Chickens from lines selected for high (HA) and low (LA) antibody response to sheep red blood cell (SRBC) antigen were Used to study the role of genetic factors involved in resistance to Eimeria tenella and Newcastle disease virus (NDV). Routes of administration were intravenous for SRBC antigen and intranasal for NDV. Chicks were exposed to E. tenella either through a natural challenge via the litter or a controlled oral administration. Differences between lines were observed in resistance to cecal coccidiosis, with line HA chicks being more resistant than those from line LA. Results were similar. for both natural and controlled exposures, Differences in resistance to E. tenella were found among alleles for the I alloystern with degree of additivity from the I⁴ allele on resistance depending on the background genome. Chicks from line HA exhibited higher antibody titers to SRBC and lower titers to NDV than did those from line LA. This pattern was the same regardless of whether antigens were given together or alone. Correlations within lines for birds which received both antigens were positive and significant in line HA and not different from zero in line LA. When NDV was given at the time birds received a booster dose of SRBC, antibody titers for NDV and for primary and booster SRBC were higher in. line HA than line LA. Correlations between all titers were positive and significant in both lines. The data suggest that relationships between titers for SRBC and NDV are influenced by both genetic and nongenetic factors. / M.S.

Thesis (Ph.D.). / Written for the Institute of Parasitology. Title from title page of PDF (viewed 2008/02/12). Includes bibliographical references.

Les transfusions de globules rouges représentent le traitement principal de l’anémie. La décision de transfuser représente un vrai dilemme clinique. L’anémie et les transfusions de globules rouges sont toutes les deux associées à des risques et à un moins bon devenir des patients, alors que le bénéfice des transfusions sanguines reste difficile à démontrer. C’est pour cela que la décision de transfuser ne doit pas être pris à la légère et qu’elle doit tenir compte de la balance antre les risques des transfusions de globules rouges et les risques de l’anémie. L’anémie, définie comme un taux d’hémoglobine sous la moyenne pour l’âge, est fréquente chez les enfants en péri-opératoire de chirurgie cardiaque. Les conséquences de l’anémie sont une diminution du transport en oxygène vers les cellules. Le taux d’hémoglobine sous lequel la demande tissulaire en oxygène est compromise n’est pas connue et dépend de l’état de santé du patient et de ses comorbidités. Les causes peropératoires de l’anémie sont surtout le saignement et l’hémodilution. Une diminution de la production d’érythropoïétine endogène, une dérégulation du métabolisme du fer, une production défectueuse de la moelle et la répétition des prélèvements sanguins contribuent à l’anémie postopératoire. L’anémie est associée à des évènements indésirables et un moins bon devenir, mais cette association semble en grande partie expliquée par la pathologie sous-jacente, elle-même associée à l’anémie. Les transfusions en globules rouges sont fréquentes en chirurgie cardiaque pédiatrique. Le rapport bénéfice-risque des transfusions sanguines reste difficile à évaluer. Alors que les études rapportant des bénéfices clairs des transfusions sanguines restent rares, plusieurs travaux observent une association entre les transfusions en globules rouges et une augmentation de la morbidité et mortalité. En outre, les transfusions sanguines demeurent une ressource rare et chère. <p>Le but de ce travail est de contribuer à une meilleure utilisation des transfusions sanguines chez les patients de chirurgie cardiaque pédiatrique. Dans la première partie du travail, nous avons étudié les déterminants des transfusions en globules rouges et du saignement, qui représentent une des causes principales de transfusion sanguine chez ces patients. Une meilleure identification et une prise en charge adéquate des facteurs qui mènent aux transfusions sanguines devraient diminuer le nombre de transfusions inappropriées. Dans la deuxième partie de ce travail, nous nous sommes penchés sur l’association entre les transfusions sanguines et le mauvais pronostic des patients en étudiant deux approches :l’âge des globules rouges transfusés et l’indication transfusionnelle. Une meilleure compréhension des facteurs associés à un moins bon pronostic devrait permettre de mieux définir les patients qui bénéficieraient réellement de transfusions en globules rouges. <p>En ce qui concerne les déterminants des transfusions sanguines, nous avons démontré que l’anémie préopératoire était significativement associée aux transfusions sanguines péri-opératoires. Les enfants qui saignent reçoivent beaucoup de produits sanguins. Nous avons déterminé les patients à risque de saignement afin de les reconnaître et les soumettre à des tests de coagulation rapides pour orienter le type de produits sanguins à transfuser en fonction des anomalies de coagulation mises en évidence. Puisque l’anticoagulation par héparine est systématique chez les patients opérés sous circulation corporelle, nous avons étudié si notre protocole de neutralisation de l’héparine avec de la protamine était adéquat. En effet, la persistance d’héparine circulante ainsi qu’un surdosage en protamine sont associés à des saignements postopératoires. Un ratio protamine-héparine de 1:2 semble permettre une neutralisation adéquate de l’héparine chez la majorité des patients sans les exposer à un surdosage en protamine. Finalement, nous avons démontré qu’une stratégie transfusionnelle restrictive en postopératoire permettait de diminuer l’exposition aux