Caractérisation des microparticules dans les concentrés érythrocytaires : une analyse des antigènes de groupe sanguins par cytométrie de flux

Cette thèse a pour but de caractériser les microparticules isolées à partir des concentrés érythrocytaires et plus précisément de déterminer la présence d'antigènes de groupes sanguins à leur surface. Elle est divisée en trois parties, sous forme d'articles publiés à partir d'un travail de recherche mené au Centre de Transfusion Sanguine d'Epalinges. Dans l'article « Microparticles in stored red blood cells : an approach using flow cytometry and proteomic tools » publié dans Vox Sanguinis en 2008, il est question de la lésion de stockage des globules rouges. Grâce à des techniques alliant la cytométrie de flux et la protéomique, il a été montré que la génération de microparticules augmente au cours du stockage des concentrés érythrocytaires et que leur composition se modifie au cours du temps. L'article de revue « Analysis and clinical relevance of microparticles from red blood cells » publié dans Current Opinion in Hematology en 2010, explique les mécanismes de formation et d'élimination des microparticules de globules rouges. Il fait une revue des implications cliniques liées à la génération de microparticules et discute leur conséquences potentielles dans le domaine de la médecine transfusionnelle. L'article « Red blood cell microparticles and blood group antigens : an analysis by flow cytometry » publié dans Blood Transfusion en 2012, décrit l'étude des antigènes de groupe sanguins à la surface des microparticules générées à partir de concentrés érythrocytaires après ajout de calcium ionophore. Les résultats de cette étude indiquent que les antigènes de groupes sanguins appartenant aux systèmes RH, KEL, JK, FY, MNS, LE et LU sont présents à la surface des microparticules. Ces antigènes pourraient potentiellement être source d'allo-immunisation après transfusion.

Pre-analytical and methodological challenges in red blood cell microparticle proteomics.

Microparticles are phospholipid vesicles shed mostly in biological fluids, such as blood or urine, by various types of cells, such as red blood cells (RBCs), platelets, lymphocytes, endothelial cells. These microparticles contain a subset of the proteome of their parent cell, and their ready availability in biological fluid has raised strong interest in their study, as they might be markers of cell damage. However, their small size as well as their particular physico-chemical properties makes them hard to detect, size, count and study by proteome analysis. In this review, we report the pre-analytical and methodological caveats that we have faced in our own research about red blood cell microparticles in the context of transfusion science, as well as examples from the literature on the proteomics of various kinds of microparticles.

Produção de micropartículas de alginato contendo Flavobacterium columnare inativada pelo método de emulsão para vacinação de peixes por via oral

Alginate microparticles were prepared by an emulsion method aiming oral controlled release of antigens to fish. The effects of emulsification temperature and impeller type on particle morphology, average diameter, and size distribution were evaluated. Microparticles contaning formalin-killed Flavobacterium columnare cells (a model antigen) were prepared and characterized regarding bacterial release and particle stability when exposed to Nile tilapia (Oreochromis niloticus) typical gastrointestinal conditions. This methodology allowed the production of microparticles containing up to 14.3 g/L of bacterin, stable at a pH range from 2.0 to 9.0 for 12 h and smaller than 35 μm.

Utilização do acetato de celulose produzido a partir da celulose extraída do caroço de manga como matriz para produção de sistemas microparticulados

Cellulose acetate produced from mango seed fibers cellulose was used as a matrix for preparation of microparticles empty and load with acetaminophen (Paracetamol) in order to evaluate the incorporation of an active agent during the formation of microparticles. The microparticles are characterized by Fourier Transformed Infrared spectroscopy (FTIR), Differential Scanning Calorimetry (DSC) and Scanning Electron Microscopy (SEM). The incorporation of paracetamol can be confirmed by the change in value of glass transition temperature (Tg). The formation of microparticles spherical was observed by SEM and showed an average diameter of 1.010 and 0.950 mm for empty and load microparticles respectively.

