L’hémoglobine est une protéine contenue dans les globules rouges dont la principale fonction est le transport de l’oxygène. Chaque molécule d’hémoglobine est un tétramère constitué de deux paires de globines identiques de type α et β. La β-thalassémie est une maladie génétique hématopoïétique provenant de mutations du gène encodant l'hémoglobine. Ce désordre se caractérise par une diminution ou une absence totale de la synthèse de la chaîne β-globine résultant principalement en une anémie hémolytique sévère ainsi que des complications multisystémiques, telles que la splénomégalie, des déformations osseuses et une dysfonction hépatique et rénale. Actuellement, les transfusions sanguines chroniques représentent le traitement standard des patients β-thalassémiques. Cette thérapie nécessite l’administration conjointe d’un traitement chélateur de fer puisqu’elle entraîne une accumulation pathologique du fer, considéré à ce jour comme la source principale des complications cardiovasculaires de la β-thalassémie. Néanmoins, malgré le traitement efficace de la surcharge de fer transfusionnelle, l’insuffisance cardiaque demeure encore la principale cause de mortalité chez les patients atteints de β-thalassémie. Cette observation indique possiblement la présence d’un mécanisme complémentaire dans le développement de la physiopathologie cardiaque β-thalassémique. L’objectif du présent projet consistait donc à étudier les altérations cardiovasculaires de la β-thalassémie indépendamment de la surcharge de fer transfusionnelle. En utilisant un modèle murin non-transfusé de la β-thalassémie majeure, nous avons d’abord évalué in vivo, par méthode d’imagerie novatrice échographique à haute fréquence, les propriétés hémodynamiques vasculaires. Nos résultats d’index de Pourcelot ainsi que de résistance vasculaire périphérique totale ont démontré une perturbation de l’écoulement microcirculatoire chez les souris β-thalassémiques non-transfusées. Subséquemment, nous avons étudié la fonction endothéliale de régulation du tonus vasculaire de vaisseaux mésentériques isolés. Nos résultats ont révélé un dysfonctionnement de la réponse vasodilatatrice dépendante de l’endothélium chez les souris β-thalassémiques malgré une augmentation de l’expression de l’enzyme de synthèse du monoxyde d’azote ainsi qu’un remodelage de la carotide commune caractérisé par un épaississement de la paroi vasculaire. Finalement, notre étude échocardiographique de la fonction et la morphologie cardiaque a montré, chez les souris β-thalassémiques, le développement d’une hypertrophie et une dysfonction ventriculaire gauche en l’absence de transfusions sanguines chroniques ou de dépôts directs de fer dans le myocarde. L’ensemble des résultats présentés dans le cadre de cette thèse indique la présence d’une pathologie cardiovasculaire chez les souris β-thalassémiques non-transfusés. Nos travaux permettent de proposer un mécanisme de la pathophysiologie cardiovasculaire β-thalassémique, indépendant de la charge de fer transfusionnelle, impliquant les effets compensatoires d’une anémie chronique combinés à une vasculopathie complexe initiée par les érythrocytes endommagés et l’hémolyse intravasculaire. / Hemoglobin is the major protein in red blood cells and is responsible of the oxygen transport. Each hemoglobin molecule is a tetramer consisting of two identical α- and β-globin subunits. β-thalassemia is a genetic hematopoietic disease caused by mutations in hemoglobin genes. This disorder is characterized by a decrease or absence of production of β-globin chain leading mainly to a severe hemolytic anemia and several systemic manifestations, including splenomegaly, skeletal deformities as well as hepatic and renal dysfunctions. Chronic blood transfusions remain the standard treatment for β-thalassemic patients. This therapy requires iron chelating management since it leads to pathological iron accumulation which is currently considered the main cause of cardiovascular complications of β-thalassemia. However, despite adequate control of transfusional iron loading, heart failure remains the leading cause of mortality in β-thalassemia. This issue is possibly indicative of additional pathogenic mechanisms underlying the development of the β-thalassemic cardiac pathology. The objective of the present research project was to study cardiovascular alterations of β-thalassemia independently of transfusional iron overloading. Using an untransfused murine model of β-thalassemia major, we have evaluated in vivo, by non-invasive high-frequency ultrasound imaging, vascular hemodynamic properties. Our results of Pourcelot indices and total peripheral vascular resistance have shown microcirculatory flow disturbances in untransfused β-thalassemic mice. Consequently, we have studied ex vivo the endothelial vasomotor function in isolated mesenteric arterioles. Our findings have pointed out endothelium-dependent vasodilator dysfunction in β-thalassemic mice despite increased expression of nitric oxide synthase, as well as remodeling of the common carotid artery wall. Lastly, our echocardiography studies of heart morphology and function in β-thalassemic mice have demonstrated the development of left ventricle hypertrophy and dysfunction in the absence of chronic blood transfusions or direct myocardial iron deposits. In conclusion, findings presented in this thesis have demonstrated for the first time development of severe cardiovascular complications in untransfused β-thalassemic mice. Based on our results, we have proposed a novel mechanism, independent of direct myocardial iron deposition, responsible for the cardiovascular complications in β-thalassemia. This model combines compensatory effects of chronic anemia with a complex vasculopathy initiated by abnormal erythrocytes and intravascular hemolysis.