transfusions sanguines sans augmenter la morbidité et mortalité de ces enfants. Cela signifie qu’on pourrait éviter des transfusions en globules rouges en prenant en charge l’anémie préopératoire, en développant un algorithme de prise en charge précoce du saignement peropératoire et en diminuant le seuil transfusionnel postopératoire. <p>La deuxième partie de ce travail avait pour but de préciser l’association qu’il existe entre les transfusions en globules rouges et la morbidité et mortalité postopératoire. L’âge du sang n’a pas l’air d’être un facteur influençant le pronostic des enfants opérés de chirurgie cardiaque. Par contre, ce travail a permis de montrer que c’est probablement l’indication transfusionnelle ou la raison qui mène à la transfusion, plutôt que la transfusion en elle-même qui est associée à un moins bon pronostic. L’association entre les transfusions sanguines et un moins bon pronostic est probablement surestimée par la présence de facteurs confondants comme l’indication transfusionnelle. Les transfusions en globules rouges seraient plutôt un marqueur de risque qu’un facteur de risque de mauvais pronostic.<p>En conclusion, ce travail contribue au développement de stratégies transfusionnelles plus rationnelles en chirurgie cardiaque pédiatrique. Reposant sur une approche multidisciplinaire, elles assurent une prise en charge structurée et orientée permettant de diminuer l’exposition des enfants aux produits sanguins, avec pour objectif une amélioration du pronostic et une réduction des coûts de prise en charge de ceux-ci. / Doctorat en Sciences médicales / info:eu-repo/semantics/nonPublished

La pollution croissante des milieux aquatiques nécessite la mise au point de nouvelles technologies permettant d’optimiser la surveillance de la qualité de l’eau. Dans ce contexte, nous avons développé un biomarqueur de susceptibilité du degré de la pollution globale des milieux aquatiques intitulé « Sentinelle ». Le principe du biomarqueur Sentinelle est basé sur le niveau de coexpression de deux protéines « Multidrug Resistance » (MDR), la protéine ABCG2-like et la P-gp, dans les érythrocytes de la truite Salmo trutta fario. Le biomarqueur sentinelle a été validé en conditions in vitro grâce au développement des cultures primaires d’érythrocytes de truite. Après l’exposition des globules rouges de truites à des concentrations croissantes d’un polluant modèle, le Benzo-a-pyrène, l’expression de la protéine ABCG2-like et de la P-gp augmente d’une manière dose dépendante. Le biomarqueur Sentinelle a ensuite été validé en milieu naturel sur des truites fario en provenance de différents cours d’eau d’Auvergne. En milieu naturel, les deux protéines MDR sont exprimées différemment dans les érythrocytes de truites fario selon le degré de contamination du cours d’eau. En effet, dans une rivière où la pollution est faible voire nulle, seule la protéine ABCG2-like est exprimée, alors que dans une rivière présentant une contamination plus importante, la P-gp et l’ABCG2-like sont toutes les deux coexprimées par une réponse de type relais. Les expériences menées en conditions in vitro et en milieu naturel, laissent supposer que la protéine ABCG2-like assure une fonction de garde alors que la P-gp assurerait une fonction de protection défensive. En conséquence, selon le niveau d’expression de la protéine de garde et de la protéine de défense, le degré de contamination de la rivière pourrait être évalué. L’intérêt de l’utilisation du biomarqueur Sentinelle a aussi été validé sur des Salmonidés en provenance de pisciculture. Ce nouvel outil biologique apporte des informations plus intégratives et plus précoces sur la qualité des milieux aquatiques, informations essentielles pour une meilleure gestion des ressources en eau. / Increasing aquatic pollution requires the development of new technologies for to optimize the monitoring of water quality. In this context, we have developed a “biomarker of susceptibility” designating the degree of global pollution in aquatic medium, entitled "Sentinel". The Sentinel biomarker is based on the co-expression level of two major "Multidrug resistance" (MDR) proteins, such as ABCG2-like protein and P-gp, in erythrocytes of brown trout’s in response to aquatic pollution. After developing a primary erythrocyte culture, the Sentinel biomarker was validated in a controlled medium. Trout erythrocytes exposure to increasing concentrations of Benzo-a-pyrene, a model pollutant, induced an increase expression of ABCG2-like protein and P-gp by a dose-dependent response. The Sentinel biomarker was then developed in a natural environment, using the erythrocytes of brown trout collected from the various rivers located in the Auvergne region of France. In the natural environment, both MDR proteins are differentially expressed in the erythrocytes of brown trout depending on the degree of contamination of rivers. Indeed, wild brown trout erythrocytes in an uncontaminated river, expressed only the ABCG2-like protein, whereas, in a river presenting a higher contamination, P-gp and ABCG2-like were both coexpressed with a relay response. Experiments in vitro conditions and natural environment, suggest that ABCG2-like protein acts as a vanguard protective protein, in complement to P-gp which acts as a “defensive” protective protein. Consequently, according to the expression level of the vanguard and defense proteins, the degree of contamination of the river could be evaluated. The use advantage of the Sentinel biomarker has also been validated on Salmonidae erythrocytes form farmed fish. This new tool provides biological information more early and integrative on the quality of aquatic environments. These informations are essential for better management of water resources.