Caractérisation et évaluation de textiles antifongiques

Hypothèse: L’impression sur textile d’une formulation de microparticules lipidiques avec un principe actif (éconazole nitrate) permet de conserver ou d’améliorer son activité pharmaceutique ex vivo et in vitro. Méthode: Une formulation de microparticules d’éconazole nitrate (ECN) a été formulée par homogénéisation à haut cisaillement, puis imprimée sur un textile LayaTM par une méthode de sérigraphie. La taille des microparticules, la température de fusion des microparticules sur textile et la teneur en éconazole du tissu ont été déterminées. La stabilité de la formulation a été suivie pendant 4 mois à 25°C avec 65% humidité résiduelle (RH). L’activité in vitro des textiles pharmaceutiques a été mesurée et comparée à la formulation commerciale 1% éconazole nitrate (w/w) sur plusieurs espèces de champignons dont le C. albicans, C. glabrata, C. kefyr, C. luminisitae, T. mentagrophytes et T. rubrum. La thermosensibilité des formulations a été étudiée par des tests de diffusion in vitro en cellules de Franz. L’absorption cutanée de l’éconazole a été évaluée ex vivo sur la peau de cochon. Résultats: Les microparticules d’éconazole avaient des tailles de 3.5±0.1 μm. La température de fusion était de 34.8°C. La thermosensibilité a été déterminée par un relargage deux fois supérieur à 32°C comparés à 22°C sur 6 heures. Les textiles ont présenté une teneur stable pendant 4 mois. Les textiles d’ECN in vitro ont démontré une activité similaire à la formulation commerciale sur toutes ii espèces de Candida testées, ainsi qu’une bonne activité contre les dermatophytes. La diffusion sur peau de cochon a démontré une accumulation supérieure dans le stratum corneum de la formulation textile par rapport à la formulation Pevaryl® à 1% ECN. La thermo-sensibilité de la formulation a permis un relargage sélectif au contact de la peau, tout en assurant une bonne conservation à température ambiante.

A zona de contato entre as formações Motuca e Sambaíba, Permo-Triássico da bacia do Parnaíba, regiões de Filadélfia (TO), Riachão (MA) e Loreto (MA)

O intervalo que compreende o final do Paleozóico e início do Mesozóico foi marcado por mudanças globais paleogeográficas e paleoclimáticas, em parte atribuídas a eventos catastróficos. A intensa continentalização do supercontinente Pangéia, com a implantação de extensos desertos, sucedeu os ambientes costeiros-plataformais do início do Permiano. Os registros desses eventos no norte do Brasil são encontrados nas bacias intracratônicas, particularmente na Bacia do Parnaíba, representados pela zona de contato entre as formações Motuca e Sambaíba. A Formação Motuca é constituída predominantemente por pelitos vermelhos laminados com lentes de gipsita, calcita e marga. Na porção leste da Bacia do Parnaíba, as fácies da Formação Motuca tornam-se mais arenosas com a ocorrência expressiva de arenitos com estratificação cruzada sigmoidal. A Formação Sambaíba consiste em arenitos de coloração creme alaranjada com estratificação plano-paralela e estratificação cruzada de médio a grande porte. Em geral, o contato entre as unidades é brusco, representado pela passagem de arenitos finos com laminação cruzada cavalgante e acamamento flaser/wavy da Formação Motuca para arenitos médios com falhas/microfalhas sinsedimentares e laminações convolutas da Formação Sambaíba. Foram individualizadas 14 fácies sedimentares, agrupadas em quatro associações: AF1 – Lacustre raso / Planície de lama (mudflat), AF2 – “Panela” salina (saline pan), AF3 – Lençol de areia e AF4 – Campo de dunas. A AF1 foi depositada dominantemente por processos de decantação em um extenso ambiente lacustre raso de baixa energia, influenciado por influxos esporádicos de areias oriundos de rios efêmeros. Este sistema lacustre foi, provavelmente, influenciado por períodos de contração e expansão, devido às variações das condições climáticas predominantemente áridas. Os mais expressivos períodos de contração ocorreram na porção oeste da Bacia do Parnaíba, representados pelo desenvolvimento de planícies de lama (mudflats) associadas a lagoas efêmeras saturadas em carbonatos e a “panelas” salinas (saline pans- AF2). Os lençóis de areia (AF3) são planícies arenosas extensas, localmente com área úmidas, intensamente retrabalhadas por processos eólicos. A AF4 é interpretada como parte de um erg composto por dunas/draas em zona saturada em areia, com interdunas secas subordinadas. Intervalos deformados lateralmente contínuos por centenas de quilômetros ocorrem na zona de contato entre as formações Motuca e Sambaíba. Pelitos com camadas contorcidas e brechadas (Formação Motuca) e arenitos com falhas/microfalhas sinsedimentares, laminação convoluta e diques de injeção preenchidos por argilitos (Formação Sambaíba) são interpretados como sismitos induzidos por terremotos de alta magnitude (>8 na escala Ritcher). Anomalias geoquímicas de elementos traços como Mn, Cr, Co, Cu e Ni na zona de contato entre as formações, juntamente com a presença de micropartículas de composição metálica na matriz argilosa dos sismitos, corroboram com impactos de meteoritos no limite c, possivelmente do astroblema Riachão.