Στην παρούσα διπλωματική εργασία θα γίνει μελέτη και εφαρμογή μεθόδων επίλυσης του προβλήματος αναγνώρισης γεωμετρικών χαρακτηριστικών ανθρώπινων ερυθρών αιμοσφαιρίων από προσομοιωμένες εικόνες σκέδασης ΗΜ ακτινοβολίας ενός He-Ne laser 632.8 μm. Στο πρώτο κεφάλαιο γίνεται μια εισαγωγή στις ιδιότητες και τα χαρακτηριστικά του ερυθροκυττάρου καθώς, επίσης, παρουσιάζονται διάφορες ανωμαλίες των ερυθροκυττάρων και οι μέχρι στιγμής χρησιμοποιούμενοι τρόποι ανίχνευσής των. Στο δεύτερο κεφάλαιο της εργασίας γίνεται μια εισαγωγή στις ιδιότητες της ΗΜ ακτινοβολίας, περιγράφεται το φαινόμενο της σκέδασης και παρουσιάζεται το ευθύ πρόβλημα σκέδασης ΗΜ ακτινοβολίας ανθρώπινων ερυθροκυττάρων. Το τρίτο κεφάλαιο αποτελείται από δύο μέρη. Στο πρώτο μέρος γίνεται εκτενής ανάλυση της θεωρίας των τεχνητών νευρωνικών δικτύων και περιγράφονται τα νευρωνικά δίκτυα ακτινικών συναρτήσεων RBF. Στη συνέχεια, αναφέρονται οι μέθοδοι εξαγωγής παραμέτρων και, πιο συγκεκριμένα, δίνεται το θεωρητικό και μαθηματικό υπόβαθρο των μεθόδων που χρησιμοποιήθηκαν οι οποίες είναι ο αλογόριθμος Singular Value Decomposition (SVD), o Angular Radial μετασχηματισμός (ART) και φίλτρα Gabor. Στο δεύτερο μέρος περιγράφεται η επίλυση του αντίστροφου προβλήματος σκέδασης. Παρουσιάζεται η μεθοδολογία της διαδικασίας επίλυσης όπου εφαρμόστηκαν ο αλογόριθμος συμπίεσης εικόνας SVD, o περιγραφέας σχήματος ART και ο περιγραφέας υφής με φίλτρα Gabor για την εύρεση των γεωμετρικών χαρακτηριστικών και νευρωνικό δίκτυο ακτινικών συναρτήσεων RBF για την ταξινόμηση των ερυθροκυττάρων. Στο τέταρτο και τελευταίο κεφάλαιο γίνεται δοκιμή και αξιολόγηση της μεθόδου και συνοψίζονται τα αποτελέσματα και τα συμπεράσματα που εξήχθησαν κατά τη διάρκεια της εκπόνησης αυτής της διπλωματικής. / In this thesis we study and implement methods of estimating the geometrical features of the human red blood cell from a set of simulated light scattering images produced by a He-Ne laser beam at 632.8 μm. Ιn first chapter an introduction to the properties and the characteristics of red blood cells are presented. Furthermore, we describe various abnormalities of erythrocytes and the until now used ways of detection. In second chapter the properties of electromagnetic radiation and the light scattering problem of EM radiation from human erythrocytes are presented. The third chapter consists of two parts. In first part we analyse the theory of neural networks and we describe the radial basis function neural network. Then, we describe the theoritical and mathematical background of the methods that we use for feature extraction which are Singular Value Decomposition (SVD), Angular Radial Transform and Gabor filters. In second part the solution of the inverse problem of light scattering is described. We present the methodology of the solution process in which we implement a Singular Value Decomposition approach, a shape descriptor with Angular Radial Transform and a homogenous texture descriptor which uses Gabor filters for the estimation of the geometrical characteristics and a RBF neural network for the classification of the erythrocytes. In the forth and last chapter the described methods are evaluated and we summarise the experimental results and conclusions that were extracted from this thesis.

CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A pré-eclâmpsia (PE) é classificada em de início (EOPE) e tardio (LOPE) quando presente antes ou após 34 semanas de gestação, respectivamente. Este estudo transversal investigou as diferenças e possíveis associações existentes entre os perfis antropométricos, hemodinâmicos, hematológicos e bioquímicos na pré-eclâmpsia entre os grupos EOPE e LOPE. O estudo incluiu 65 voluntárias admitidas em um hospital universitário no Brasil sendo 29 normotensos (grupo C) e 36 com pré-eclâmpsia (13 com EOPE e 23 com LOPE). O grupo LOPE apresentou um aumento significante de peso e aumento limítrofe no índice de massa corporal ao final da gestação em relação aos outros grupos, o que é compatível com a origem metabólica, associada à obesidade, atribuída a esta forma da doença. As mulheres grávidas do EOPE apresentaram uma redução limítrofe no número de eritrócitos e uma diminuição signi-ficante no número de plaquetas e um aumento significante de reticulócitos, ferro sérico e fer-ritina quando comparados ao grupo C e LOPE. O grupo EOPE demonstrou um aumento significante na estabilidade osmótica dos eritrócitos em relação aos demais gru-pos. A análise hemodinâmica por ultrasonografia Doppler da artéria oftálmica mostrou que ambos os grupos de mulheres grávidas com PE apresentaram alterações compatíveis com a ocorrência de hiperfluxo no território orbital. Essas alterações hemodinâmicas foram associa-das a alterações nos índices hematimétricos. / Preeclampsia (PE) is classified in early- (EOPE) and late-onset PE (LOPE) when pre-sent before or after 34 gestation weeks, respectively. This transversal study aimed to investi-gate the differences and possible associations existing in the anthropometric, hemodynamic, hematologic and biochemical profiles of late- and early-onset preeclampsia. The study in-cluded 65 volunteers admitted to a tertiary hospital in Brazil, 29 normotensive and 36 with preeclampsia (13 with EOPE and 23 with LOPE). Pregnant women with LOPE presented greater weight gain and borderline increase in body mass index at the end of gestation in rela-tion to the other groups, which is compatible with the metabolic origin, associated with obesi-ty, attributed to this form of the disease. Pregnant women with EOPE presented a borderline reduction in the number of erythrocytes and a significant decrease in the number of platelets, in addition to a significant increase in reticulocytes, serum iron and ferritin when compared to normotensive pregnant women and pregnant women with LOPE. A significant increase in osmotic stability of erythrocytes was observed in the EOPE group in relation to groups. He-modynamic analysis by Doppler ultrasonography of the ophthalmic artery showed that both groups of pregnant women with PE presented alterations compatible with the occurrence of hyperflow in the orbital territory. These hemodynamic changes were associated with changes in hematimetric indices / Tese (Doutorado)

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Os ácidos graxos poli-insaturados n-3 derivados do óleo de peixe estão associados a benefícios cardiovasculares, que podem ser decorrentes da ativação da óxido nítrico sintase (NOS). Assim como as células endoteliais, os eritrócitos possuem NOS endotelial (eNOS) e induzível (iNOS) e, portanto, são capazes de sintetizar óxido nítrico (NO). O presente estudo testou a capacidade que diferentes concentrações de óleo de peixe tem de ativar a via L-arginina-NO e, em seguida, alterar os níveis de guanosina monofosfato cíclica (GMPc) em eritrócitos de camundongos alimentados com dieta hiperlipídica. Além disso, foram analisados os marcadores de estresse oxidativo nos eritrócitos, objetivando investigar a biodisponibilidade do NO. O transporte de L-arginina, avaliado através da incubação com L-[3H]-arginina, mostrou-se ativado quando da administração de dietas contendo elevadas concentrações de óleo de peixe, em comparação com as dietas contendo baixas concentrações e controle. A atividade da NOS, medida pela conversão de L-[3H]-arginina em L-[3H]-citrulina, e a expressão da eNOS também aumentaram nos animais que se alimentaram com dietas ricas em óleo de peixe. Apesar da ativação da via L-arginina-óxido nítrico observada em nossos experimentos, os níveis de GMPc intraeritrocitário não foram afetados. O dano oxidativo nos eritrócitos aumentou linearmente conforme o óleo de peixe era acrescido na dieta, sem afetar a atividade das enzimas antioxidantes. Além do endotélio, os eritrócitos contribuem para o metabolismo do NO. Desta forma, a ativação da via L-arginina-NO nessas células pode ser benéfica para saúde cardiovascular. Estudos futuros poderão investigar outros marcadores de estresse oxidativo durante o consumo de óleo de peixe para assegurar que o seu uso não resulta em efeitos prejudiciais secundários e para garantir a biodisponibilidade de NO. / The n-3 polyunsaturated fatty acids derived from fish oil are associated with cardiovascular benefits and it has been suggested that the activation of nitric oxide synthase (NOS) would be a potential mechanism responsible for its effects. Beside endothelial cells, red blood cells (RBC) possess endothelial NOS (eNOS) and inducible NOS (iNOS), and thus are capable of synthesizing their own nitric oxide (NO). The present study tested the capacity of different amounts of fish oil to activate L-arginine-NO pathway and therefore alter cyclic guanosine monophosphate (cGMP) levels in RBC from mice fed on a high fat diet. Additionally, the oxidative status in RBC was performed to investigate NO bioavailability. L-arginine transport, assessed by incubation with L-[3H]-arginine, was activated by higher doses of fish oil, compared to control diet and to lowest doses of fish oil. RBC NOS activity, measured by the conversion of L-[3H]-arginine into L-[3H]-citrulline, and eNOS expression were also enhanced by diets rich in fish oil. Despite the L-arginine-NO activation, no effect on intra RBC cGMP basal levels was seen among the groups. Oxidative damage of RBC rises linearly with increasing amounts of fish oil in the diet without affecting the activity of antioxidant enzymes. Besides endothelium, red blood cells also contribute regulating the NO bioactivity. Therefore, the activation of L-arginine-NO pathway in RBC by fish oil would be beneficial in cardiovascular health. Future studies testing other oxidant markers during dietary fish oil supplementation will be necessary to verify that its consumption does not result in detrimental secondary effects and to ensure NO bioavailability.