La drépanocytose est une maladie génétique touchant l'hémoglobine, de transmission autosomique récessive, caractérisée par trois grandes manifestations cliniques :anémie hémolytique chronique, phénomènes vaso-occlusifs et susceptibilité accrue aux infections. Dans diverses régions du monde et particulièrement en Afrique subsaharienne, cette maladie est très fréquente et constitue un problème crucial de santé publique. Sa physiopathologie complexe est basée sur la polymérisation de l’hémoglobine anormale (Hb S) qui provoque une falciformation et une déshydratation du globule rouge. Les hématies falciformées sont impliquées dans les phénomènes de vaso-occlusion. Le traitement et la prise en charge de la maladie reste toujours problématique. A l’heure actuelle, le seul traitement curatif est la transplantation de moelle osseuse mais les donneurs compatibles sont assez rares et les coûts élevés. Des traitements symptomatiques et préventifs (principalement la transfusion et l’hydroxyurée) existent toutefois.<p>Des études in vitro et in vivo ont démontré les possibilités thérapeutiques de certaines molécules dont les cibles sont les transports membranaires impliqués dans la déshydratation cellulaire.<p>Depuis les années 1990, le cromoglycate de sodium, un médicament anti-allergique et anti-asthmatique, a montré un intérêt potentiel dans le traitement de la drépanocytose. Néanmoins, son mode d’action n’est actuellement pas connu. Notre travail a pour but de contribuer à l’étude du mécanisme d’action anti-drépanocytaire de la molécule.<p>Dans un premier temps, des globules rouges drépanocytaires préincubés en absence ou présence de cromoglycate ont été désoxygénés par un flux d’azote. Ensuite, les concentrations intracellulaires en Na+ et en K+ ont été mesurées. Les résultats de ces investigations ont montré un effet inhibiteur du cromoglycate sur l’efflux de K+ et l’influx du Na+ provoqués par la désoxygénation.<p>Sur base de ces observations, des expériences testant l’action du cromoglycate sur le canal K+ dépendant du Ca2+ (canal de Gardos) ont été effectuées. Dans des globules rouges normaux et drépanocytaires, ce canal a été activé par augmentation de la concentration intra-cellulaire en Ca2+. L'effet du cromoglycate a été comparé à celui d'un inhibiteur connu, le clotrimazole. Les résultats ont montré que 1e cromoglycate n'exerce pas d'effet inhibiteur sur le canal de Gardos, au contraire du clotrimazole. Il est également sans effet significatif sur la Ca2+-ATPase.<p>Nous avons ensuite investigué l’implication du Ca2+ dans les perturbations du flux des ions K+ et Na+. Des globules rouges drépanocytaires ont été incubés en absence et présence d’EGTA 5 mmol/l ou de BAPTA 10 µmol/l, respectivement chélateurs du Ca2+ extra et intracellulaire. Après désoxygénation, les concentrations intracellulaires en Na+ et K+ ont été mesurées. Les résultats de ces expériences montrent que seul le chélateur du Ca2+ extracellulaire bloque les perturbations ioniques causées par la désoxygénation. Ces résultats viennent donc confirmer les observations d’autres auteurs quant à l’implication du Ca2+ extracellulaire dans la fuite de K+ des globules drépanocytaires soumis à la désoxygénation. <p>Enfin, l’effet du cromoglycate sur l’influx de Ca2+ extracellulaire et sur la falciformation induits par le métabisulfite a été mesuré et comparé à celui du clotrimazole. Des globules rouges drépanocytaires, prélablement chargés en Fura Red, un indicateur fluorescent du Ca2+, ont été exposés au métabisulfite, un puissant réducteur provoquant une falciformation rapide. L’influx de Ca2+ a été mesuré par la diminution de la fluorescence du Fura Red. Parallèlement, la falciformation a été suivie en mesurant la lumière diffractée à 90° par les cellules. Les résultats de ces investigations montrent que le cromoglycate (1 µmol/l) et le clotrimazole (10 µmol/l) ont des effets inhibiteurs comparables sur la falciformation mais que le cromoglycate freine significativement plus l'influx de Ca2+ au cours de ce processus.<p>En conclusion, sur base de ces différents tests in vitro, le cromoglycate inhibe la falciformation induite par la désoxygénation. Cette inhibition résulte du blocage des perturbations ioniques induites par la désoxygénation en empêchant l’influx du Ca2+ extracellulaire et secondairement la fuite du K+ intracellulaire, ce qui inhibe la déshydratation cellulaire.<p>La diminution des crises vaso-occlusives observée chez les patients drépanocytaires traités par le cromoglycate s’expliquerait donc par ces effets. En présence de cromoglycate, les globules rouges sont moins déshydratés et falciforment moins rapidement. Ils sont dès lors moins impliqués dans les phénomènes de vaso-occlusion, ce qui améliore l’état des patients.<p> / Doctorat en Sciences biomédicales et pharmaceutiques / info:eu-repo/semantics/nonPublished

