Atualmente, as nanopartículas superparamagnéticas despertam enorme interesse científico devido sua grande variedade de aplicações em biomedicina, tanto na área de diagnóstico quanto no tratamento de enfermidades. Embora muitos materiais vem sendo estudados, os óxidos de ferro (magnetita e maghemita) apresentam maiores avanços nos estudos para aplicações em medicina. A preferência por óxidos de ferro se deve a baixa toxicidade destas partículas quando comparado as nanopartículas metálicas ou ligas. Entretanto, as nanopartículas destes óxidos possuem baixas magnetizações de saturação que diminuem ainda mais com as sucessivas etapas de recobrimento necessárias para conferir funcionalidade a estas partículas. Desse modo, há uma necessidade atual para o desenvolvimento de nanopartículas superparamagnéticas com elevada magnetização, baixa toxicidade e maior facilidade de funcionalização da sua superfície com biopolímeros e agentes funcionalizantes. Neste trabalho, nanopartículas superparamagnéticas de cobalto metálico e ferrita de cobalto foram sintetizadas e suas propriedades magnéticas foram comparadas com a magnetita. Nanopartículas de cobalto foram escolhidas, pois seu elevado comportamento ferromagnético é menor apenas que o ferro metálico, além do baixo custo de seus reagentes. As nanopartículas magnéticas foram sintetizadas pelos métodos de microemulsão e decomposição térmica (baseado no método poliol) e suas composições química, estrutural, tamanho e distribuição de tamanho foram devidamente determinadas. Além disso, as nanopartículas de cobalto metálico e ferrita de cobalto foram recobertas com ouro utilizando o método de crescimento mediado por semente. Os sistemas microemulsionados utilizados neste trabalho não foram eficientes nem na síntese de nanopartículas estáveis de cobalto metálico nem no seu esperado controle morfológico. Já o método de decomposição térmica resultou em um rigoroso controle de composição química, estrutural e morfológico para as diferentes nanopartículas sintetizadas. O recobrimento com ouro foi efetivo na proteção do núcleo magnético e adicionalmente conferiu estabilidade, baixa toxicidade e bifuncionalidade às nanopartículas magnéticas através do seu fenômeno de ressonância plasmônica de superfície o qual foi preservado na nanoestrutura core@shell. O comportamento superparamagnético das nanopartículas de cobalto metálico recobertas com ouro e sua elevada magnetização de saturação foram expressivamente intensificadas quando comparadas as nanopartículas de magnetita sem recobrimento. Portanto, as nanopartículas sintetizadas neste trabalho apresentam propriedades de superfície e magnéticas otimizadas demonstrando um bom potencial para aplicações em biomedicina como sensores bifuncionais óptico-magnético. / Superparamagnetic nanoparticles have been extensively studied because its wide range of biomedical applications in both diagnostic and therapy areas. Although different materials are currently investigated, superparamagnetic iron oxides nanoparticles (SPION), magnetite and maghemite, are the most extensively studied for applications in medicine. The lower toxicity profile of the SPION becomes the most attractive than metal or alloys nanoparticles. Nevertheless, iron oxides nanoparticles have low saturation magnetization, which further decreases due to successive coats to provide their functionality, leading the actual demand to develop superparamagnetic nanoparticles with high magnetization, low toxicity and easy surface functionalization with biocompatible agents. In this work, superparamagnetic nanoparticles of metallic cobalt and cobalt ferrite were synthesized and their magnetic properties were compared with the magnetite SPION. Cobalt nanoparticles were chosen because present high ferromagnetic behavior among chemical elements, second only to iron, besides their low cost. The magnetic nanoparticles were synthesized by both microemulsion and thermal decomposition (based on the polyol process) methods and their chemical composition, structure, size and size distribution were properly characterized. In addition, the ferrite and metallic cobalt nanoparticles were coated with gold by using the seed-mediated growth method. The used microemulsion systems were not efficient enough to synthesize stable metallic nanoparticles and to promote the expected morphological control even to ferrites. Instead, the thermal decomposition processes resulted in rigorous control of chemical compositional, structure and morphology in all different prepared samples. Au-coating process was effective to protect the magnetic nuclei also giving additional stability, low toxicity and a bifunctionality to the magnetic nanoparticle since their surface plasmon resonance phenomenon was preserved in the core@shell nanostructure. The superparamagnetic behavior of the Au-coated cobalt nanoparticle was preserved and their saturation magnetization was significantly increased compared with the naked magnetite SPION. In conclusion, the synthesized nanoparticles present enhanced magnetic and surface properties showing good potential to be used in biomedical application as bifunctional optical-magnetic sensor.