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Introdução: o óxido nítrico (NO) é um gás inorgânico com uma meia-vida curta e tem um papel crítico na manutenção da homeostase vascular e fluidez sanguínea. O NO é sintetizado a partir do aminoácido L-arginina por uma família de enzimas NO sintases (NOS). Estudos têm mostrado que eritrócitos expressam NOS endotelial (eNOS) funcional, que serve como uma fonte de NO intraluminal. Além disso, eritrócitos participam da defesa antioxidante removendo os radicais livres e prevenindo o dano oxidativo às membranas biológicas e a destruição do NO. Dietas hiperlípidicas estão associadas a um risco aumentado de doença cardiovacular e síndrome metabólica, mas os exatos mecanismos não estão completamente esclarecidos. O objetivo deste estudo foi investigar os efeitos de diferentes dietas hiperlípidicas na via L-arginina-NO e o estresse oxidativo em eritrócitos de camundongos. Metodologia: camundongos machos C57BL/6 de três meses de idade receberam diferentes dietas por 10 semanas: dieta normolipídica ou dieta hiperlipídica contendo banha de porco (HB), óleo de oliva (HO), óleo de girassol (HG) ou óleo de canola (HC). Foram analisados o transporte de L-arginina mediado pelos transportadores catiônicos y+ e y+L, a atividade da NOS, a expressão da eNOS e da NOS induzível (iNOS), a formação de substâncias reativas ao ácido tiobarbitúrico (TBARS) e a atividade das enzimas antioxidantes catalase (CAT) e superóxido dismutase (SOD). Resultados: o transporte total de L-arginina estava aumentado no grupo HO em comparação aos controles e aos outros grupos com dieta hiperlipídica. Quando o transporte foi fracionado, o sistema y+ estava mais ativado no grupo HO em relação aos controles e outros grupos que receberam dieta hiperlipídica. O transporte de L-arginina via sistema y+L estava maior nos grupos HO, HG e HC comparados aos grupos controle e HB. Adicionalmente, a atividade basal da NOS e a expressão de eNOS estavam aumentadas em eritrócitos independente do tipo de dieta hiperlípidica insaturada. Observou-se uma maior expressão da iNOS no grupo HO comparado ao controle. Em contraste, o grupo HB apresentou uma inibição da via L-arginina-NO. A análise da peroxidação lipídica, através da formação de TBARS, e da atividade da enzima antioxidante CAT não revelou diferenças entre os grupos, ao contrário do grupo HO, que induziu uma ativação de outra enzima antioxidante, a SOD. Conclusões: o presente estudo proporciona a primeira evidência de que os sistemas y+ e y+L regulam o transporte aumentado de L-arginina em eritrócitos de camundongos do grupo HO. Além disso, todas as dietas hiperlipídicas insaturadas induzem um aumento da atividade basal da NOS associada a uma expressão elevada da eNOS. É possível que diferentes mudanças na composição lipídica da membrana plasmática induzidas pelas dietas possam afetar transportadores e enzimas nos eritrócitos. Além disso, a inibição da via L-arginina-NO no grupo HB pode contribuir para o desenvolvimento da aterosclerose, enquanto dietas hiperlipídicas insaturadas podem ter um efeito protetor via aumento da geração de NO. / Introduction: nitric oxide (NO) is an inorganic gas with a short half life that plays a critical role in maintaining vascular homeostasis and blood fluidity in physiological conditions. NO is synthesized from the cationic amino acid L-arginine by a family of enzymes: nitric oxide synthase (NOS). Studies have shown that red blood cells (RBCs) express functional endothelial NOS (eNOS), which potentially serves as an intraluminal NO source. Moreover, circulating RBCs participate in antioxidant defence, scavenging oxygen free radicals and preventing oxidative damage to biological membranes and NO destruction. High fat diets are associated with an increased risk of cardiovascular disease and metabolic syndrome, but the exact mechanisms are not completely clear. The objective of this study was to investigate the effects of different high fat (HF) diets in the RBC L-arginine-NO pathway and in oxidative stress in C57BL/6 mice. Methods: three-month-old male C57BL/6 mice were fed different diets for a 10-week period: a standard diet or high-fat (HF) diet containing lard oil (HF-L), olive oil (HF-O), sunflower oil (HF-S) or canola oil (HF-C). Studies of L-arginine transport, mediated by cationic transport systems y+ and y+L, basal activity of NOS, expression of eNOS and inducible NOS (iNOS), thiobarbituric acid reactive substances (TBARS) formation, and antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) activities in RBCs were analysed in these groups. Results: total L-arginine influx into RBCs was upregulated in the HF-O group compared to controls and other HF diet groups. When transport systems were fractionated, there was a higher activation of system y+ in the HF-O group in relation to controls and other HF diet groups. L-arginine transport via system y+L in RBCs was increased in the HF-O, HF-S and HF-C groups compared to controls and the HF-L group. In addition, NOS activity and eNOS expression were enhanced in RBCs, independent of unsaturated HF diets. An overexpression of iNOS was observed in the HF-O group compared with controls. In contrast, the HF-L group showed an inhibition of the RBC L-arginine-NO pathway. The analysis of lipid peroxidation and antioxidant enzyme catalase activity revealed no differences among the groups studied. On the other hand, HF-O induced activation of another antioxidant enzyme, superoxide dismutase (SOD). Conclusions: this study provides the first evidence that systems y+ and y+L mediate increased L-arginine transport into mice RBCs from the HF-O group. Moreover, all unsaturated high-fat diets can induce an increase in basal NOS activity associated with an overexpression of eNOS. It is possible that changes in the lipid composition of the plasmatic membrane induced differently by HF diets could affect transporters and enzymes in RBCs. An inhibition of the L-arginine-NO pathway in HF-L group could contribute to the development of atherosclerosis, while HF unsaturated diets may have a protector effect via enhanced generation of NO.

