nous avons exposé également l'ADN de plasmide en solution aqueuse et complexe aux deux autres acides aminés basiques (lysine et histidine) par rayons X ultra-mous. Nous avons constaté que la présence de ces acides aminés augmente également la détérioration de l'ADN et suggère que les acides aminés sont donc également susceptibles de produire des effets secondaires. Enfin, nous avons étudié Ie rôle de nanoparticules d'or (NPO) dans le renforcement de la sensibilisation de la radio de l'ADN. En utilisant de nouveaux protocoles expérimentaux, nous avons fini à interpréter l'interaction du (NPOs) avec ionisation rayonnement dans des solutions aqueuses oxygénées. Pour obtenir une meilleure compréhension des mécanismes de sensibilisation de radio par NPOs, nous avons utilisé une variété de espèces détritivores, spécifiques et réactives d'oxygène et l'ADN plasmatique comme sonde afin de favoriser les voies donnees et de determiner laquelle d'entre elles est la plus efficace dans l'ADN dommageable. Cette étude a démontré pour la première fois que les NPOs contribuent en permanence à l'amélioration de la production de OH en utilisant un stock potentiel maintenu sous la forme d'HO2 • / 02 • - et H202 produits de radiolyse de l'eau primaire. Nous avons montre en effet que H202 peut réagir avec NPOs pour produire • OH radicaux. / This thesis aims at studying the gold nanoparticles (GNPs) as radiosensitizer and nanocatalyst in order to enhance the generation of reactive oxygen species (ROSs) and at providing insight into the potential protective effect of basic amino acids against ionizing radiation effects in order to study the mechanism of DNA damage. Also, it aims at improving our knowledge of direct and indirect effect on plasmid DNA which is an important and sensible target when studying ionizing radiation effects. This study required both the good distribution and control of the DNA deposited on HOPG (Highly Ordered Pyrolytic Graphite). We carried out the preparation of films (nanometer scaled layers) made of complex of DNA with diamines as wel as basic amino acids. We have characterized dry deposited by AFM (Atomic Force Microscopy) and by XPS (X-ray photoelectron spectroscopy). We have shown that the layers containing basic amino acids are very dense and that the protection against DNA damage after re-dissolution in water is very effective. Then, we have deposited dry plasmid DNA alone on thin mylar foils and exposed those films to a proton beam at various energies. We have shown that in the Bragg-peak energy range i.e. at proton energies lower than 500 keV there is a high efficiency of protons to damage DNA. The indirec effect was thus studied by exposing plasmid DNA in aqueous solution by (USX). We carried out such experiments by using 100 pit of plasmid DNA solution with and without different concentrations of 2-Amino-2-hydroxymethyl-propane-1,3-diol (Tris) and arginine (Arg) at ambient temperature and under stirring. A very good agreement was found with the rare data dealing with DNA plasmid exposed to Al Ka photons at low temperature (T < 277 K), which therefore validated the experimental procedure. We showed that the yields for DNA single strand breaks determined enabled the ratio of indirect to direct effects to be determined at 96.2%; in good agreement with the value of 97.7% stemming from a study based on y-ray irradiation of frozen solutions of plasmid DNA. We analyze our results and suggest that secondary products of Arginine react with OH radicals to produce side effects; notably due to the fact that contrary to Tris, Arg is capable of binding DNA outside but also inside the double-helix. We exposed also the plasmid DNA in aqueous solution and complexed to the two other basic amino acids (histidine and lysine) by USX. We found that the presence of tilos amino acids also increases DNA damage and suggest that those amino acids are therefore also prone to produce side effects. Lastly, we studied the role of Gold Nano Particles (GNPs) in enhancing DNA radio sensitization. By using new experimental protocols, we achieved to interpret the interaction of GNPs with ionization radiation in oxygenated aqueous solutions. To get better insight into the mechanisms of radiosensitization by GNPs, we used a variety of specific reactive oxygen species scavengers and plasmid DNA as a probe in order to favor given pathways and to determine which of them is the most effective in damaging DNA. This study has demonstrated for the first time that the GNPs contribute permanently to the enhancement of ■OH production by using a potential stock kept under the form of 1102•/02•- and H202 primary water radiolysis products. We showed indeed that H202 ca react with GNPs to produce •0H radicals.