Aim of this thesis was to design and prepare polymer-coated monodisperse Fe3O4 nanoparticles as a safe and non-toxic contrast agent for magnetic resonance imaging (MRI) and heat mediator for hyperthermia. Uniform superparamagnetic Fe3O4 nanoparticles were synthesized by thermal decomposition of Fe(III) oleate, mandelate, or glucuronate in high- boiling solvents at temperature >285 řC. Size of the particles was controlled in the range of 8- 27 nm by changing reaction parameters, i.e., temperature, type of iron precursor, and concentration of stabilizer (oleic acid and/or oleylamine), while preserving uniformity of the nanoparticles. Because particles contained hydrophobic stabilizer on the surface, they were dispersible only in organic solvents. To ensure water dispersibility, oleic acid on the particle surface was replaced by hydrophilic and biocompatible methoxy-poly(ethylene glycol) (PEG) and poly(3-O-methacryloyl-α-D-glucopyranose) by ligand exchange. Polymers were previously terminated with anchoring-end groups (hydroxamic or phosphonic) to provide firm bonding to iron atoms on the particle surface. Fe3O4 nanoparticles were also hydrophilized by encapsulation into a silica shell by reverse microemulsion method. Tetramethyl orthosilicate was used to prepare Fe3O4@SiO2 nanoparticles, which were...

DNA detection has an increasing importance in our everyday lives, with applications ranging from microbial diagnostics to forensic analysis. Currently, as the associated costs decrease, DNA diagnostic techniques are routinely used not only in research laboratories, but also in clinical and forensic practice. The present thesis aims to unravel the potential of cellulose filter paper to be a viable candidate for DNA array support. There are two papers in this study. In Paper I, we studied the method of functionalizing the surface of filter paper and the possibility to detect DNA on acitve paper using fluorescence. In Paper II, we investigated visualization and throughput of DNA detection with magnetic beads on active filter papers, an assay which requires no instrumentation (scanner). The findings in Paper I show that XG-NH2 and PDITC can functionalize the cellulose filter paper and that the activated filter papers can covalently bind oligonucleotides modified with amino groups to detect DNA. The detection limit of the assay is approximately 0.2 pmol. In Paper II, visualization of DNA detection on active paper is achieved without instrumentation, based on the natural color of magnetic beads. Furthermore, successful multiplex detection supports the potential to increase the throughput of DNA detection on active papers. In summary, these studies show that active cellulose filter paper is a good DNA array support candidate as it provides a user-friendly and cost-efficient DNA detection assay. The methods described in Paper I and II are possible sources of development to a point-of-care device for on-site analysis of DNA contents in a sample. / <p>QC 20131111</p>

Superparamagnetic -Fe2O3 nanoparticles were synthesized by coprecipitation of ferric and ferrous salts with a base. Resulting nanoparticles were coated with shells, such as poly(N,N- dimethylacrylamide) (PDMAAm), neat and functionalized silica (SiO2 and SiO2-NH2), and polyaniline (PANI). PDMAAm shell was introduced by modification of iron oxide nanoparticle surface with an initiator and N,N-dimethylacrylamide was polymerized producing -Fe2O3&PDMAAm core-shell particles. In case of SiO2-NH2 shell, tetramethyl orthosilicate was used to yield -Fe2O3&SiO2 nanoparticles, which were subsequently modified by (3-aminopropyl)triethoxysilane to prepare γ-Fe2O3&SiO2-NH2 particles. Oxidation of aniline hydrochloride with ammonium persulfate in an aqueous solution of poly(N-vinylpyrrolidone) in the presence of iron oxides produced -Fe2O3&PANI nanoparticles. Finally, the last type of the particles was based on thionin-modified poly(carboxymethyl methacrylate) (PCMMA&Th). The particles were characterized by techniques, such as scanning and transmission electron microscopy (SEM and TEM) and dynamic light scattering (DLS) to determine the particle morphology and hydrodynamic diameter. The presence of the functional groups, chemical composition, and the iron content were investigated by Fourier-transform...