O presente estudo teve como objetivo determinar a capacidade antioxidante, antimicrobiana e anti-hemolítica do gengibre (Zingiber officinale) utilizando diferentes solventes extratores. Para tal, rasuras secas de gengibre foram trituradas e usadas na proporção 1:50 (p/v) nas quatro diferentes extrações: água, etanol (70%), acetona/ácido acético (70%/2%) ou acetona (70%). Após centrifugação, o extrato foi utilizado pra determinar o conteúdo de compostos fenólicos por Folin-Ciocalteau e ação sobre os radicais ABTS+. e DPPH.. Foi selecionado o melhor solvente extrator pelas propriedades antioxidantes e facilidade de sua remoção da matriz. Este extrato foi submetido à rotaevaporaçao, liofilizaçao e ressuspenção em tampão fosfato salino (PBS) e, então, utilizado nos estudos biológicos de captura de NO, ação antioxidante sobre hemólise induzida e ação sobre o crescimento de S. aureus. Os resultados foram expressos como média e desvio padrão. Para análise estatística empregou o software Minitab® com comparação entre grupos por ANOVA, diferenças significativas por Tukey e P < 0,05. O extrato de gengibre obtido em acetona apresentou maior conteúdo de compostos fenólicos (8,52 ± 1,24 mg equivalente de ácido gálico/g de gengibre). O extrato acetona apresentou menor valor de EC50 e maior ação antioxidante mediantes o ensaio com ABTS+., sendo respectivamente 0,07 ± 0,01 mg de massa seca do extrato/mL de ensaio e 10,93 ± 0,79 mg equivalentes de trolox (ET)/g de gengibre, sem diferença significativa com os valores obtidos nos extratos etanol e acetona/ácido. No ensaio do DPPH., o extrato acetona apresentou menor valor de EC50 (0,15 ± 0,01 mg de massa seca de extrato/mL de ensaio) e maior ação antioxidante (8,35 ± 0,60 mg ET/g de gengibre), sem diferença significativa com relação ao extrato etanólico. Dentre todos os ensaios, a extração em água apresentou resultados menos satisfatórios. A acetona foi selecionada como solvente extrator dos componentes do gengibre para os estudos biológicos. O extrato de gengibre (17,25 mg de massa seca/mL) inibiu 50% da formação de produtos no ensaio de captura de NO, valor similar ao obtido pela presença do antioxidante ácido gálico. Concentrações menores de extrato também agiram sobre o NO, sendo que 1,25 mg/mL refletiu em 33% de inibição. A hemólise foi evitada em 100% pelo extrato de gengibre em concentrações acima de 113 µg de massa seca de extrato/mL, resultado similar ao encontrado no ensaio contendo ácido ascórbico. A lise das hemácias foi evitada em 44% na presença de extrato 57 µg/mL. O extrato de gengibre não apresentou ação antimicrobiana sobre S. aureus. Conclui-se o extrato de gengibre, nas condições deste estudo, apresenta atividade antioxidante sobre ABTS+. e DPPH.; bem como sobre sistemas biológicos modelos como na captura de NO e lise de membrana celular. / The aim of this study was to determinate antioxidant capacity, antimicrobial activity and anti hemolytic action of ginger (Zingiber officinale) extract obtained by different solvents. For this purpose, dried ginger flakes were powdered and used in ratio 1:50 (w/v) in the four different extractions: water, ethanol (70%), acetone/acetic acid (70%/2%) and acetone (70%). The extract was centrifuged and used for phenolic compounds determination by Folin-Ciocalteau and action on ABTS+. and DPPH. radicals. The best extractor solvent was selected by its antioxidant properties and easier elimination from the matrix. The select extract was submitted on evaporation and lyophilization, and subsequent re-suspension on phosphate buffer saline (PBS). Then, this suspension was used in biologic studies as NO scavenging, antioxidant action on induced hemolysis and effect on S. aureus growth. The values were express as mean and standard deviation. Statistical analysis carried out by Minitab® using ANOVA and Tukey to p< 0.05. Acetone ginger extract showed highest value of phenolic content (8.52 ± 1.24 mg galic acid equivalent /g of ginger). The acetone extract showed the lowest EC50 value and higher antioxidant action on ABTS+., 0.07 ± 0.01 mg of dry weight of the extract/mL of assay and 10.93 ± 0.79 mg trolox equivalents (TE)/g of ginger, respectively. These values did not show significant difference in relation to ethanol and acetone/acid extracts. For DPPH. assay, acetone extract showed lower value of EC50 (0.15 ± 0.01 mg dry weight of extract/mL assay) and higher antioxidant action (8.35 ± 0.60 mg TE/g of ginger), without significant difference in relation to ethanolic extract. Among all extracts, aqueous extract showed lesser satisfactory results. Acetone extract was selected for biological assays. Ginger extract (17.25 mg of dry weigth/mL) inhibited 50% of products formation on NO scavenging assay, similarly to galic acid results. Lower extract concentrations as 1.25 mg/mL inhibited 33% of NO. The 100% of hemolysis prevention was obtained by concentrations of ginger extract higher than 113 µg of dry weight of extract/mL of assay. Similar result was observed by presence of ascorbic acid. Erythrocytes lysis was avoided in 44% by 57 µg/mL of ginger extract. Ginger extract did not show antimicrobial action on S. aureus. In conclusion, ginger extract, in present conditions, showed antioxidant action on ABTS+. and DPPH. assays and in biological model systems as in NO scavenging assay and cell membrane lysis.