Made available in DSpace on 2014-12-17T14:13:22Z (GMT). No. of bitstreams: 1 SebastiaoDSF.pdf: 475002 bytes, checksum: d28adb2821adc67501d7e19f42e9b6a3 (MD5) Previous issue date: 2007-08-16 / Conselho Nacional de Desenvolvimento Cient?fico e Tecnol?gico / Several clinic evaluations have been possible with radiobiocomplexes labeled with technetium-99m (99mTc). Some natural and synthetic drugs are capable of to interfere on the labeling of blood constituents with 99mTc, as well as on the biodistribution of radiobiocomplexes. Authors have also reported about the toxicity of several natural products. The aim of this study was to compare the effects of the Mentha crispa (hortel?) and of the Hypericum perforatum (hip?rico) in different experimental models. On the labeling of red blood cells (RBC) and plasma and cellular proteins with 99mTc, both extracts were capable of to decrease the radioactivity percentage on the cellular compartment and on the fixation on plasma and cellular proteins. On the morphometry of the RBC, only the hortel? was capable to alter the shape and the perimeter/area ratio of the RBC. On the biodistribution of the radiobiocomplex sodium pertechnetate (Na99mTcO4), the hortel? increased the Na99mTcO4 distribution in the kidney, spleen, liver and thyroid, meanwhile the hip?rico decreased the Na99mTcO4 distribution in the bone, stomach, lungs and thyroid, and increased the Na99mTcO4 distribution in the pancreas. On the bacterial cultures survival, the hip?rico was capable of to protect the bacteria against the stannous chloride (SnCl2) effect. The hip?rico did not alter the topology of plasmidial DNA and did not protect the plasmidial DNA against the SnCl2 action. Probably, the effects presented by both extracts could be due to chemical compounds of the extracts that could alter the morphology of the RBC and the plasma membrane ions transport, and/or by phytocomplexes that could be formed with different effects dependent on the biological system considered / Avalia??es cl?nicas t?m sido poss?veis com radiobiocomplexos marcados com tecn?cio-99mTc (99mTc). Drogas naturais ou sint?ticas s?o capazes de interferir na marca??o de estruturas sangu?neas com 99mTc, assim como na biodistribui??o de radiobiocomplexos. Tamb?m tem sido descrita a toxicidade de v?rios produtos naturais. O objetivo deste estudo foi comparar o efeito dos extratos de Mentha crispa (hortel?) e de Hypericum perfloratum (hip?rico) em diferentes modelos experimentais. Na marca??o de estruturas sang??neas com 99mTc verificou-se que ambos os extratos foram capazes de diminuir a radioatividade no compartimento celular, nas prote?nas plasm?ticas e celulares. Na morfometria das hem?cias, apenas a hortel? foi capaz de alterar a forma e a rela??o per?metro/?reas das hem?cias. Na biodistribui??o do radiobiocomplexo pertecnetato de s?dio (Na99mTcO4) a hortel? aumentou a capta??o do Na99mTcO4 no rim, no ba?o, no f?gado e na tire?ide, enquanto que o hip?rico diminuiu a capta??o do Na99mTcO4 no osso, no est?mago, no pulm?o e na tire?ide, e aumentou no p?ncreas. Na sobreviv?ncia de culturas bacterianas o hip?rico foi capaz de proteger a bact?ria do efeito danoso do cloreto estanoso (SnCl2). O hip?rico n?o alterou a topologia nem protegeu o DNA plasmidial da a??o do SnCl2. Provavelmente os efeitos apresentados por ambos os extratos poderiam ser explicados por subst?ncias presentes nos extratos que poderiam alterar a morfologia das hem?cias, o transporte de ?ons pela membrana e/ou formar fitocomplexos. O estudo teve car?ter multidisciplinar com a participa??o das seguintes ?reas do conhecimento: Radiobiologia, Bot?nica, Endocrinologia, Fitoterapia e Hematologia

Made available in DSpace on 2014-12-17T14:13:38Z (GMT). No. of bitstreams: 1 DanielleAT.pdf: 281978 bytes, checksum: c61d07a033e83fa024963234eef23f5d (MD5) Previous issue date: 2008-06-11 / Artemisia vulgaris L..is used in folk medicine and in Traditional Chinese Medicine (TCM). This medicinal plant has been utilized as anticonvulsive, analgesic, antispasmodic effect, rheumatic pains, menstrual dyspepsia, asthenia, epilepsy, hepatitis, fevers, anemia and to expel parasites. In nuclear medicine, blood constituents are labeled with technetium-99m (99mTc) and used as radiopharmaceuticals (radiobiocomplexes). Authors have been described that synthetic and/or natural drugs could modify the labeling of blood constituents with 99mTc. The aim of this work was to evaluate the effects of an aqueous extract of Artemisia vulgaris L. on the labeling of blood constituents with 99mTc. Blood samples withdrawn of Wistar rats were incubated with Artemisia vulgaris L, stannous chloride and 99mTc, as pertechnetate ion. Aliquots of plasma (P) and blood cells (BC) were isolated. Aliquots of P and BC were also precipitated with trichloroacetic acid and soluble (SF) and insoluble (IF) fractions were separated. The radioactivity in each fraction was counted and the percentages of radioactivity (%ATI) were calculated. Artemisia vulgaris L. extract decreased