Les particules superparamagnétiques constituent un outil puissant pour de nombreuses applications biomédicales, ce potentiel est souvent restreint à cause de leur stabilité limitée dans les milieux biologiques ou du piégeage orientationnel sous champ magnétique. Dans cette thèse, ces problèmes ont été résolus en créant une nouvelle génération de particules à interfaces liquides fonctionnalisées. Ces particules sont formulées en utilisant des émulsions de ferrofluides qui incorporent des phospholipides fonctionnalisés, notamment biotinylés pour permettre la capture de streptavidine. La taille est contrôlée grâce à la microfluidique, permettant la production d'émulsions uniformes. L'utilisation de streptavidine fluorescente révèle que la capture est influencée par les propriétés du cosurfactant et du ligand ainsi que par le nombre de ligands disponibles. La mobilité des ligands est démontrée par l'adhésion observée entre les gouttelettes liées par de la streptavidine et le mouvement des billes couvertes de streptavidine capturées à l'interface. Enfin, le potentiel de ces particules est exploré en créant un dosage pour le diagnostic. La présence d'analytes en solution est indiquée par l’agglutination. Dans ce travail l'agglutination est provoquée par la complexation entre des émulsions biotinylés et  la streptavidine (ou des billes couvertes de streptavidine). L’utilisation de gouttelettes de taille calibrée permet de compter avec précision des agrégats spécifiques par cytométrie de flux, la limite de détection étant dans la gamme femtomolaire. Cela surpasse la gamme picomolaire atteint généralement par des billes solides. / Colloidal superparamagnetic particles are a powerful tool in biotechnology, yet their applications are often hindered by limited stability in biological media or by orientation trapping under applied magnetic fields. In this thesis, these problems are addressed by developing novel magnetic particles bearing ligands at a liquid interface. Magnetic particle analogues are formulated using ferrofluidic emulsions, which incorporate functionalised phospholipids. Droplet size is controlled using microfluidic membrane emulsification to produce highly uniform populations. Ligands are modelled using biotinylated lipids, permitting the capture of streptavidin at the droplet interface. Fluorescently labelled proteins reveal that capture efficiency is influenced by the cosurfactant interfacial activity and the polymer spacer length of the ligand. Overall, capture saturation is found to be related to the number of ligands available at the interface. Ligand mobility is demonstrated by the formation of adhesion plaques between streptavidin cross-linked droplets and the motion of streptavidin coated beads caught at the interface. Finally, an application is explored by creating a new immunoassay. Polyvalent proteins or beads crosslink ligand functionalised droplets forming aggregates. Using size calibrated droplets specific aggregates can be accurately counted using flow cytometry and the limit of detection is found to be in the femtomolar range, this surpasses the picomolar range typically achieved using solid beads.

Mesenchymal stem cells (MSCs) derived from human adipose tissue and umbilical cord (Wharton’s jelly, UCB) represent a useful source of adult stem cells for cellular therapy and tissue engineering. The biggest concern with the use of MSCs therapeutically relates to their isolation and growth/manipulation ex vivo. This study aimed to establish methods for the routine isolation and characterization of MSCs from human tissues. The objectives were (1) to show that MSCs could be isolated from different human tissues, namely adipose tissue, Wharton’s jelly, and UCB; (2) to confirm the MSC phenotypic profile over at least 10 passages; and (3) to show the multilineage differentiation capacity of the isolated cells. The minimal criteria as defined by the International Society for Cellular Therapy (ISCT) were used to determine whether MSCs were successfully isolated from various human tissues. Two different techniques involving enzymatic digestion or explant cultures were utilized, and compared for isolating MSCs from Wharton’s jelly. Umbilical cord blood has been suggested as another source of MSCs. However, we were unable to grow MSCs from UCB. Proliferation kinetics of isolated MSCs revealed that cords, either from digested cords or cord pieces had a mean PDT from passage 1 to 4 that was approximately 3 fold lower than for the ASCs. Mesenchymal stem cells from adipose tissue and Wharton’s jelly expressed the classical MSC phenotype (CD73+, CD90+, CD105+, CD34-, and CD45-). The cells from Wharton’s jelly showed a more uniform MSC profile over passages, with higher levels of marker expression when compared to ASCs. Variability in phenotype was observed in early ASC passages, whereas WJ-MSCs seemed to attain the MSC phenotype as early as passage 0 for both isolation