Made available in DSpace on 2016-03-15T19:35:52Z (GMT). No. of bitstreams: 1 DENIS PAVEL CABEZAS HUAMAN.pdf: 1718725 bytes, checksum: b98f52c6b4dac9b37a33cc5bc62d871e (MD5) Previous issue date: 2015-03-26 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The project aims to study the disturbances (responses) of the low solar atmosphere caused by solar flares, using H&#945;, 30 THz (10 &#956;m), UV/EUV, soft and hard X-rays observations, and a wide range of radio waves emissions (microwaves, millimeter, sub-millimeter). This set of multispectral data enabled us to (i) examine in detail various aspects of the phenomena, (ii) determine the origin of the radio emission during the gradual phase, and therefore (iii) understand the mechanism of the particles acceleration. The data we used for the completion of this study are based on H&#945; and 30 THz (10 &#956;m) observations, made by the high cadence flare imaging system installed at OSM4 and CASLEO5 observatories, additionally data from HASTA (H-Alpha Solar Telescope for Argentina) telescope installed at OAFA6 observatory. In the sub-millimeter and millimeter domain at 212, 405 GHz and 45, 90 GHz, were from SST (Solar Sub-millimeter Telescope) and from POEMAS (POlarization Emission of Millimeter Activity at the Sun) solar radio telescopes, respectively. Complementary radio observations in the microwave range (0.2-15 GHz) from RSTN (Radio Solar Telescope Network) were considered. In addition, solar observations obtained by RHESSI (Reuven Ramaty High Energy Solar Spectroscopic Imager), Fermi and GOES (Geostationary Operational Environmental Satellite) satellites in X-ray channels, and from AIA (Atmospheric Imaging Assembly) and HMI (Helioseismic and Magnetic Imager) instruments, onboard the SDO (Solar Dynamics Observatory) satellite, respectively. / O trabalho tem como objetivo estudar as perturbações da baixa atmosfera solar produzidas pelas explosões solares, usando observações em H&#945;, 30 THz (10 &#956;m), UV/EUV, raios X e uma ampla faixa de emissões em ondas de rádio (microondas, milimétrica, submilimétrica); com o propósito de determinar a natureza dos processos físicos envolvidos. Este conjunto de dados multiespectrais nos permitiu (i) analisar os diferentes aspectos das explosões tanto na fase impulsiva como gradual, (ii) determinar a origem da emissão em rádio durante a fase gradual, e consequentemente (iii) entender melhor os mecanismos de aceleração das partículas. Os dados em H&#945; e 30 THz foram fornecidos pelo sistema de aquisição de dados com alta resolução temporal, instalado nos observatórios OSM1 e CASLEO2, adicionalmente dados do telescópio HASTA (H-Alpha Solar Telescope for Argentina) instalado no observatório OAFA3. Enquanto as observações em rádio, os dados foram do rádio polarímetro POEMAS (POlarization Emission of Millimeter Activity at the Sun) nas frequências de 45 e 90 GHz, do telescópio SST (Sub-millimeter Solar Telescope) nas frequências 212 e 405 GHz, ambos instalados no CASLEO. Dados na faixa de microondas (0,2-15 GHz) obtidos pela rede RSTN (Radio Solar Telescope Network). Além disso, foram considerados observações do Sol obtidas pelos satélites RHESSI (Reuven Ramaty High Energy Solar Spectroscopic Imager), Fermi e GOES (Geostationary Operational Environmental Satellite) para raios X, e dos instrumentos AIA (Atmospheric Imaging Assembly), HMI (Helioseismic and Magnetic Imager), a bordo do satélite SDO (Solar Dynamics Observatory), respectivamente.