Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Evidências crescentes têm demonstrado que o exercício prejudica a estrutura da membrana eritrocitária, como consequência do aumento do estresse físico e químico. O óxido nítrico (NO) derivado dos eritrócitos afeta a fluidez da membrana e a sua biodisponibilidade depende do equilíbrio entre a sua síntese e eliminação por espécies reativas de oxigênio. Nós investigamos se o exercício realizado em diferentes intensidades afetaria biodisponibilidade do NO eritrocitário e se levaria a um quadro de estresse oxidativo. Dez homens (26 4 anos, VO2pico 44,1 4,3 mL.kg-1.min-1) realizaram um teste cardiopulmonar máximo em esteira e um teste de exercício submáximo a 70% VO2pico durante 30 min. O sangue foi coletado em repouso e imediatamente após os exercícios para isolamento dos eritrócitos. O exercício máximo aumentou a contagem de eritrócitos, hemoglobina e hematócrito, sem levar a qualquer alteração na massa corporal que pudesse sugerir hemoconcentração devido a uma redução do volume plasmático. Observou-se uma diminuição do influxo de L-arginina depois do teste submáximo, mas não no teste máximo. No entanto, a atividade da óxido nítrico sintase, ou seja, a produção de NO, foi aumentada após o teste máximo. Os níveis de GMPc não se alteraram após ambos os teste de exercício. Em relação aos biomarcadores de estresse oxidativo, o exercício submáximo reduziu a oxidação proteica e aumentou a atividade da catalase e a expressão da glutationa peroxidase, enquanto que o exercício máximo levou a uma maior peroxidação lipídica e diminuição da atividade da SOD. Nem a atividade glutationa peroxidase ou a expressão NADPH oxidase foram afetadas pelo exercício. Estes resultados sugerem que o exercício induziu alterações no estresse oxidativo de eritrócitos, que parecem estar mais associadas com a intensidade do que a duração do execicio. Além disso, nas intensidades recomendadas para a promoção da saúde, o exercício mostrou ser protetor, aumentando a atividade e a expressão de enzimas antioxidantes importantes e reduzindo os danos oxidativos. / Growing evidence has shown that exercise impairs erythrocyte membrane structure as a consequence of increased physical and chemical stress. Erythrocyte-derived nitric oxide (NO) affects membrane fluidity, and its bioavailiability depends on the balance between its synthesis and scavenging by reactive oxygen species. Here, we investigated whether aerobic exercise performed at different intensities would affect erythrocyte NO bioavailability and oxidative stress. Ten men (26 4 years old, VO2peak 44.1 4.3 mL.kg-1.min-1) performed a treadmill maximal cardiopulmonary exercise test, and a submaximal exercise at 70% VO2peak during 30 min. Blood was collected at rest and immediately after exercises for erythrocytes isolation. Maximal exercise increased erythrocytes count, haemoglobin and haematocrit levels, without any change in body mass that could suggest haemoconcentration due to a plasma volume reduction. It was observed a decrease in L-arginine influx after moderate, but not maximal exercise. Yet, nitric oxide synthase activity, and thus, NO production, was increased after maximal exercise. Cyclic GMP levels did not change after both exercise bouts. In relation to biomarkers of oxidative stress, moderate exercise reduced protein oxidation, and increased catalase activity and glutathione peroxidise expression; whereas maximal exercise led to greater lipid peroxidation, and diminished SOD activity. Neither glutathione peroxidase activity nor NADPH expression were affected by exercise. These findings suggest that exercise induced changes in erythrocyte oxidative stress are more associated with intensity than duration. Furthermore, at intensities recommended for health promotion, exercise was shown to be protective, increasing the activity and expression of important antioxidant enzymes and reducing oxidative damage.