techniques. Low levels of CD34 positive cells remained in the ASCs. Oil red O staining was used for identifying the lipid droplets in adipogenic differentiation cultures. A colorimetric assay as well as image analysis was used to quantify the differentiation. For the cord samples, both assays produced positive results. Histological examination, however, revealed that the cords did not form lipid droplets. The ASCs showed a statistically significantly greater differentiation capacity into adipocytes compared with the cords (pooled digested and pieces data). Alizarin red S staining was used for identifying calcium deposition during matrix mineralization in osteogenic differentiation cultures. No significant differences in osteogenic differentiation were observed between ASCs and WJ-MSCs. Chondrogenic differentiation was observed for both MSC sources by positive staining of glycosaminoglycans using toluidine blue O. The main findings of the study showed that MSCs, according to the ISCT guidelines, were successfully harvested from adipose tissue. However, due to the lack of adipogenic differentiation of WJ-derived cells, they did not meet the ISCT guidelines to be classified as MSCs, and were referred to as MSC-like cells. Regardless of the isolation technique used, Wharton’s jelly yielded cells with similar proliferation capacity, phenotype, and differentiation capacity. This study did, however, reveal that biological differences do exist between stem cells from different sources. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Immunology / unrestricted

La nouvelle distribution et classification des Coffea (124 espèces Psilanthus incluses, en Afrique, îles de l‟océan Indien (IOI), Inde, Asie du Sud-Est et Australasie) pose la question de leur origine. Des données sont disponibles pour les caféiers africains mais pas pour les Mascarocoffea (caféiers des IOI représentant la moitié des espèces du genre). Les objectifs de cette thèse sont i)- estimer la divergence morphologique, génétique (13 SSR) et génomique des Mascarocoffea, ii)- établir la phylogénie moléculaire du genre (20 COSII) et iii) reconstituer l‟histoire évolutive des caféiers.La diversité morphologique bien que sous forme de continuum, permet la caractérisation de groupements et de stratégies reproductives originales. Les tailles de génome varient de 0,96 à 1,41 pg/2C, avec tendance à l‟augmentation du Nord au Sud-Est de Madagascar.Les nombres moyens d‟allèles par locus et par grande région sont élevés et 20% des allèles totaux sont partagés par ces régions. La phylogénie moléculaire place Psilanthus à la base de l‟arbre et indique une nette divergence entre les Mascarocoffea et les espèces africaines. Il n‟y a pas de structuration hiérarchisée indiquant une divergence indépendante de chaque grand clade.Nos résultats et des données de la littérature nous permettent de proposer un scenario de l‟histoire évolutive des caféiers dont l‟origine est non africaine. Deux centres de diversification primaire sont mis en évidence, situé l‟un en Afrique dans la région Kenya – Ethiopie (à l‟origine de diversifications secondaires vers le Centre, l‟Ouest et l‟Est) et l‟autre dans le Nord Madagascar (à l‟origine des diversifications secondaires à Madagascar et Maurice). / The new classification and repartition of the genus Coffea (124 species including ex-genus Psilanthus native to Africa, Indian Ocean islands (IOIs), India, southeast Asia and Australasia) rises the question on its origin. Genetic and cytogenetic data are available for African species but not for Mascarocoffea (coffees from IOIs including half part of the species). The aims of this thesis were i) to estimate the morphologic, genetic (13 SSRs) and cytogenetic divergence of and among Mascarocoffea; ii) to produce a molecular phylogeny and iii) to reconstruct the coffees evolutionary history.Although the global morphological diversity appears as a continuum, it permits the identification of groupings and obvious specific reproductive strategies. Genomes size varied from 0.96 to 1.41 pg/2C with an increasing global trend from north to southeast Madagascar.Average alleles number per locus is high whatever the region and 20% total alleles are shared by African and IOIs coffees.The molecular phylogeny shows a basal position for Psilanthus, clear divergence between African and IOIs species. Clearly, IOIs clade is not nested within the African. Our results and information from other works permit us to elaborate a new scenario for the evolutionary history of coffees. The centre of origin should be located in India. Then two primary centres of diversification occurred; one in Kenya- Ethiopia, Africa the other in north Madagascar. From these centres, migrations towards centre and west and towards east Africa led to secondary centres of diversification while in Madagascar, migrations towards southeast and towards the other islands led to radiative speciation.