Par ce travail expérimental, nous essayons d’établir un lien entre les propriétés mécaniques de gels d’actine branchés de levure et la composition biochimique des réseaux. L’actine est un polymère semi-flexible qui fait partie du cytosquelette. De nombreux partenaires protéiques de l’actine (notés ABPs par la suite) se lient aux filaments d’actine et les agencent en différents types de réseaux. Arp2/3 est un complexe protéique qui génère la croissance de réseaux d’actine branchés. Les réseaux d’actine branchés en croissance intéressent tout particulièrement physiciens comme biologistes car ils sont capables de développer des forces nécessaires à de nombreux processus vitaux pour la cellule, comme l’endocytose. Nous avons ici étudié les propriétés mécaniques de gels d’actine branchés reconstitués in vitro, en nous focalisant sur le rôle d’un type d’ABPs en particulier, les protéines de réticulation. Il nous a été possible de quantifier et de comparer l’effet de trois protéines de réticulation différentes sur la mécanique des réseaux d’actine branchés de levure.Afin de mener à bien cette étude, nous avons combiné deux puissantes techniques expérimentales.Nous avons utilisé une technique de mesure des propriétés mécaniques basée sur l’utilisation de colloïdes superparamagnétiques développée au laboratoire. Cette technique permet de réaliser des mesures quantitatives et à haut débit sur des gels polymères très fins (quelques centaines de nanomètres d’épaisseur). Les réseaux ont été reconstitués in vitro grâce à la fonctionnalisation des billes superparamagnétiques avec Las17, une protéine que notre collaborateur biologiste a identifiée comme suffisant à activer Arp2/3 chez la levure. Nous avons de plus combiné deux approches complémentaires en travaillant à la fois sur des extraits cellulaires de levure contenant toutes les ABPs des réseaux Arp2/3 et à la fois sur des mélanges de quelques protéines purifiées.L’approche « top-down » est basée sur l’utilisation d’extraits cellulaires de mutants de la levure n’exprimant pas une ou des protéine(s) d’intérêt(s), et l’approche « bottom-up » sur l’addition de la protéine étudiée dans le système simplifié de quelques protéines purifiées. / In this experimental work we tried to quantify the mechanical properties of yeast branchedactin networks with regard to their biochemical composition. Actin is a semi-flexible biopolymerthat is assembled as part of the cytoskeleton. Proteins partners of actin (ABPs) shape itsfilaments into different type of networks. Arp2/3 is a protein complex that has the propertyto generate branched actin gels. Growing branched actin networks are of particular interest forboth biologists and physicists because of their ability to generate forces necessary to many vitalprocesses such as endocytosis. Here we study in vitro the mechanical properties of such networks,and we focus on the role of one type of actin binding proteins, the crosslinkers. This family ofproteins appears to play a role in both the elastic, viscous and plastic properties of the gels. Weare able to quantify and to compare the impact of three different crosslinkers on branched actinnetworks in yeast.In order to conduct said study, we combined two powerful experimental methods. We used asuperparamagnetic particle-based mechanical measurement technique that was developed in thelab and allows quantitative, high-throughput measurements on very thin gels. And the networkswere reconstituted in vitro by functionalization of the magnetic particles with Las17, which hasbeen showed to activate Arp2/3 for the yeast by our biologist collaborator. We furthermoreworked on both yeast extracts containing all the ABPs of the Arp2/3 networks, and with setsof a few purified proteins, in order to combine a « top-down » (use of mutations in yeast toprevent the expression of protein(s) of interest) and a « bottom-up » (addition of a protein ofinterest in a simplified system) approaches.

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<p> Aqueous ferrofluid has been prepared via precipitation of iron oxide into a polysaccharide gel matrix followed by degradation of the polymer to form a stable magnetic colloid. Nanocrystalline particles of iron oxide were formed in an alginate network by the alkaline hydrolysis and oxidation of the crosslinking agent, Fe2+, used to bind the linear polysaccharide chains. Methanol was used to inhibit the degradation of alginate by Fe2+ and oxygen during the precipitation and growth of iron oxide particles. In addition, the structural integrity of the gel was maintained in part by interaction between the iron oxide particulate and the alginate matrix. Controlled chemical degradation of the matrix resulted in a aqueous suspension of alginate-stabilized magnetic iron oxide particles. The resulting fluid is orange-brown in color, optically transparent, superparamagnetic and stable between 2.8<pH<10 </p> <p> The magnetic gels were isolated as 2mm beads containing ~2-20 mmole Fe. X-ray and electron diffraction patterns of the composite correspond to maghemite (γ-Fe2O3) and/or magnetite (Fe3O4). At room temperature, the composite material is superparamagnetic with saturation magnetizations in excess of 20 emu g^-1 at 30kOe. TEM photomicrographs of sectioned beads and of the magnetic fluid revealed the presence of spherical nanocrystalline oxide particles with diameters ranging from 3 nm to 6 nm. The iron oxide-alginate colloid has a diameter of 54nm with an average zeta potential of -51.6 mV.</p> / Thesis / Master of Science (MSc)

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No description available.