Síntese e caracterização de micropartículas de etilcelulose e seu uso na encapsulação do fungicida tebuconazole

Pós-graduação em Química - IQ

Síntese e caracterização de hidrogéis de amido retrogradado e goma gelana utilizados como matriz em sistemas de liberação cólon específica de fármacos

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Preparação e Modificação Química de Nanoestruturas de Octa(aminopropil) silsesquioxano para Aplicações Analíticas

Pós-graduação em Ciência dos Materiais - FEIS

Hyperverzweigte Polyphenylene und redoxaktive Dendrimere

Die Doktorarbeit gliedert sich in zwei Abschnitte. Das erste Kapitel beschäftigt sich mit den hyperverzweigten Polyphenylenen. Es wurden Untersuchungen des Molekulargewichts in Abhängigkeit von der Monomerkonzentration und der Reaktionszeit durchgeführt. Die synthetisierten Polymere haben große Polydispersitäten, die durch fraktioniertes Fällen herabgesetzt werden können. Die Funktionalisierung der hyperverzweigten Polyphenylene mit unterschiedlichen Methoden führt zu verschiedenen Funktionen auf der Oberfläche der Polymere. Die chlormethylierten hyperverzweigen Polymere können als Makroinitiator für den Aufbau von Kern-Schale-Systemen genutzt werden. Mit Hilfe der ATRP-Polymerisation wurde Methylmethacrylat anpolymerisiert. Als Charakterisierungsmethode zur Bestimmung des freien Volumens findet die Positronenauslöschungsspektroskopie Anwendung. Im zweiten Teil der Arbeit stehen die Synthese und Charakterisierung von redoxaktiven Dendrimeren mit Triphenylamin- bzw. Naphthalinkern im Mittelpunkt. Den Einfluß der Dendrimerhülle auf die Redoxaktivität zeigen cyclovoltammetrische Untersuchungen. Die Zunahme der dendritischen Hülle führt zu einer Abschirmung des Redoxzentrums gegen die Elektrode und damit zu einer Inhibierung des Elektronentransfers. Das spiegelt sich in der Abnahme der Geschwindigkeitskonstanten sowie in der Ausdehnung der cyclovoltammetrischen Kurve wieder. Die Funktionalisierung der Triphenylamin-Dendrimere mit Chromophoren auf der Oberfläche führt zu einer Änderung der optische Eigenschaften, die mit Hilfe von Absorptions- und Emissionsmessungen untersucht wurden.

1-, 2- and 3-dimensional phenylene-based materials for optoelectronic applications

A series of oligo-phenylene dendronised conjugated polymers was prepared. The divergent synthetic approach adopted allowed for the facile synthesis of a range of dendronised monomers from a common intermediate, e.g. first and second generation fluorene. Only the polymerisation of the first generation and alkylarylamine substituted dendronised fluorene monomers yielded high molecular weight materials, attributed to the low solubility of the remaining dendronised monomers. The alkylarylamine substituted dendronised poly(fluorene) was incorporated into an organic light emitting diode (OLED) and exhibited an increased colour stability in air compared to other poly(fluorenes). The concept of dendronisation was extended to poly(fluorenone), a previously insoluble material. The synthesis of the first soluble poly(fluorenone) was achieved by the incorporation of oligo-phenylene dendrons at the 4-position of fluorenone. The dendronisation of fluorenone allowed for a polymer with an Mn of 4.1 x 104 gmol-1 to be prepared. Cyclic voltammetry of the dendronised poly(fluorenone) showed that the electron affinity of the polymer was high and that the polymer is a promising n-type material. A dimer and trimer of indenofluorene (IF) were prepared from the monobromo IF. These oligomers were investigated by 2-dimensional wide angle x-ray spectroscopy (2D-WAXS), polarised optical microscopy (POM) and dielectric spectroscopy, and found to form highly ordered smetic phases. By attaching perylene dye as the end-capper on the IF oligomers, molecules that exhibited efficient Förster energy transfer were obtained. Indenofluorene monoketone, a potential defect structure for IF based OLED’s, was synthesised. The synthesis of this model defect structure allowed for the long wavelength emission in OLED’s to be identified as ketone defects. The long wavelength emission from the indenofluorene monoketone was found to be concentration dependent, and suggests that aggregate formation is occurring. An IF linked hexa-peri-hexabenzocoronene (HBC) dimer was synthesised. The 2D-WAXS images of this HBC dimer demonstrate that the molecule exhibits intercolumnar organisation perpendicular to the extrusion direction. POM images of mixtures of the HBC dimer mixed with an HBC with a low isotropic temperature demonstrated that the HBC dimer is mixing with the isotropic HBC.