Philosophiae Doctor - PhD / Natural products have been a source of many pharmaceutical drugs and a number of drugs that are currently used in the treatment of cancer are derivatives of compounds originally isolated from natural products. There is evidence that extracts of Tulbaghia violacea can be used to treat cancer. The activation of apoptosis in cancer cells is a target for the development of novel anti-cancer drugs since one of the characteristics of cancer cells is resistance to apoptosis due to the deregulation of biochemical pathways leading to apoptosis. In fact, many current anti-cancer drugs exert their effects through the activation of apoptosis. Previous studies showed that extracts of T.violacea induce apoptosis in cancer cells and one study reported on the isolation of a compound (methyl-ԃ-D-glucopyranoside), which is responsible for the pro-apoptotic activity of the T.violacea extract. Therefore the aim of this study was to investigate the anti-cancer activity of methyl-ԃ-Dglucopyranoside and extracts prepared from T.violacea. In this study the pro-apoptotic activity of methyl-ԃ-D-glucopyranoside and extracts prepared from T.violacea were investigated on a panel of human cancer cell lines, which included HepG2, MCF7, H157, HT29 and the non-cancerous cell line, KMST6. The induction of apoptosis was evaluated by flow cytometry using several bioassays which measures biochemical events (caspase activation, phosphatidylserine externalisation and reactive oxygen species (ROS) production that is associated with the induction of apoptosis. The results demonstrated that the effects of methyl--D-glucopyranoside on cultured cells are transient and that the cells recover from the effects of methyl--D-glucopyranoside. This suggested thatmethyl-ԃ-D-glucopyranoside is not the compound responsible for the pro-apoptotic bioactivity in the T.violacea extract. This study also showed that cytotoxic and pro-apoptotic bioactivity of the leaf-extract was significantly higher in comparison to the tuber-extract. The bioactivity of the organic solvent extracts (dichloromethane, hexane, methanol and 50% methanol/water) of T.violacea leaves was also significantly higher than water extracts of T.violacea leaves. A comparison of the different organic extracts prepared from the T.violacea leaves showed that the highest activity was observed for the dichloromethane and hexane extracts. In an effort to identify the bioactive compound(s) the dichloromethane extract was subjected to Versaflash® column chromatography. However, due to problems experienced with the solubility of the dichloromethane sub-fractions, these compounds could not be tested for their bioactivity. Palmitone (16-hentriacontanone) was identified as one of the major compounds present in the dichloromethane sub-fractions. This compound was previously shown to have anticonvulsant bioactivity but there is no evidence in the literature that it has anti-cancer or pro-apoptotic activities. Fingerprinting of the methanol extract showed the presence of long chain fatty acid derivatives, flavonoids and allicin derivatives in the methanol extract. Although, this study failed to isolate the pro-apoptotic bioactive compound(s) present in the extracts of T.violacea, it confirmed that extracts of this plant induce apoptosis in cultured human cancer cell lines.