The photobiomodulation therapy (PBMT) has many demonstrated applications in the health area including anti-inflammatory and wound healing effects. The main objective of this work is to verify if the PBMT causes measurable changes in the mechanical properties of cells, specifically in red blood cells, epithelial cells and fibroblasts. In addition, to contribute to the knowledge of the action mechanisms of the PBMT, this study intends to support applications of the PBMT during invasive procedures, such as the direct photo-treatment of the blood in surgical procedures with cardiopulmonary bypass, regarding security of the cellular integrity. For this analysis, three experimental techniques were used: optical magnetic twisting cytometry (OMTC), defocusing microscopy and confocal laser-scanning microscopy. Human bronchial epithelial cells were evaluated with OMTC. The epithelial cell culture was either photo-treated or not, with red laser (lambda=660 nm), and fixed power and time (power density of 153 mW/cm2, time 300 s). It was not possible to observe significant differences between photo-treated and control epithelial cells, for the hysteresivity (ratio between the cell loss and elastic shear moduli). The defocusing microscopy, similar to a phase contrast microscopy, was used to study human red blood cells from fresh blood. The red blood cells were either photo-treated or not, with red laser (lambda=660 nm), and different powers and times (power densities from 0 to 510 mW/cm2, times from 0 to 180 s). Some morphological and mechanical characteristics of individual red blood cells were evaluated, such as volume, radial profile of cell thickness, lateral and vertical membrane fluctuations, for the photo-treated and control red blood cells. It was not possible to detect differences between the two groups, for any of the parameters analyzed. For both techniques, the absence of detectable differences might be due to several factors, such as the non-action of the PBMT, with the parameters used, in the epithelial cells and red blood cells or to the small sensitivity of each technique. Confocal laser-scanning microscopy was used to evaluate the actin filaments of mouse fibroblasts. The fibroblast cell culture was either photo-treated or not, with red (lambda=625 nm) or infrared (lambda=808 nm) light and fixed power and time (power density from 113 to 158 mW/cm2, time 300 s). The nucleus and cell areas increased slightly when comparing photo-treated and control cells. On the other hand, the total actin, total actin density and the number of filaments decreased. These changes were detected for a short time after treatment, however, after 24 h they are not anymore detectable. The total branch length does not seem to suffer any modifications. In summary, with the data acquired with the three techniques, it was found that the PBMT, in the red range, with the parameters used, could not cause noticeable changes in red blood cells and epithelial cells, in vitro. On the other hand, the PBMT in the red and near-infrared range, with the power and times used, cause changes in actin filaments of fibroblasts, in vitro, in particular the decrease of the total actin density. / A terapia por fotobiomodulação tem muitas aplicações na área de Saúde devido a sua ação anti-inflamatória e de reparação tecidual. O objetivo geral desse trabalho é verificar se a terapia por fotobiomodulação provoca mudanças nas propriedades mecânicas de células, em particular em hemácias, células epiteliais e fibroblastos. Além de contribuir com o conhecimento dos mecanismos de ação da terapia por fotobiomodulação, este estudo pretende subsidiar aplicações da terapia por fotobiomodulação durante procedimentos mais invasivos, como a iluminação direta do sangue em procedimentos cirúrgicos com circulação extracorpórea, sob o ponto de vista da segurança quanto à integridade celular. Para essa análise foram utilizadas três técnicas experimentais: citometria óptica magnética de oscilação (OMTC), microscopia de desfocalização e microscopia confocal. Com a técnica de OMTC foram avaliadas células epiteliais brônquicas humanas em cultura, foto-tratadas com laser vermelho (lambda=660 nm), com potência e tempo fixos (densidade de potência de 153 mW/cm2, tempo 300 s). Não foi possível constatar diferenças significativas entre as células epiteliais foto-tratadas e as células controle, para a histerisividade (razão entre os módulos viscoso e elástico das células). Com a técnica de microscopia de desfocalização, semelhante a uma microscopia de contraste de fase, foram estudadas hemácias humanas de sangue recém coletado. As