Multimetallic complexes based on phosphine- and phosphine oxide- appended p-hydroquinones

Presented here, is the work done with a series of binucleating ligands based on phosphine and phosphine oxide appended p-hydroquinones and their reactions towards various metals sources. The long term goal of the project was to produce coordination polymers that would have novel electronic, magnetic, and optical properties which would be of use in the field of molecular electronics. Binucleating ligands contained a p-hydroquinone motif in which various phosphine- and phosphine oxide substituents have been placed in the ortho position relative to each of the hydroxy position were synthesized. A previously published synthetic method for such lugands utilized n-BuLi to form a phenyl lithium intermediate which was quenched with chlorodiphenylphosphine. This technique was also used to produce a ligand with diisopropylphosphine groups. Phosphine ligands, containing the same structural motif, were also generated using LDA as the lithiating agent. This technique was found to be higher yielding. Phosphine chalcogenide ligands were accessed by further oxidizing the low valent phosphorous centers with either hydrogen peroxide or with elemental sulfur. These ligands were characterized using multinuclear NMR, low and high resolution mass spectroscopy, FTIR, and single crystal X-ray diffraction. Their electrochemical properties were explored with cyclic voltammetry. The phosphine appended ligands were used in the synthesis of a several bimetallic complexes. It was found that the ligands readily reacted with NiCp2 and NiCp*2, displacing one of the cyclopentadiene (Cp) or pentamethylcyclopentadiene (Cp*) rings. A cyclopentadiene complexes, containing diisopropylphine, was readily oxidized by[FeCp2]PF6 to give a NMR silent mixed valence complex. Cyclic voltammetry of these complexes showed a number of reversible waves with a large potential separation. The mixed valence compounds also showed a large absorbance band in the NIR region which was assigned to be an intervalence charge transfer. The cyclic voltammetry and NIR spectroscopy suggest that these systems are very capable of efficient metal-to-metal charge transfer. These complexes were characterized by multinuclear NMR, single crystal X-ray diffraction, UV/VIS-NIR spectroscopy and elemental analysis. The phosphine oxide ligands were reacted with a variety of different metal sources but limited success was gained in obtaining single crystals, allowing structural characterization of these compounds. Single crystals were obtained from products generated by reacting the diphenylphosphine oxide ligand with (Bipy)Cu(NO3)2 and Cu(NO3)2. In all cases the ligand had been further oxidized to a 2,5-dihydroxy-1,4-benzoquinone motif. In the reaction between the diphenylphosphine oxide ligand and (Bipy)Cu(NO3)2 it was found that the phosphine oxide moiety was involved with intermolecular coordination leading to the formation of a one-dimensional polymer composed of a series of bimetallic complexes tethered together. When NaSbF6 was present in the reaction with (Bipy)Cu(NO3)2 a unique tetrametallic complex was formed. Here the phospine oxide moiety was oriented so that two bimetallic complexes were bound together. If only Cu(NO3)2 was present, a two-dimensional polymeric sheet was formed where the ligand was present in two different coordination modes. The electronic properties of these complexes remained to be assessed.

Destruction, Alteration, Simples and World-Stuff

When a tree is chopped to bits, or a sweater unraveled, its matter still exists. Since antiquity, it has sometimes been inferred that nothing really has been destroyed: what has happened is just that this matter has assumed new form. Contemporary versions hold that apparent destruction of a familiar object is just rearrangement of microparticles or of 'physical simples' or 'world stuff'. But if destruction of a familiar object is genuinely to be reduced to mere alteration of something else, we must identify an alternation proper to the career, the course of existence, of this something else; relatedly, the alteration must be characterizable without asserting the existence of the familiar object. All contemporary views fail one of these requirements.

Particle concentration and size distribution in the NGRIP ice core

A novel laser microparticle detector used in conjunction with continuous sample melting has provided a more than 1500 m long record of particle concentration and size distribution of the NGRIP ice core, covering continuously the period approximately from 9.5-100 kyr before present; measurements were at 1.65 m depth resolution, corresponding to approximately 35-200 yr. Particle concentration increased by a factor of 100 in the Last Glacial Maximum (LGM) compared to the Preboreal, and sharp variations of concentration occurred synchronously with rapid changes in the delta18O temperature proxy. The lognormal mode µ of the volume distribution shows clear systematic variations with smaller modes during warmer climates and coarser modes during colder periods. We find µ ~ 1.7 µm diameter during LGM and µ ~ 1.3 µm during the Preboreal. On timescales below several 100 years µ and the particle concentration exhibit a certain degree of independence present especially during warm periods, when µ generally is more variable. Using highly simplifying considerations for atmospheric transport and deposition of particles we infer that (1) the observed changes of µ in the ice largely reflect changes in the size of airborne particles above the ice sheet and (2) changes of µ are indicative of changes in long range atmospheric transport time. From the observed size changes we estimate shorter transit times by roughly 25% during LGM compared to the Preboreal. The associated particle concentration increase from more efficient long range transport is estimated to less than one order of magnitude.