Le développement des bactéries sporulées dans les aliments peut être responsable d’intoxication alimentaire ou d’altérations des produits. Trois leviers ont été identifiés pour prévenir le développement de ce microbiote : les conditions de sporulation, l’intensité du traitement appliqué pour inactiver les spores et les conditions d’incubation. Ce travail de thèse a pour objectif (i) de quantifier l’impact des conditions de sporulation, de traitement et d’incubation sur la capacité des spores à former des colonies, et (ii) de quantifier l’impact des conditions de sporulation, de traitement et d’incubation sur les cinétiques de germination et de reprise de croissance. Dans un premier temps, un modèle mathématique a été développé pour décrire et quantifier l’impact des conditions d’incubation sur la capacité des spores à former des colonies après un traitement thermique. Ce modèle intègre uniquement des paramètres physiologiques, les limites de croissance des souches étudiées. La germination et la reprise de croissance est un processus complexe au cours duquel les spores passent par plusieurs stades successifs : spores dormantes, spores germées et cellules végétatives. Afin de quantifier l’impact des conditions de sporulation, de traitement thermique et d’incubation sur chacun de ces stades, une méthode par cytométrie en flux a été développée. Elle a permis de suivre l’évolution de chaque stade au cours du temps et un modèle primaire a été proposé afin de décrire l’évolution de chacun de ces stades. A partir de ce modèle il a été possible de décrire l’impact des différentes conditions de sporulation, de traitement thermique et d’incubation sur cette évolution et un modèle secondaire a été développé pour quantifier l’impact de ces facteurs sur les cinétiques de germination et de reprise de croissance. Afin de corréler les différences de comportement avec la composition protéique des spores, une analyse protéomique a été réalisée sur des spores produites dans différentes conditions. Ces travaux permettent de mieux appréhender le comportement de germination et de reprise de croissance des spores bactériennes. De plus, les résultats apportés ainsi que les modèles mathématiques développés dans cette thèse pourront permettre de mieux contrôler le développement des bactéries sporulées en industrie agro-alimentaire, connaissant l’impact des conditions de stockage et de formulation des produits, comme la température et le pH, sur le comportement des spores. / The development of spore forming bacteria in foods can be responsible for food poisoning or food spoilage. Three levers allowing the development of this microbiota were identified: the conditions of sporulation, the conditions of heat treatment and the conditions of incubation. This PhD work objectives were (i) to quantify the impact of sporulation conditions, heat treatment intensity and recovery conditions of the ability of spores to form colonies, and (ii) to quantify the impact of sporulation conditions, heat treatment intensity and recovery conditions on germination and outgrowth kinetics. Firstly, a mathematical model was developed to describe and quantify the impact of recovery conditions on the spore ability to form colonies a heat treatment. This model integrated only physiological parameters, the growth limits. The germination and outgrowth is a complex process made of successive physiological stages the spores pass through: the dormant spores, the germinated spores and the vegetative cells. A flow cytometry method was developed in order to quantify the impact of sporulation conditions, the heat treatment intensity and the incubation conditions on each physiological stage. This method allowed monitoring the evolution of each stage over time and a primary model was proposed to describe these evolutions. Thanks to this model, the impact of sporulation conditions, the heat treatment intensity and the incubation conditions were quantified and a secondary model was developed to quantify the impact of these factors on germination and outgrowth kinetics. In order to correlate the differences of behavior with the proteome of spores, proteomic analysis were performed on spores produced in different conditions. This work allows a better comprehension of germination and outgrowth behavior. Moreover, the results and the mathematical models provided by this work can be applied in food industry to improve the control of spores forming bacteria development knowing the impact of storage conditions and the product formulation, like temperature and pH, on spore behavior.