Ce travail de thèse s'inscrit dans le cadre d'une collaboration avec le groupe Guerbet. Il visait à caractériser la lésion qui fait suite à un accident vasculaire cérébral (AVC) ischémique en imagerie par résonance magnétique (IRM) grâce à des agents de contraste novateurs. Guerbet et leurs collaborateurs ont développés des USPIO ciblés (ultrasmall superparamagnetic iron oxide), particules de fer couplées à des peptides reconnaissant spécifiquement un biomarqueur. Dans un modèle d’ischémie cérébrale avec reperfusion réalisé chez la souris, nous avons recherché la capacité de ces agents à caractériser la lésion d’une part en terme de type de mort cellulaire par le ciblage de la phosphatidylsérine (PS), marqueur cellulaire externalisé au cours de l’apoptose, et d’autre part en terme de déficit vasculaire par le ciblage de VCAM-1, molécule d’adhésion impliquée dans le processus inflammatoire. En ce qui concerne l’apoptose, nous avons tout d’abord montré par immunohistochimie l’expression de caspase-3 active, marqueur apoptotique, dès 6 heures et jusqu’à 72 heures après l’ischémie. Cependant, en IRM, l’utilisation d’USPIO ciblant la PS (le P03234 et le P03675) n’a pas permis la détection du phénomène apoptotique. Actuellement d’autres agents de contraste de ce type sont en cours de développement chez Guerbet. En ce qui concerne l’inflammation vasculaire, l’étude de l’expression de VCAM-1 par immunohistochimie a montré l’apparition d’un marquage dès 6 heures après l’ischémie avec un maximum à 24 heures. L’utilisation d’un USPIO-VCAM-1 (le P03011) a permis de mettre en évidence sur les images IRM des zones d’hypointensités dans la lésion, ce qui correspond à la présence de particules de fer. L'analyse histologique de ces cerveaux a montré une colocalisation de l’USPIO avec sa cible VCAM-1, ce qui établit la preuve de concept. Ces travaux ont permis mettre en évidence la capacité d’USPIO développés par Guerbet à cibler des marqueurs biologiques, notamment VCAM-1, à la suite d’une ischémie cérébrale. Ceci suggère que ce type d’agent de contraste pourrait être un bon outil clinique pour la caractérisation de la lésion ischémique chez les patients victimes d’AVC. / This work is a collaboration with Guerbet group. It aimed to characterize the lesion that follows an ischemic stroke with magnetic resonance imaging (MRI) by innovative contrast agents. Guerbet developped targeted USPIO (ultrasmall superparamagnetic iron oxide), which are iron particles coupled to peptides which specifically bind a biomarker. In a mouse model of cerebral ischemia-reperfusion, we studied the capacity of these agents to characterize the lesion on the one hand in terms of cellular death by targeting phosphatidylserin (PS), a cellular marker externalized during apoptosis, and on the other hand in terms of vascular deficit by targeting VCAM-1, an adhesion molecule implied in the inflammatory process. Concerning apoptosis, we showed by immunohistochemistry the expression of active caspase-3, an apoptotic marker, between 6 and 72 hours after ischemia. Nevertheless on MRI, the use of USPIO targeting PS (both P03234 and P03675) did not allow us to detect the apoptotic phenomenon. Currently, other PS-targeted contrast agents are developed by Guerbet. Concerning vascular inflammation, the study of VCAM-1 by immunohistochemistry showed an up-regulated expression 6 hours after ischaemia which reached a maximum at 24 hours. VCAM-1-USPIO (P03011) induced a decrease of the MRI signal appearing as hypointense foci in the lesion, which correspond to iron particles. The histological analysis of these brains showed a colocalisation of the USPIO with its target VCAM-1, which establishes the proof of concept. This work showed the capacity of USPIO developed by Guerbet to target biological markers, particularly VCAM-1, following cerebral ischemia. This suggests that this kind of contrast agent could be a good clinical tool to characterize the ischemic lesion in patients suffering from stroke.