Made available in DSpace on 2014-08-20T13:49:19Z (GMT). No. of bitstreams: 1 CatiaFLeite.pdf: 1303895 bytes, checksum: f2a907bfd8b3da1ab25067969c014119 (MD5) Previous issue date: 2011-03-24 / Fatigue can be caused by depletion of carbohydrates and by the action of reactive oxygen species. Thus, carbohydrates are recognized as important to improve performance, minimize the decline in immune activity and reduce oxidative stress. Objective: To assess the occurrence of oxidative damage and abnormal levels of blood leukocytes in trained and sedentary rats supplemented with maltodextrina or water. Methods: Male Wistar rats (60 days) were divided into six groups: sedentary non-supplemented (n = 12) and supplemented (n = 12), trained in EEML - Maximum Lactate Steady State - not supplemented (n = 11) and supplemented (n = 11), trained at high intensity non-supplemented (n = 12) and supplemented (n = 11). The training protocol consisted of 8 weeks of swimming in a continuous pattern in EEML (60min.day-1) or intermittent (two periods of 30 minutes, with an interval of 10min), with loads corresponding to 5% and 10% of body weight,respectively. During 37 days the animals were supplemented with a daily dose of 0.48 g.kg-1 maltodextrin dissolved in water or pure water. Concentrations of blood lactate, hepatic glycogen content and muscle lipid peroxidation, protein oxidation and total and differential leukocytes were measured. Results: Despite the lack of statistical significance in malondialdehyde concentration and total and differential count of leukocytes between the groups, both exercise models have resulted in increases in protein carbonyl (p˂0.001). Aerobic exercise and supplementation with maltodextrin resulted in increased muscle glycogen content when compared to sedentary groups receiving water (p=0.007) or maltodextrin (p=0.008). Conclusions: The exercise did not cause damage to the lipid layers or abnormal levels of circulating leukocytes, however, both standards of training provided important protein loss. Maltodextrin supplementation was effective in sparing muscle glycogen stores during aerobic exercise. / A fadiga pode ser causada pela acidose metabólica, a depleção de substratos e a ação das espécies reativas de oxigênio (EROs). A hipoglicemia, por exemplo, também resulta em imunossupressão. Sendo o carboidrato um nutriente importante para retardar a acidose metabólica, melhorar o desempenho esportivo, minimizar a queda na atividade imunológica e reduzir a ocorrência de estresse oxidativo, além de haver uma escassez de estudos nesta área se reforçam a necessidade de novos estudos. Objetivo: verificar a ocorrência de dano oxidativo e alterações nas concentrações de leucócitos sanguíneos em ratos treinados e suplementados ou não com maltodextrina. Materiais e Métodos: Ratos machos (69 no total), Wistar, com 60 dias serão divididos em seis grupos experimentais: SN (sedentário não suplementado, n=12), SS (sedentário suplementado, n=12), TEN (treinado em EEML - Estado Estável Máximo de Lactato - não suplementado, n=11), TES (treinado em EEML suplementado, n=11), TAN (treinado em alta intensidade não suplementado, n=12) e TAS (treinado em alta intensidade suplementado, n=11). O protocolo de treinamento consistirá de oito semanas de exercícios de natação em padrão contínuo em EEML (60min.dia-1) ou intermitente (2 períodos de 30min, com intervalo de 10min), com sobrecargas correspondentes a 5% e 10% do peso corporal, respectivamente. Os animais serão suplementados por oito semanas com uma dose diária de 0,48g.kg-1 de maltodextrina dissolvida em água ou receberão somente água pura. As concentrações de lactato sanguíneo, conteúdo de glicogênio hepático e muscular, peroxidação lipídica através de TBARS, oxidação de proteínas e contagem total e diferencial de leucócitos serão analisadas