Long term management plans for restoration of natural flow conditions through the Everglades increase the importance of understanding potential nutrient impacts of increased freshwater delivery on coastal biogeochemistry. The present study sought to increase understanding of the coastal marine system of South Florida under modern conditions and through the anthropogenic changes in the last century, on scales ranging from individual nutrient cycle processes to seasonal patterns in organic material (OM) under varying hydrodynamic regime, to century scale analysis of sedimentary records. In all applications, carbon and nitrogen stable isotopic compositions of OM were examined as natural recorders of change and nutrient cycling in the coastal system. High spatial and temporal variability in stable isotopic compositions were observed on all time scales. During a transient phytoplankton bloom, ä15N values suggested nitrogen fixation as a nutrient source supporting enhanced productivity. Seasonally, particulate organic material (POM) from ten sites along the Florida Reef Tract and in Florida Bay demonstrated variable fluctuations dependent on hydrodynamic setting. Three separate intra-annual patterns were observed, yet statistical differences were observed between groupings of Florida Bay and Atlantic Ocean sites. The POM ä15N values ranged on a quarterly basis by 7‰, while ä13C varied by 22‰. From a sediment history perspective, four cores collected from Florida Bay further demonstrated the spatial and temporal variability of the system in isotopic composition of bulk OM over time. Source inputs of OM varied with location, with terrestrial inputs dominating proximal to Everglades freshwater discharge, seagrasses dominating in open estuary cores, and a marine mixture of phytoplankton and seagrass in a core from the boundary zone between Florida Bay and the Gulf of Mexico. Significant shifts in OM geochemistry were observed coincident with anthropogenic events of the 20th century, including railroad and road construction in the Florida Keys and Everglades, and also the extensive drainage changes in Everglades hydrology. The sediment record also preserved evidence of the major hurricanes of the last century, with excursions in geochemical composition coincident with Category 4-5 storms.

Le striatum est une région du cerveau impliquée dans d'importantes fonctions physiologiques telles que l'apprentissage par renforcement ou le contrôle du mouvement, mais aussi dans des pathologies comme l'addiction. Le fonctionnement de ce système s'appuie sur deux types de neurones de projection, appelés " neurones épineux de taille moyenne ". Les uns expriment le récepteur de la dopamine de type 1 (D1R) et les autres expriment le récepteur de type 2 (D2R). L'objectif de cette thèse est de caractériser ces deux types de neurones au niveau épigénétique, en conditions basales et après traitement à la cocaïne. Il a été nécessaire de développer de nouvelles méthodes utilisant la cytométrie de flux pour distinguer les populations de neurones exprimant D1R ou D2R. La première méthode utilise des souris transgéniques L10a-GFP et du tissu non fixé, la seconde répond aux limitations de la précédente et utilise du tissu fixé. Nous avons montré que la cocaïne régule de nombreuses modifications post-traductionnelles d'histones, de façon spécifique de populations neuronales. Par ailleurs, nous avons identifié plus d'une centaine de gènes différemment méthylés ou hydroxyméthylés entre les deux types neuronaux. Certains gènes sont déjà connus pour avoir un rôle fonctionnel important dans l'une des populations. La comparaison des neurones exprimant D1R ou D2R est un bon modèle pour explorer les liens entre méthylation de l'ADN, hydroxyméthylation et transcription. Par exemple, nous observons une association très claire entre l'augmentation de la méthylation de l'ADN et la répression de la transcription, ainsi qu'une corrélation entre modifications de méthylation et d'hydroxyméthylation. / The striatum is a brain region implicated in physiological functions such as reinforcement learning or movement selection but also in pathologies such as addiction or Parkinson’s disease. It relies on two types of projecting neurons, named “medium spiny neurons” because of their morphology. They are very similar but have a complementary and opposite role. One type expresses the dopamine receptor type 1 (D1R) and the other type expresses the dopamine receptor type 2 (D2R). The aim of this work was to characterize this two neuronal types epigenetically, in basal conditions and after cocaine treatment. We have developed new flow cytometry techniques to be able to distinguish the two cell types. The first method uses transgenic L10-eGFP mice and fresh tissue, the second one goes beyond the limitations of the first one and uses fixed tissue. We have shown that cocaine regulates many post-translational histone modifications, dynamically, and differently between the two populations. Moreover, we have identified more than 100 genes differentially methylated or hydroxymethylated between the two neuronal types. Some of these genes are already known for having a functional role in one of the populations. The comparison between D1R and D2R neurons is a good model to explore the links between DNA methylation, hydroxymethylation and transcription. For example, we have observed a strong association between an increase in DNA methylation and a transcriptional repression, as well as a correlation between DNA methylation and hydroxymethylation.