hemácias foram tratadas com laser vermelho (lambda=660 nm), com potências e tempos variados (densidade de potência de 0 a 510 mW/cm2, tempo de 0 a 180 s). Foram avaliadas algumas características morfológicas e mecânicas das hemácias individualmente, como o volume, perfil radial de espessura, flutuações lateral e vertical da membrana, tanto para hemácias foto-tratadas quanto para hemácias controle. Não foi possível detectar diferenças entre as hemácias foto-tratadas e controle para nenhum dos parâmetros avaliados. Para ambas as técnicas, a falta de mudanças observáveis poderia ser devida a diversos fatores, como a não ação da terapia por fotobiomodulação nas células epiteliais e nas hemácias, com os parâmetros aqui empregados, ou à falta de sensibilidade de cada uma das técnicas usadas. A microscopia confocal foi utilizada para avaliar os filamentos de actina de fibroblastos de camundongo em cultura, os quais foram foto-tratados com luz vermelha (lambda=625 nm) ou infravermelha (lambda=808 nm) e potência e tempo fixos (densidade de potência de 113 a 158 mW/cm2, tempo 300 s). Foi possível constatar ligeiro aumento nas áreas nuclear e celular das células foto-tratadas em relação aos fibroblastos controle. Também foi possível verificar a diminuição da quantidade total de actina, densidade de actina e do número de filamentos de actina nos fibroblastos foto-tratados. Essas mudanças são detectadas para tempos curtos após o tratamento, sendo que depois de 24 h elas desaparecem. O tamanho total dos filamentos parece não sofrer alterações. A partir dos dados coletados com as três técnicas, foi possível constatar que a terapia por fotobiomodulação, com os parâmetros utilizados, não consegue provocar mudanças perceptíveis em hemácias e em células epiteliais, in vitro. Porém, causa mudanças nos filamentos de actina de fibroblastos, in vitro, em particular a diminuição da densidade de actina total.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A maneira mais difundida na observaÃÃo de cÃlulas sanguineas (hemÃcias) à aquela que utiliza microscopia Ãtica convencional. Devido ao limite de resoluÃÃo dos instrumentos Ãticos, novas tÃcnicas de microscopia colocam-se como alternativas para o estudo de cÃlulas, tais como: a microscopia eletrÃnica (de varredura e transmissÃo) e as tÃcnicas de varredura por sonda. Inclui-se neste Ãltimo grupo a microscopia de forÃa atÃmica (AFM). Este trabalho discute as possibilidades de uso da Microscopia de ForÃa AtÃmica ($emph{Atomic Force Microscopy}$ - AFM) em ciÃncias da vida, para ser mais especÃfico, na caracterizaÃÃo de eritrÃcitos. Cinco experimentos envolvendo hemÃcias e AFM estÃo aqui descritos: diferenciaÃÃo entre os grupos sanguÃneos AB+ e O+; anÃlise do perfil da membrana eritrocitÃria de indivÃduos sadios e portadores de SMD; preparaÃÃo de cÃlulas vermelhas para anÃlise em microscopia de forÃa atÃmica; anÃlise volumÃtrica de cÃlulas vermelhas; e monitoramento do envelhecimento de um eritrÃcito ao ar usando o AFM. No primeiro experimento, a rugosidade das membranas celulares dos grupos AB+ e O+ mostraram-se diferentes. Jà no segundo experimento, depressÃes foram encontradas sobre a membrana de pacientes com SMD e indivÃduos sadios, contudo, aparentemente mais profundas no primeiro grupo. O terceiro estudo trouxe à tona a importÃncia da preparaÃÃo adequada dos eritrÃcitos para medidas especÃficas em AFM. A quarta experiÃncia comprovou a capacidade da tÃcnica AFM de fornecer informaÃÃo de volume, o que tambÃm foi utilizado no Ãltimo experimento para monitorar o envelhecimento de uma hemÃcia ao ar. / The optical microscopy is the most employed technique used for visualize red blood cells (RBCs). But, due to its resolution limit, it is necessary to use other complementary techniques to study the cells, such as: the scanning and transmission electron microscopy, and the scanning probing microscopy. The Atomic Force Microscopy (AFM) is included in the last group. This work refers to the possibilities of using AFM in life science, focusing on the erythrocytes characterization. Five experiments involving red blood cells and AFM were carried out: AB+ and O+ blood types differentiation; RBCs membrane study of donors and patients with MDS (Myelodysplastic Syndrome); suitable preparation of red blood cells for AFM analysis; volume study of erythrocytes; and finally aging process observation of RBC in air. The first experiment determined the cell membrane roughness for AB+ and O+ groups, which were different. For the second one, depressions were found on the cell surface of both MSD patients and healthy people. These "holes" were deeper in the first group. The third experiment showed the importance of sample (RBCs) preparation for each AFM specific analysis. The fourth experimental procedure showed the AFM technique capability for providing volume information, which was also used in the last experiment to monitor the aging process of RBCs in air.