Rasa3 est un membre de la famille GAP1 des GTPase activating proteins. Cette protéine stimule la fonction GTPase intrinsèque des petites protéines G telles que Ras et Rap. Rasa3 présente une haute affinité pour l’Ins(1,3,4,5)P4 et le PtdIns(4,5)P2, et est principalement exprimée dans les cellules du système hématopoïétique chez la souris.<p>Dans le but d’étudier chez la souris le rôle physiologique de Rasa3 dans les cellules hématopoïétiques, nous avons d’une part généré et analysé des souris transgéniques surexprimant une forme normale (TgN) ou mutée et catalytiquement inactive (TgM) de la protéine Rasa3 dans les lymphocytes B et/ou T. D’autre part, nous avons reçu des souris Rasa3+/- exprimant une protéine Rasa3 tronquée et catalytiquement inactive. Comme les souris Rasa3-/- meurent pendant la gestation, des transferts de cellules hématopoïétiques souches de foie fœtal d’embryons Rasa3-/- ont été réalisés dans des souris SCID. Les souris SCID après reconstitution développent le système hématopoïétique des souris Rasa3-/-.<p>Nous avons découvert que l’expression de Rasa3 est régulé au niveau de l’ARNm et de la protéique après activation des lymphocytes B par la voie du récepteur aux antigènes (BCR).<p>L’analyse des souris Rasa3 TgN a permis de mettre en évidence une diminution de la survie des lymphocytes B in vitro et une diminution de la réponse immune à un antigène T-indépendant (TI) in vivo. Les souris TgM présentent un phénotype inverse aux souris TgN.<p>Six à 8 semaines après reconstitution, les souris SCID Rasa3-/- présentent un phénotype opposé à celui des souris TgN :leurs lymphocytes B ont une meilleure survie in vitro et une meilleure réponse immune à un antigène TI in vivo. On observe également chez ces souris SCID Rasa3-/- une augmentation du nombre de lymphocytes B dans certaines sous-populations spléniques.<p>L’observation prolongée des souris SCID Rasa3-/- a mis en évidence une diminution de leur survie. A l’autopsie, 90% d’entre elles développent une splénomégalie reflétant une hématopoïèse extra-médullaire. Soixante pourcents d’entre-elles présentent de l’ostéosclérose dans de la cavité médullaire du fémur, associée à une augmentation du pourcentage de mégacaryocytes. D’un point de vue sanguin, les souris SCID Rasa3-/- développent une thrombocytopénie sévère. Des hémorragies et une anémie régénérative ont également été observées. Les causes exactes de la thrombocytopénie doivent encore être investiguées dans le cadre de ce travail.<p>Enfin, environ 15 % des souris SCID Rasa3-/- ont montré une infiltration massive de la rate et de la moelle osseuse par des cellules progénitrices CD117+, suggérant qu’une fraction d’entre-elles développent une leucémie myéloïde aigue.<p> / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Cette étude a pour objectifs 1) d'évaluer l'activité érythrocytaire de l'inosine triphosphate pyrophosphatase (ITPA) et de l'inosine monophosphate deshydrogénase (IMPDH) en lien avec le suivi des métabolites thiopuriques et le phénotypage de la thiopurine S-méthyltransférase (TPMT), 2) d'étudier le lien entre l'activité de l'ITPA et la survenue d'effets indésirables observés sous azathioprine (AZA).L'étude rétrospective réalisée sur une large population de sujets adultes et enfants, sains et atteints de maladies auto-immunes, a permis d'identifier une distribution quadrimodale de l'activité de l'ITPA à l'aide d'un modèle de mélange gaussien ainsi qu'une faible variabilité intra-individuelle de cette activité. Dans la population pédiatrique, une activité de l'ITPA basse est corrélée à une augmentation des dérivés méthylés suggérant un risque d'hépatotoxicité. Le lien observé entre l'activité ITPasique, le volume globulaire moyen et les gammaglobulines chez les enfants atteints de maladie inflammatoire chronique de l'intestin sous AZA suggère que la mesure de l'activité de l'ITPA permettrait d'anticiper la persistance d'un syndrome inflammatoire chez les sujets à activité élevée.L'étude phénotypique de l'IMPDH montre une importante variabilité inter-individuelle de l'activité de cette enzyme et l'absence d'influence de l'âge, du sexe et du traitement par AZA sur cette activité. Une distribution bimodale de l'activité de l'IMPDH érythrocytaire a été observée ainsi qu'un lien entre cette activité et les dérivés méthylés.En perspective, l'étude combinée des activités de l'ITPA, l'IMPDH et de la TPMT sur la variabilité de la réponse aux traitements thiopuriques sera envisagée / The aims of this study are 1) to evaluate inosine triphosphate pyrophosphatase (ITPA) and inosine monophoshate dehydrogenase (IMPDH) activities in red blood cells (RBCs) in relation to the monitoring of thiopurine metabolites and the phenotyping of thiopurine S-methyltransferase (TPMT), 2) to investigate a possible link between ITPA activity and the occurence of