La qualité de l'eau est une préoccupation majeure pour la santé publique et la préservation de l'environnement. Nous proposons d’utiliser les phages comme des biosenseurs pour détecter les pathogènes humains ou animaux présents dans les eaux de surface, un outil nommé Phagosensor.La mise au point d’un phagosensor prototype a permis d’optimiser la détection des bactéries par cytométrie en flux. Les résultats montrent que cet outil est extrêmement rapide, sensible, et très spécifique. L’outil phagosensor permet de détecter des bactéries cibles présentes dans un environnement complexe tel que l’eau de mer.Le prototype fonctionnel a servi de base à la construction de phagosensors spécifiques de bactéries pathogènes. Les résultats montrent que la stratégie développée à partir du prototype peut être rapidement transposée à la détection de pathogènes tel que Salmonella. Le séquençage de novo et l’annotation des génomes de trois phages isolés de l’environnement permettront la construction de nouveaux phagosensors spécifiques de souches d’E. coli pathogènes.Cette stratégie a été adaptée à la détection d’un signal luminescent post-infection en utilisant les phages recombinants portant l’opéron luxCDABE. Les bactéries infectées sont rapidement détectables.L’ensemble de ces résultats démontre que la stratégie développée est applicable à la construction de phagosensor sur demande pour une détection rapide, sensible et spécifique des bactéries d’intérêt que ce soit en fluorescence ou en luminescence. La détection dans l’eau de mer suggère, qu’à terme, des outils pourront être conçus pour la détection de bactéries pathogènes dans d’autres matrices telles que le sang ou les aliments. / Water quality is a major concern for public health and natural environment preservation. We propose to use phages to develop biosensor tools able to detect human and animal pathogens present in water. The construction of a phagosensor prototype using an optimized genetic engineering strategy, infection and detection conditions, allowed the specific detection of bacteria. The results show that detection is fast, specific and highly sensitive. Moreover, the phagosensor tool detects target bacteria in a complex environment such as seawater. Phagosensors specific of pathogenic bacteria were constructed following the strategy developed for the prototype. Results show that the strategy we designed can be successfully transposed to detect pathogens such as Salmonella. De novo sequencing and genomes annotation of three phages isolated from the environment were carried out to develop phagosensors that are specific of pathogenic E. coli. This technology was then adapted to detect a luminescent signal arising post-infection using genetically modified phages carrying the entire luxCDABE operon. The bacteria infected with the lux recombinant phages were rapidly detected by luminescence emission. Together, these results demonstrate that our technology can be applied to construct various phagosensors adapted to the detection of different bacterial species of interest and using at least two output signals. These tools allow a rapid, specific and highly sensitive detection that are close to the European guideline. Efficient bacterial detection in seawater suggests that phagosensors could be developed to detect pathogenic bacteria in other matrices such as blood or food.

Orientadores: Nicola Amanda Conran Zorzetto, Fernando Ferreira Costa / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciencias Medicas / Made available in DSpace on 2018-08-14T12:14:33Z (GMT). No. of bitstreams: 1 Proenca-Ferreira_Renata_M.pdf: 950238 bytes, checksum: 21ee0800d218d95b35b72ea1eeaab7e3 (MD5) Previous issue date: 2009 / Resumo: Anemia falciforme (AF) é uma doença causada por uma mutação de ponto, que resulta na formação de hemoglobina S (HbS). A polimerização de HbS desoxigenada resulta na deformação, enrijecimento e fragilização das células vermelhas, anemia hemolítica e eventos vasos-oclusivos, principal causa de morbidade nos pacientes com AF. A adesão anormal das células brancas e vermelhas ao endotélio diminui o fluxo de sangue na circulação micro-vascular, principal fator envolvido na vaso-oclusão. O objetivo deste trabalho foi comparar as propriedades adesivas de plaquetas de indivíduos sadios (AA) com as plaquetas de pacientes com AF (SS) e em terapia com hidroxiuréia (HU), e quais moléculas de adesão e sinalização estão envolvidas nesta adesão. A adesão basal de plaquetas ao fibrinogênio (FB) do grupo de pacientes SS foi significativamente maior em relação às plaquetas AA, entretanto, as plaquetas de