significantly (p<0.05) the %ATI on BC and on IF-BC. The analysis of the results indicates that the extract could have substances that could interfere on the transport of stannous through the erythrocyte membrane altering the labeling of blood cells with 99mTc. Working in this study was a multidisciplinary group, with Phisical therapists, Biomedicals, Physicals, Pharmacists, Biologists, Statistics and Physicians. / Artemisia vulgaris L. ? usada na medicina tradicional como anticonvulsivante, analg?sico, antiespasm?dico, indicada para dores reum?ticas, dispepsia menstrual, astenia, epilepsia, hepatite, febre, anemia e para expelir parasita. Na medicina nuclear, constituintes do sangue s?o marcados com tecn?cio-99m (99mTc) e usados em procedimentos cl?nicos como radibiocomplexos. Estudos t?m demonstrado que drogas sint?ticas ou naturais podem modificar a marca??o dos constituintes sangu?neos com 99mTc. O presente estudo teve como objetivo avaliar os efeitos de um extrato de Artemisia vulgaris L. na marca??o dos constituintes sangu?neos com 99mTc. Amostras de sangue foram incubadas com o extrato vegetal, cloreto estanoso e 99mTc na forma de pertecnetato de s?dio. Plasma e c?lulas sang??neas foram isolados por centrifuga??o. Al?quotas de plasma e c?lulas sang??neas foram tamb?m precipitadas com ?cido tricloroac?tico para isolamento de fra??es sol?vel e insol?vel. Em cada fra??o a radioatividade foi contada e as porcentagens de radioatividade (%ATI) calculadas. O extrato de Artemisia vulgaris L diminuiu significantemente (p<0,05) a %ATI nas c?lulas sangu?neas e nas prote?nas celulares. A an?lise dos resultados indica que o extrato de Artemisia vulgaris L apresentaria subst?ncias que poderiam interferir no transporte de ?ons estanoso e/ou pertecnetato atrav?s da membrana do eritr?cito alterando a marca??o das c?lulas sangu?neas com 99mTc. A realiza??o deste trabalho envolveu uma equipe multidisciplinar, incluindo fisioterapeutas, biom?dicos, f?sicos, farmac?uticos, bi?logos, estat?sticos e m?dicos

Medical devices are used widely at every stage of disease diagnosis and treatment. To eradicate certain infectious diseases, the development of highly sensitive diagnostic tools and techniques is essential. The work reported in this thesis presents a novel approach, which can be used for the diagnosis of various diseases in the field of clinical cytology. The central theme of this approach was to develop a simple, holistic and completely automated system for point-of-care (POC) diagnostics. This is realized through the Development of an Absorption Flow-Cytometer with Synergistic Integration of Microfluidic, Optics and simple Electronics. Quantitative diagnosis of malaria has been taken as test case for the characterization and validation of the developed technology. Malaria is a life-threatening disease widely prevalent in developing countries. Approximately half the world population undergoes a test of malaria and it kills close to half a million people every year. Early detection and treatment will reduce the number of fatalities and also decrease its transmission rate. In the recent past, several diagnostic tools have been developed to detect malaria but there are varied demands on diagnostic instruments in healthcare settings and endemic contexts. The objective of this thesis is to develop an instrument capable of identifying malaria-infected red blood cells (i-RBCs) from a given few micro-liters of whole blood. The optical absorption properties of blood cells were measured at a single-cell level to diagnose malaria. The proof-of-concept for the instrument was established in four stages, after which a prototype was also developed and validated. In the first stage, a system capable of simultaneously imaging cells and also measuring their optical absorbance properties was developed. The developed system was employed to characterize absorption properties of red blood cells (malaria-infected and healthy ones) on blood-smear. A custom-made bright-field transmission microscope in combination with a pair of laser diode and photo-detector was used to simultaneously image and measure transmittance of infected and uninfected RBCs. In the second stage, the technique was extended to enable high-throughput measurements with the use of microfluidic sample handling and synchronous data acquisition. Using this technique, the optical absorbance and morphology of infected and healthy RBCs have been characterized in statistically significant numbers. The correlation between cell morphology (from images) and single-cell optical absorbance level helped to establish the thresholds for differentiating healthy and infected cells. In the third stage, a portable prototype capable of assessing optical absorbance levels of single cells was fabricated. The developed prototype is capable of assessing cells at throughputs of about 1800 cells/ second. It was initially validated with sample suspensions containing infected and healthy RBCs obtained from malaria cultures. For the device to be usable at