adverse effects observed under azathioprine (AZA) treatment.The retrospective study was carried out on a large population of healthy adults and children as well as those suffering from immunological diseases. A quadrimodal distribution of ITPA was identified among the population using a gaussian mixture model. A weak intraindividual variability of ITPA activity was found. In the paediatric population, a low ITPA activity is correlated with increased levels of methyl nucleotides, suggesting a risk of hepatotoxicity. The relationship observed between ITPA activity and both mean corpuscular volume and gammaglobulin levels in IBD children on AZA therapy suggests that the determination of ITPA activity may allow the prediction of a persistent inflammatory process in subjects with elevated ITPA activity.The phenotypic study of IMPDH shows no influence of age, sex and AZA therapy on the activity of IMPDH. Moreover a large interindividual variability in the activity of IMPDH was found. A bimodal distribution of IMPDH activity in RBCs was observed as well as a relation between this activity and the methyl nucleotides.Further study on the combined effect of the three enzymes ITPA, IMPDH and TPMT on the variability of response to thiopurine therapy will be considered

A lack of information is identified on the ability of professional nurses working in a neonatal intensive care unit to make competent clinical decisions pertaining to the employment of bedside blood product filters during neonatal transfusion. In addition, a lack of complete guidelines existed to aid professional nurses in instances where they are faced with such a decision. A descriptive survey was conducted to determine and describe the knowledge professional nurses working in neonatal intensive care unit have regarding the employment of bedside blood product filters during the transfusion of a neonate with blood or blood products, as well as their ability to make competent clinical decisions in the regard. A questionnaire was designed, which was validated by experts in neonatal intensive care nursing and blood transfusion. The questionnaires were distributed to 10 nominated public and private hospitals with neonatal intensive care units in Gauteng, South Africa, for completion. Participants were self-nominated; they participated in the study of their own free will. Neither the participants’ nor the hospitals’ names were revealed at any stage. Numbers were used to identify the questionnaires. 120 questionnaires were completed and were analysed using descriptive statistics. In the following step, the results obtained from the questionnaires together with literature were used to recommend guidelines for neonatal transfusion utilisation by professional nurses working in neonatal intensive care units. The recommended guidelines were divided into two categories: General guidelines and Specific blood products guidelines. Validity and reliability was enhanced by using staff from ten neonatal intensive care units from the private and public health care sector in Gauteng, South Africa; obtaining a sufficient sample size (n=120); involvement of experts in the field of neonatal nursing science and blood transfusion, as well as a statistician from the University of Pretoria; and verification of results with literature. Ethical principles were adhered to: confidentiality was maintained as no names of any of the hospitals or the participants were disclosed. All information regarding the study was provided to the relevant parties and the participants voluntarily signed an informed consent form. Permission to conduct the study was obtained from the selected hospitals’ management. Approval to conduct the study was obtained from the Ethics Committee of the Faculty of Health Science, University of Pretoria. There were no known risks involved in the study. Recommendations were made for more research on the same topic to be conducted and their outcomes be compared to the results yielded by this study and research to be conducted to related topics. The findings of this study were meant to improve the clinical practice of nursing in neonatal intensive care units. It was therefore recommended that these guidelines be implemented by neonatal intensive care units, training institutions and the South African Blood Transfusion Services. AFRIKAANS : Die navorser het ’n intensiewe oorsig van die bestaande literatuur rakende die kliniese besluitneming van professionele verpleegkundiges in neonatale intensiewe sorgeenhede gedoen. Dit het duidelik uit dié navorsingsoorsig geblyk dat daar baie min gedoen is om die vermoëns vas te stel van professionele verpleegkundiges wat in die neonatale intensiewe sorgeenheid werk met betrekking tot kliniese besluitneming wat die aanwending van bloedprodukfilters tydens neonatale transfusie betref. Aanvullend het die oorsig onthul dat daar geen volledige riglyne bestaan, of voorsien word, vir professionele verpleegkundiges in gevalle waar hul met so ’n besluit gekonfronteer word. Die studie is in twee fases gedoen. In Fase een is ’n vraelys ontwerp gebaseer op konsepte en temas uit die literatuur geїdentifiseer, waarna dit gevalideer is deur kundiges. Die vraelyste is uitgegee aan 10 genomineerde privaat- en publieke hospitale met neonatale inesiewe sorgeenhede in Gauteng vir voltooiing. Die voltooide vraelyste is gesorteer en geanaliseer. Deelnemers is self-genomineerd; die navorser het hulle toegelaat om self te besluit of hulle wou deelneem aan die studie of nie. Deelnemers en hospitale se name is op geen stadium bekend gemaak nie. Nommers is gebruik om die vraelyste te identifiseer. In Fase twee is die uitslae van die vraelyste gebruik om riglyne te ontwerp om gebruik te word in neonatale intensiewe sorgeenhede deur professionele verpleegkundiges vir neonatale transfusie. Riglyne vir neonatale transfusie is geformuleer volgens die uitkoms van die vraelyste in kombinasie sowel as die oorsig van die reeds bestaande literatuur deur middel van induktiewe en deduktiewe beredenering. Aangesien professionele verpleegkundiges kennis moet dra van algemene riglyne om spesifieke riglyne te kan toepas, is die riglyne in twee kategorieë verdeel: Algemene riglyne en Spesifieke bloedprodukte riglyne. Deur hierdie studie in twee verskillende omgewings, naamlik privaat- en publieke instansies te doen, en 10 eenhede van een area (Gauteng) in Suid- Afrika daarby te betrek, sowel as om ’n relatief groot steekproef te gebruik, is die oordraagbaarheid van die uitkoms van die studie na ander streke verhoog. Die navorser het aanbeveel dat verdere studies oor dieselfde onderwerp gedoen word, en die uitkomste daarvan vergelyk word met die resultaat van hierdie studie. Die uitkoms die studie sal dan meer oordraagbaar wees na ander streke in Suid-Afrika. Die uitgebreide literatuuroorsig, die betrokkenheid van kundiges en die navorser se eie ondervinding en kennis in die neonatale intensiewe sorgverpleegkunde, het bygedra tot die vertrouenswaardigheid van die studie. Kundiges op die gebied van neonatale verpleegkunde en bloedtransfusie was betrokke, sowel as ’n biostatistikus verbonde aan die Universiteit van Pretoria. Etiese beginsels is gevolg. Die navorser was nie bewus van, en is ook nie gekonfronteer met enige etiese dilemmas of probleme tydens die studie nie. Konfidensialiteit is deurgaans gehandhaaf in die studie deurdat geen name van die deelnemers of hospitale genoem is nie. Alle inligting met betrekking tot die studie is aan die relevante partye verstrek en die deelnemers het almal uit eie vrye wil ’n ingeligte toestemmingsvorm onderteken. Toestemming om die studie te doen is ook verkry van die genomineerde hospitale se bestuur. Toestemming om die studie te doen is verkry van die Etiese Komittee van die Fakulteit van Gesondheidswetenskappe, Universiteit van Pretoria. Daar was geen risiko’s betrokke aan die studie nie, slegs voordele, aangesien die doel van die studie was om die professionele verpleegkundiges in die neonatale intensiewe sorgeenhede behulpsaam te wees met besluitneming rakende die gebruik van bloedprodukfilters tydens transfusie van bloed of bloedprodukte aan ’n neonaat. Die bevindings van die studie is bedoel om die kliniese praktyk van verpleging in neonatale intensiewe sorgeenhede te verbeter. Gevolglik word ’n aanbeveling gemaak dat hierdie riglyne geïmplimenteer word in neonatale intensiewe sorgeenhede, opleidingsinstansies sowel as die Suid-Afrikaanse Bloedoortappingsdiens. / Dissertation (MCur)--University of Pretoria, 2011. / Nursing Science / unrestricted