pacientes SSHU mostraram uma adesão similar às plaquetas AA. As plaquetas AA, SS e SSHU quando estimuladas com trombina (TB), apresentaram uma adesão significativamente maior em relação às suas adesões basais. Por outro lado, não houve diferenças significativas entre as adesões basais das plaquetas AA, SS e SSHU ao colágeno como ligante. A citometria de fluxo foi utilizada para comparar a expressão e ativação das principais moléculas de adesão nestas plaquetas, e identificou-se um aumento de expressão da integrina aIIbß3 na sua conformação de ativação, na superfície das plaquetas SS, em relação às plaquetas AA e SSHU. A molécula P-selectina (CD62P) foi encontrada com maior expressão também na superfície das plaquetas SS. Ensaios de adesão utilizando anticorpos específicos para as moléculas de adesão indicaram um possível papel para a integrina aIIbß3 na adesão de plaquetas ao FB. O AMPc é um importante inibidor de ativação plaquetária, e os níveis intraplaquetários de adenosina manofosfato cíclica (AMPc) das plaquetas SS foram significativamente menores em relação às plaquetas AA e SSHU. A co-incubação das plaquetas com TB reduziu significativamente os níveis de AMPc nas plaquetas AA, e SSHU, e nas plaquetas SS essa redução não foi significativa. Além disso, foi interessante notar que os níveis de hemoglobina fetal (HbF) em pacientes SS e SSHU apresentaram uma significativa correlação com os níveis de AMPc. Em relação aos níveis intraplaquetários de guanosina monofosfato cíclica (GMPc), importante inibidor de agregação plaquetária, das plaquetas AA, SS e SSHU não houve diferenças estatisticamente significativas, entretanto com estímulo de trombina houve um aumento significativo de GMPc nas plaquetas AA. A incubação das plaquetas com o cilostazol (inibidor específico da fosfodiesterase 3A, PDE3A) levou a uma redução da adesão de plaquetas SS, e sugere-se que as plaquetas SS possuem a atividade de fosforilação da PDE3A aumentada, e esteja relacionada com a degradação de AMPc nessas plaquetas. Além disso, dados indicam que as vias de sinalização dependentes de PKA, PKG e PKC não estão envolvidas na adesão alterada das plaquetas SS. A atividade funcional nos ensaios com agregação plaquetária de pacientes com AF está alterada, mas será necessário que novos experimentos sejam realizados para maiores conclusões. Os resultados sugerem que as plaquetas de pacientes AF circulam num estado ativado e que elas possuem maior capacidade de aderirem às proteínas que podem ser encontradas na matriz extracelular (MEC) e na superfície da parede vascular. Esta adesão aumentada está associada com os níveis diminuídos de AMPc intraplaquetários e ativação da integrina aIIbß3. Estas mesmas alterações parecem ser revertidas nos pacientes em uso de HU. Resultados sugerem que as plaquetas podem ter um papel importante no processo de vaso-oclusão. Quando estão ativadas, estas plaquetas servem como fonte importante para mediadores inflamatórios que, por sua vez, podem levar à exacerbação da inflamação e ativação celular no local. / Abstract: Sickle cell disease (SCD) is caused by a point mutation that results in the formation of Hemoglobin S (HbS). The polymerization of deoxygenated HbS causes deformation of red cells, which then adopt a sickle shape and become rigid and fragile. Hemolytic anemia and vaso-occlusive events are the main cause of morbidity in SCD; abnormal adhesion of white and red cells to the endothelium decreases the blood flow in the microcirculation and appears to be the main factor involved in vaso-occlusion. The objective of this study was to compare the adhesive properties of platelets from healthy individuals (AA platelets) with those of platelets from SCD patients (SS platelets) and from patients on HU therapy (SSHU platelets), as well as the adhesion molecules and signaling pathways that may be involved in this adhesion. The basal adhesion of SS platelets to fibrinogen (FB) was significantly higher than that of AA platelets; in contrast, SSHU platelets demonstrated a similar adhesion to that of AA platelets. Platelets from AA, SS and SSHU individuals, when stimulated with thrombin (TB), all presented significantly higher adhesions when compared with their basal adhesions. In contrast, there were no significant differences between the adhesions of AA, SS and SSHU platelets when collagen was used as a ligand. Flow cytometry was utilized to compare the expression and activation of the main adhesion molecules on AA, SS and SSHU platelets and identified a significantly higher expression of the ?IIb?3 integrin in its activated conformation on the surface