the field-level, it has to function in the presence of all other cellular components of whole blood. The optical absorbance of other cellular components of blood like white blood cells and platelets, were characterized. The device was finally tested with blood samples spiked with malaria-infected RBCs validating the overall proof-of-concept and the developed prototype. The deployment of such cost-effective, automated POC system would enable malaria diagnosis at remote locations and play a crucial role in the ongoing efforts to eradicate malaria. In future, the presented technology can be extended to develop POC diagnostic tool for other diseases as well. As it enables quantitative estimation of malaria, the present optical absorption flow analyzer would also find application in disease prognosis monitoring, anti-malarial drug development and other studies requiring measurements on a single-cell basis. The hyper-imaging system can be used to characterize and validate the threshold information, and can be incorporated in the prototype. Thus, it is a continuous process to characterization and implementation in the prototype. The optofluidic absorption flow analyzer will help enable affordable clinical diagnostic testing in resource limited settings. This approach will be extended to diagnose other diseases, using differences in optical absorption as criteria for differentiating healthy and infected cells.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Os ácidos graxos poli-insaturados n-3 derivados do óleo de peixe estão associados a benefícios cardiovasculares, que podem ser decorrentes da ativação da óxido nítrico sintase (NOS). Assim como as células endoteliais, os eritrócitos possuem NOS endotelial (eNOS) e induzível (iNOS) e, portanto, são capazes de sintetizar óxido nítrico (NO). O presente estudo testou a capacidade que diferentes concentrações de óleo de peixe tem de ativar a via L-arginina-NO e, em seguida, alterar os níveis de guanosina monofosfato cíclica (GMPc) em eritrócitos de camundongos alimentados com dieta hiperlipídica. Além disso, foram analisados os marcadores de estresse oxidativo nos eritrócitos, objetivando investigar a biodisponibilidade do NO. O transporte de L-arginina, avaliado através da incubação com L-[3H]-arginina, mostrou-se ativado quando da administração de dietas contendo elevadas concentrações de óleo de peixe, em comparação com as dietas contendo baixas concentrações e controle. A atividade da NOS, medida pela conversão de L-[3H]-arginina em L-[3H]-citrulina, e a expressão da eNOS também aumentaram nos animais que se alimentaram com dietas ricas em óleo de peixe. Apesar da ativação da via L-arginina-óxido nítrico observada em nossos experimentos, os níveis de GMPc intraeritrocitário não foram afetados. O dano oxidativo nos eritrócitos aumentou linearmente conforme o óleo de peixe era acrescido na dieta, sem afetar a atividade das enzimas antioxidantes. Além do endotélio, os eritrócitos contribuem para o metabolismo do NO. Desta forma, a ativação da via L-arginina-NO nessas células pode ser benéfica para saúde cardiovascular. Estudos futuros poderão investigar outros marcadores de estresse oxidativo durante o consumo de óleo de peixe para assegurar que o seu uso não resulta em efeitos prejudiciais secundários e para garantir a biodisponibilidade de NO. / The n-3 polyunsaturated fatty acids derived from fish oil are associated with cardiovascular benefits and it has been suggested that the activation of nitric oxide synthase (NOS) would be a potential mechanism responsible for its effects. Beside endothelial cells, red blood cells (RBC) possess endothelial NOS (eNOS) and inducible NOS (iNOS), and thus are capable of synthesizing their own nitric oxide (NO). The present study tested the capacity of different amounts of fish oil to activate L-arginine-NO pathway and therefore alter cyclic guanosine monophosphate (cGMP) levels in RBC from mice fed on a high fat diet. Additionally, the oxidative status in RBC was performed to investigate NO bioavailability. L-arginine transport, assessed by incubation with L-[3H]-arginine, was activated by higher doses of fish oil, compared to control diet and to lowest doses of fish oil. RBC NOS activity, measured by the conversion of L-[3H]-arginine into L-[3H]-citrulline, and eNOS expression were also enhanced by diets rich in fish oil. Despite the L-arginine-NO activation, no effect on intra RBC cGMP basal levels was seen among the groups. Oxidative damage of RBC rises linearly with increasing amounts of fish oil in the diet without affecting the activity of antioxidant enzymes. Besides endothelium, red blood cells also contribute regulating the NO bioactivity. Therefore, the activation of L-arginine-NO pathway in RBC by fish oil would be beneficial in cardiovascular health. Future studies testing other oxidant markers during dietary fish oil supplementation will be necessary to verify that its consumption does not result in detrimental secondary effects and to ensure NO bioavailability.

Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro / Introdução: o óxido nítrico (NO) é um gás inorgânico com uma meia-vida curta e tem um papel crítico na manutenção da homeostase vascular e fluidez sanguínea. O NO é sintetizado a partir do aminoácido L-arginina por uma família de enzimas NO sintases (NOS). Estudos têm mostrado que eritrócitos expressam NOS endotelial (eNOS) funcional, que serve como uma fonte de NO intraluminal. Além disso, eritrócitos participam da defesa antioxidante removendo os radicais livres e prevenindo o dano oxidativo às membranas biológicas e a destruição do NO. Dietas hiperlípidicas estão associadas a um risco aumentado de doença cardiovacular e síndrome metabólica, mas os exatos mecanismos não estão completamente esclarecidos. O objetivo deste estudo foi investigar os efeitos de diferentes dietas hiperlípidicas na via L-arginina-NO e o estresse oxidativo em eritrócitos de camundongos. Metodologia: camundongos machos C57BL/6 de três meses de idade receberam diferentes dietas por 10 semanas: dieta normolipídica ou dieta hiperlipídica contendo banha de porco (HB), óleo de oliva (HO), óleo de girassol (HG) ou óleo de canola (HC). Foram analisados o transporte de L-arginina mediado pelos transportadores catiônicos y+ e y+L, a atividade da NOS, a expressão da eNOS e da NOS induzível (iNOS), a formação de substâncias reativas ao ácido tiobarbitúrico (TBARS) e a atividade das enzimas antioxidantes catalase (CAT) e superóxido dismutase (SOD). Resultados: o transporte total de L-arginina estava aumentado no grupo HO em comparação aos controles e aos outros grupos com dieta hiperlipídica. Quando o transporte foi fracionado, o sistema y+ estava mais ativado no grupo HO em relação aos controles e outros grupos que receberam dieta hiperlipídica. O transporte de L-arginina via sistema y+L estava maior nos grupos HO, HG e HC comparados aos grupos controle e HB. Adicionalmente, a atividade basal da NOS e a expressão de eNOS estavam aumentadas em eritrócitos independente do tipo de dieta hiperlípidica insaturada. Observou-se uma maior expressão da iNOS no grupo HO comparado ao controle. Em contraste, o grupo HB apresentou uma inibição da via L-arginina-NO. A análise da peroxidação lipídica, através da formação de TBARS, e da atividade da enzima antioxidante CAT não revelou diferenças entre os grupos, ao contrário do grupo HO, que induziu uma ativação de outra enzima antioxidante, a SOD. Conclusões: o presente estudo proporciona a primeira evidência de que os sistemas y+ e y+L regulam o transporte aumentado de L-arginina em eritrócitos de camundongos do grupo HO. Além disso, todas as dietas hiperlipídicas insaturadas induzem um aumento da atividade basal da NOS associada a uma expressão elevada da eNOS. É possível que diferentes mudanças na composição lipídica da membrana plasmática induzidas pelas dietas possam afetar transportadores e enzimas nos eritrócitos. Além disso, a inibição da via L-arginina-NO no grupo HB pode contribuir para o desenvolvimento da aterosclerose, enquanto dietas hiperlipídicas insaturadas podem ter um efeito protetor via aumento da geração de NO. / Introduction: nitric oxide (NO) is an inorganic gas with a short half life that plays a critical role in maintaining vascular homeostasis and blood fluidity in physiological conditions. NO is synthesized from the cationic amino acid L-arginine by a family of enzymes: nitric oxide synthase (NOS). Studies have shown that red blood cells (RBCs) express functional endothelial NOS (eNOS), which potentially serves as an intraluminal NO source. Moreover, circulating RBCs participate in antioxidant defence, scavenging oxygen free radicals and preventing oxidative damage to biological membranes and NO destruction. High fat diets are associated with an increased risk of cardiovascular disease and metabolic syndrome, but the exact mechanisms are not completely clear. The objective of this study was to investigate the effects of different high fat (HF) diets in the RBC L-arginine-NO pathway and in oxidative stress in C57BL/6 mice. Methods: three-month-old male C57BL/6 mice were fed different diets for a 10-week period: a standard diet or high-fat (HF) diet containing lard oil (HF-L), olive oil (HF-O), sunflower oil (HF-S) or canola oil (HF-C). Studies of L-arginine transport, mediated by cationic transport systems y+ and y+L, basal activity of NOS, expression of eNOS and inducible NOS (iNOS), thiobarbituric acid reactive substances (TBARS) formation, and antioxidant enzymes catalase (CAT) and superoxide dismutase (SOD) activities in RBCs were analysed in these groups. Results: total L-arginine influx into RBCs was upregulated in the HF-O group compared to controls and other HF diet groups. When transport systems were fractionated, there was a higher activation of system y+ in the HF-O group in relation to controls and other HF diet groups. L-arginine transport via system y+L in RBCs was increased in the HF-O, HF-S and HF-C groups compared to controls and the HF-L group. In addition, NOS activity and eNOS expression were enhanced in RBCs, independent of unsaturated HF diets. An overexpression of iNOS was observed in the HF-O group compared with controls. In contrast, the HF-L group showed an inhibition of the RBC L-arginine-NO pathway. The analysis of lipid peroxidation and antioxidant enzyme catalase activity revealed no differences among the groups studied. On the other hand, HF-O induced activation of another antioxidant enzyme, superoxide dismutase (SOD). Conclusions: this study provides the first evidence that systems y+ and y+L mediate increased L-arginine transport into mice RBCs from the HF-O group. Moreover, all unsaturated high-fat diets can induce an increase in basal NOS activity associated with an overexpression of eNOS. It is possible that changes in the lipid composition of the plasmatic membrane induced differently by HF diets could affect transporters and enzymes in RBCs. An inhibition of the L-arginine-NO pathway in HF-L group could contribute to the development of atherosclerosis, while HF unsaturated diets may have a protector effect via enhanced generation of NO.