Made available in DSpace on 2016-05-02T13:54:46Z (GMT). No. of bitstreams: 1 ClaudiaUmbelinaBaptistaAndrade.pdf: 352738 bytes, checksum: 380be5e60412ee243294a4902837952a (MD5) Previous issue date: 2007-11-08 / Plants are a source of many biologically active products and nowadays they are of great interest to the pharmaceutical industry. In the present study, the mutagenic potential of the fruit peels extract from Musa paradisiaca was assessed using the single cell gel electrophoresis (Comet assay) and micronucleus assays. Animals were treated orally with three different concentrations of the extract (1000, 1500 and 2000 mg/kg of body weight). Peripheral blood cells of Swiss mice were collected 24 h after the treatment for the comet assay and 48 and 72 h for the micronucleus test. The results showed that the extract of M. paradisiaca induced statistically significant increases in the average numbers of DNA damage in peripheral blood leukocytes for the two higher doses and a significant increase in the mean of the micronucleated polychromatic erythrocytes at three tested doses. The polychromatic/normochromatic erythrocytes ratio (PCE/NCE) scored in the tested groups was not statistically different from the negative control, showing that the extract presented no cytotoxic effects. The data obtained indicate that fruit peels extract from M. paradisiaca showed mutagenic effect in the peripheral blood cells of Swiss albino mice. / As plantas em geral são fontes de muitos produtos com atividades biológicas, e atualmente são de grande interesse para a indústria farmacêutica. Musa paradisíaca é uma dessas plantas, cuja casca vem sendo utilizada para tratamento de fissuras na pele, devido ao seu poder cicatrizante, e, devido aos seus altos valores energéticos e nutritivos, também tem servido de alimentação alternativa através da farinha. Visto que nunca foi investigado o efeito da casca desta planta sobre o genoma de mamíferos, foi objetivo deste trabalho analisar o potencial mutagênico do extrato de cascas de Musa paradisíaca sobre células sangüíneas de camundongos Swiss in vivo. Para esta avaliação, foram utilizados o ensaio cometa e o teste do micronúcleo. Os animais foram separados em cinco grupos de seis animais cada, onde em três deles foram testadas, por via oral, três diferentes concentrações do extrato (1000, 1500 e 2000 mg/kg de peso corpóreo). As células do sangue periférico foram coletadas 24 horas após o tratamento para a realização do ensaio cometa e 48 e 72h para o teste do micronúcleo. Os resultados obtidos com o ensaio cometa mostraram que o extrato de Musa paradisíaca induziu aumento estatisticamente significativo na quantidade de danos no DNA dos leucócitos de sangue periférico nas duas maiores concentrações do extrato, e, pelo teste do micronúcleo, um aumento também significativo na média de eritrócitos policromáticos micronucleados nas três doses testadas. A relação de eritrócitos policromáticos e normocromáticos (PCE/NCE) em 1000 células por animal não mostrou diferença significativa em relação ao grupo controle negativo, indicando que o extrato não apresenta citotoxicidade. Com base nas condições do ensaio desenvolvido, os dados obtidos revelaram que o extrato de cascas de Musa paradisíaca apresentou efeito mutagênico em células de sangue periférico de camundongos Swiss albinos.

Human cells from acute myeloid leukemia (AML) patients are frequently transplanted into immune-compromised mouse strains to provide an in vivo environment for studies on the biology of the disease. Since frequencies of leukemia re-initiating cells are low and a unique cell surface phenotype that includes all tumor re-initiating activity remains unknown, the underlying mechanisms leading to limitations in the xenotransplantation assay need to be understood and overcome to obtain robust engraftment of AML-containing samples. We report here that in the NSG xenotransplantation assay, the large majority of mononucleated cells from patients with AML fail to establish a reproducible myeloid engraftment despite high donor chimerism. Instead, donor-derived cells mainly consist of polyclonal disease-unrelated expanded co-transplanted human T lymphocytes that induce xenogeneic graft versus host disease and mask the engraftment of human AML in mice. Engraftment of mainly myeloid cell types can be enforced by the prevention of T cell expansion through the depletion of lymphocytes from the graft prior transplantation.