of the SS platelets, in relation to the AA and SSHU. Expression of the P-selectin adhesion molecule (CD62P) was also found to be increased on the surface of SSHU platelets. Adhesion assays utilizing specific antibodies for the activated ?IIb?3 and P-selectin indicated a possible role for the ?IIb?3 integrin in the adhesion of platelets to FB. The cyclic nucleotide, cyclic adenosine monophosphate (cAMP) is an important inhibitor of platelet aggregation. Intraplatelet levels of cAMP were found to be significantly lower in SS platelets compared to AA and SSHU platelets. Co-incubation of platelets with TB significantly reduced levels of cAMP in the AA and SSHU platelets, but in SS platelets this decrease was not significant. Interestingly, levels of fetal hemoglobin (HbF) in SCD patients (both SS and SSHU) were found to correlate significantly with levels of platelet AMPc. With regard to intraplatelet levels of cyclic guanosine monophosphate (cGMP), there were no significant differences found between AA, SS and SSHU platelets, however following a TB stimulus AA platelets demonstrated a significant increase in intracellular cGMP. Phosphodiesterase 3A (PDE3A) is the main cyclic nucleotide hydrolyzing enzyme in platelets. Incubation of SS platelets, but not AA or SSHU platelets, with cilostazol (specific inhibitor of PDE3A) resulted in a significant reduction in their adhesion to FB, suggesting that PDE3A activity in SS platelets may be activated and that augmentation of cAMP is capable of reverting increased SS platelet adhesion. Additional experiments indicate that Protein kinase A (PKA), PKG and PKC dependent signaling pathways are not involved in the altered adhesion of SS platelets. The functional activity of SS platelets was found to be altered in platelet aggregation assays, however further experiments are required to draw conclusions from these data. Results of this study suggest that platelets from SCD individuals circulate in an activated state and that they present a greater ability to adhere to proteins that may be encountered on the vessel wall. This augmented adhesion is associated with decreased levels of intraplatelet cAMP and activation of the ?IIb?3 integrin. These alterations appear to be reversed in patients on HU therapy. Results suggest that platelets may have an important role in the vaso-occlusive process.When activated these cells are an important source of inflammatory mediators that may, in turn, result in an exacerbation of cellular activation at the vaso-occlusive site. / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Flow Cytometry analyzers have become trusted companions due to their ability to perform fast and accurate analyses of human blood. The aim of these analyses is to determine the possible existence of abnormalities in the blood that have been correlated with serious disease states, such as infectious mononucleosis, leukemia, and various cancers. Though these analyzers provide important feedback, it is always desired to improve the accuracy of the results. This is evidenced by the occurrences of misclassifications reported by some users of these devices. It is advantageous to provide a pattern interpretation framework that is able to provide better classification ability than is currently available. Toward this end, the purpose of this dissertation was to establish a feature extraction and pattern classification framework capable of providing improved accuracy for detecting specific hematological abnormalities in flow cytometric blood data. This involved extracting a unique and powerful set of shift-invariant statistical features from the multi-dimensional flow cytometry data and then using these features as inputs to a pattern classification engine composed of an artificial neural network (ANN). The contribution of this method consisted of developing a descriptor matrix that can be used to reliably assess if a donor’s blood pattern exhibits a clinically abnormal level of variant lymphocytes, which are blood cells that are potentially indicative of disorders such as leukemia and infectious mononucleosis. This study showed that the set of shift-and-rotation-invariant statistical features extracted from the eigensystem of the flow cytometric data pattern performs better than other commonly-used features in this type of disease detection, exhibiting an accuracy of 80.7%, a sensitivity of 72.3%, and a specificity of 89.2%. This performance represents a major improvement for this type of hematological classifier, which has historically been plagued by poor performance, with accuracies as low as 60% in some cases.