Submitted by Sandro Camargo (sandro.camargo@unipampa.edu.br) on 2015-03-09T02:33:51Z No. of bitstreams: 1 116110002.pdf: 901358 bytes, checksum: edf3d6fae4ace04065873f00039b9507 (MD5) / Made available in DSpace on 2015-03-09T02:33:51Z (GMT). No. of bitstreams: 1 116110002.pdf: 901358 bytes, checksum: edf3d6fae4ace04065873f00039b9507 (MD5) Previous issue date: 2012-11-16 / A hiperglicemia leva a uma série de fenômenos bioquímicos que estão envolvidos na gênese do estresse oxidativo. O vinho é considerado um alimento antioxidante por conter uma grande quantidade de compostos fenólicos. O objetivo desse estudo foi observar o efeito antioxidante do vinho Tannat (safra 2006), produzido em Itaqui (RS), sobre o estresse oxidativo induzido por glicose ou frutose em eritrócitos in vitro. Eritrócitos foram incubados durante 24 horas a 37°C com concentrações de 5, 10, 30 e 100 mmol/L de glicose ou frutose, na presença ou ausência de diferentes volumes de vinho (0,075, 0,15 e 0,225 mL de vinho/mL de eritrócitos). Foram determinadas espécies reativas ao ácido tiobarbitúrico, consumo de glicose e fragilidade osmótica dos eritrócitos, além de polifenóis totais, antocianinas totais, ácido gálico, ácido caféico, epicatequina, resveratrol e a capacidade antioxidante (DPPH) do vinho. Os eritrócitos incubados com glicose e frutose apresentaram um aumento significativo da peroxidação lipídica quando comparados com eritrócitos incubados com 5 mmol/L de glicose ou frutose, o que foi prevenido com a adição de vinho. O vinho tinto apresentou altas concentrações de polifenóis totais, ácido gálico, ácido caféico, epicatequina e resveratrol, além de boa capacidade antioxidante. O consumo de glicose pelos eritrócitos nas concentrações de 5 e 10 mmol/L foi significativo, o que não ocorreu para os eritrócitos incubados com 30 e 100 mmol/L de glicose. O volume de 0,075 mL de vinho foi capaz de prevenir a inibição da captação de glicose pelos eritrócitos incubados com 30 e 100 mmol/L de glicose. No teste de fragilidade osmótica concentrações hipotônicas de NaCl induziram lise progressiva dos eritrócitos, que foi significativa apenas para os eritrócitos incubados com 30 e 100 mmol/L de frutose, sendo esta revertida através do tratamento dos eritrócitos com o vinho. Podemos concluir que o vinho tinto, a partir do volume mais baixo utilizado, pode diminuir a peroxidação lipídica, prevenir a inibição da captação de glicose pelos eritrócitos, além de diminuir a fragilidade osmótica de eritrócitos incubados com frutose. Este efeito antioxidante do vinho se deve, provavelmente, às altas concentrações de polifenóis encontrados e sua boa capacidade antioxidante. / Hyperglycemia leads a series of biochemical phenomena that are involved in the genesis of oxidative stress. The wine is considered an antioxidant food to contain a large amount of phenolic compounds. The aim of this study was to observe the antioxidant effect of Tannat wine (vintage 2006), produced in Itaqui (RS), on oxidative stress induced by glucose or fructose in erythrocytes in vitro. Erythrocytes were incubated for 24 hours at 37°C with concentrations of 5, 10, 30 and 100 mmol/L glucose or fructose in the presence or absence at different volumes of wine (0.075, 0.15 and 0.225 mL of red wine/mL of erythrocytes). Were determined Thiobarbituric acid reactive species, glucose consumption and osmotic fragility of erythrocytes; in addition total phenolic, anthocyanins, gallic acid, caffeic acid, epicatechin, resveratrol and the DPPH Scavenging Activity of wine. Erythrocytes incubated with glucose and fructose showed a significant increase in lipid peroxidation compared with erythrocytes incubated with 5 mmol/L glucose or fructose, which was prevented by addition of wine. The red wine presented high concentrations of total polyphenols, gallic acid, caffeic acid, epicatechin and resveratrol, further good antioxidant potential. The consumption of glucose by the erythrocytes at concentrations of 5 and 10 mmol/L was significant, this was not observed for the erythrocytes incubated with 30 and 100 mmol/L glucose. The volume of 0.075 ml of wine was able to prevent the inhibition of glucose uptake by erythrocytes incubated with 30 and 100 mmol/L glucose. In the test of osmotic fragility hypotonic concentrations of NaCl induced lysis of erythrocytes progressive, which was significant only for erythrocytes incubated with 30 and 100 mmol/L fructose, this being reversed by treating cells with wine. We conclude that red wine, from the lower volume used, can decrease lipid peroxidation, prevented the inhibition of glucose uptake and decreased osmotic fragility of erythrocytes incubated with fructose. This antioxidant effect of wine is probably due to high concentrations of polyphenols and its good antioxidant capacity.

Efficient removal of apoptotic cells is critical for development, tissue remodelling, maintenance of homeostasis, and response to injury. Phagocytosis of apoptotic cells is mediated by many phagocytic receptors, soluble bridging molecules, and pro-phagocytic ligands on the surface of apoptotic cells. Macrophage phagocytosis in general is controlled by stimulatory and inhibitory mechanisms. An example of the latter mechanism is that mediated by the cell surface glycoprotein CD47, which by binding to the inhibitory receptor Signal Regulatory Protein alpha (SIRPα) on macrophages, is known to inhibit phagocytosis of viable host cells. The studies of the present thesis aimed at investigating possible changes to CD47 on apoptotic cells, which could influence their elimination by macrophages. The endoplasmatic protein calreticulin (CRT), in conjunction with Low density lipoprotein Receptorrelated Protein 1 (LRP1) on the phagocyte, can act as a receptor for collectin family members and mediate uptake of apoptotic cells. However, CRT itself was found to also be expressed on the surface of many viable cell types, and the CRT expression increased on apoptotic cells. By using antibodies to LRP1 or receptor‐associated protein (RAP), an antagonist blocking LRP1 ligand binding, we found that CRT on target cells could interact in trans with LRP1 on a phagocyte and stimulate phagocytosis. CD47 on the target cell inhibited LRP1‐mediated phagocytosis of viable cells (e.g. lymphocytes or erythtocytes), but not that of apoptotic cells. The inability of CD47 on apoptotic cells to inhibit LRP1‐ mediated phagocytosis could be explained in two ways: 1) Some apoptotic cell types (fibroblasts and neutrophils, but not Jurkat T cells) lost CD47 from the cell surface, or 2) CD47 is evenly distributed on the surface of viable cells, while it was redistributed into patches on apoptotic cells, segregated away from areas of the plasma membrane where the pro‐phagocytic ligands CRT and phoaphatidylserine (PS) were concentrated. Apoptotic murine thymocytes also showed a patched distribution of CD47, but no significant loss of the receptor. However, both PS‐independent and PS‐dependent macrophage phagocytosis of apoptotic CD47‐/‐ thymocytes was less efficient than uptake of apoptotic wild‐type (wt) thymocytes. This contradictory finding was explained by the fact that CD47 on apoptotic thymocytes did no longer inhibit phagocytosis, but rather mediated binding of the apoptotic cell to the macrophage. These effects could in part be dependent on the apoptotic cell type, since uptake of experimentally senescent PS+ wt or CD47‐/‐ erythrocytes by macrophage in vitro, or by dendritic cells (DC) in vivo, were the same. In vivo, PS+ erythrocytes were predominantly trapped by marginal zone macrophages and by CD8+ CD207+ DCs in the splenic marginal zone. DCs which had taken up PS+ erythrocytes showed a slight increase in expression levels of CD40, CD86 and MHC class II. These findings suggest that PS+ erythrocytes may be recognized by splenic macrophages and DCs in ways similar to that reported for apoptotic T cells. Uptake of senescent erythrocytes by DCs may serve as an important mechanism to maintain self‐tolerance to erythrocyte antigens, and defects in this function may facilitate development of AIHA. Glucocorticoids are used to treat inflammatory conditions and can enhance macrophage uptake of apoptotic cells. We found that the glucocorticoid dexamethasone time‐ and dose‐dependently stimulated macrophage cell surface LRP1 expression. Dexamethasone‐stimulated macrophages also showed enhanced phagocytosis of apoptotic thymocytes and unopsonized viable CD47‐/‐ erythrocytes. In summary, LRP1 can mediate phagocytosis of both viable and apoptotic cells by binding CRT on the target cell. Macrophage expression of LRP1 is increased by glucocorticoids, which could be one explanation for the anti‐inflammatory role of glucocorticoids. While CD47 on viable cells efficiently inhibits phagocytosis in macrophages, CD47 on apoptotic cells does not and can sometimes even promote their removal.