Chez l’homme, les erreurs innées du métabolisme des lipides sont dues à des déficits enzymatiques, entraînant une accumulation intracellulaire de substrats lipidiques. Il en résulte un large éventail de symptômes tels que des atteintes viscérales, osseuses et dans certains cas neurologiques. En outre, de nombreux patients atteints de ce type de maladie présentent des anomalies hématologiques et vasculaires attribuées à des anomalies rhéologiques du globule rouge (GR). Ces observations ont conduit à l’hypothèse de l’existence d’un lien entre les propriétés anormales du GR et sa composition lipidique. Or actuellement, le profil lipidique du GR normal reste méconnu. Cependant, le diagnostic précoce de ces troubles est d’une importance capitale pour la prise en charge des patients, notamment dans les cas où un traitement correctif est disponible. La maladie de Gaucher (MG) de type 1, qui est une maladie lysosomale caractérisée par un déficit en β-glucocérébrosidase et pour laquelle un traitement enzymatique substitutif (ERT) est proposé, en est le meilleur exemple. D’où l’intérêt de disposer d’un outil simple et rapide de diagnostic de ce type de maladie.Dans le cas de la MG, le diagnostic repose encore sur la mise en évidence, laborieuse, du déficit enzymatique. Néanmoins, des travaux récents suggèrent que les anomalies rhéologiques du GR pourraient être dues à l’accumulation de quatre sphingolipides, le glucosylcéramide, la glucosylsphingosine, la sphingosine et la sphingosine-1-phosphate, qui seraient de bons candidats biomarqueurs. Or, les méthodes actuelles de dosage de ces sphingolipides nécessitent au moins deux étapes chromatographiques, avec pour chacune une étape longue et fastidieuse de préparation de l’échantillon, ce qui ne facilite guère une approche lipidomique de ce sujet. En outre, seul le glucosylcéramide a été dosé dans le GR tandis que les trois autres sphingolipides n’ont été dosés que dans le plasma. Ces candidats biomarqueurs restent donc à valider.Dans cette thèse, nous avons développé et validé une méthode simple et rapide, par UHPLC-MS/MS, de dosage simultané des 4 sphingolipides impliqués dans la MG. L’application de cette méthode à des GR provenant de patients atteints de la MG, en collaboration avec l’Institut National de Transfusion Sanguine et la société Shire, nous a permis de : 1- valider un biomarqueur parmi les quatre proposés et de montrer que les trois autres n’étaient pas suffisamment spécifiques ; 2- vérifier l’efficacité du traitement ERT actuellement proposé et 3- confirmer l’hypothèse de départ reliant les anomalies rhéologiques du GR à sa composition lipidique.De même, une étude systématique des conditions opératoires nous a permis de généraliser la méthode proposée à l’identification et au dosage de l’ensemble des sphingolipides présents dans un GR ainsi que des phospholipides, constituants majoritaires de sa membrane. Appliquée à la quantification simultanée d’une trentaine de sphingolipides et de phospholipides dans le GR normal et celui de la MG, cette méthode nous a permis de mettre en évidence l’implication d’autres lipides polaires dans la maladie de Gaucher, outre les 4 sphingolipides jusqu’alors proposés. De même, il est prévu de l’adapter à moyen terme pour le profilage total, par classe, de tous les lipides présents dans le GR.Enfin, nous avons évalué d’autres techniques de SM telles que la haute résolution et la mobilité ionique (TWIMS et DIMS) dans le but d’affiner la recherche de nouveaux biomarqueurs, notamment par l’identification des lipides isomères non discriminables par les techniques de MS conventionnelles. Grâce à une collaboration avec le Laboratoire de Chimie Physique (LCP, CNRS UMR 8000) nous avons montré la faisabilité de cette approche en séparant en DIMS deux isomères : la galactosylsphingosine 18:1 et la glucosylsphingosine 18:1 et nous poursuivons actuellement cette étude pour séparer d’autres couples d’isomères. / In humans, hereditary disorders of lipid metabolism are due to enzyme deficiencies, resulting in intracellular accumulation of lipid substrates. This results in a wide range of symptoms such as visceral, bone and in some cases neurological disorders. Furthermore, many patients suffering such diseases have hematologic and vascular symptoms attributed to red blood cell (RBC) rheological abnormalities. These observations led to a hypothesis linking RBC abnormal properties to its lipid composition. However, the lipid profile of normal RBC remains unknown to date. Early diagnosis of these conditions is of importance notably when a therapy is available. This is the case for Gaucher disease (GD) type 1, a lysosomal disorder characterized by β-glucocerebrosidase deficiency, where an enzyme replacement therapy (ERT) is proposed. Hence, the availability of a simple and rapid tool of diagnosis of such a disorder is of great importance, notably for a better patient care and monitoring.To the best of our knowledge, standard diagnosis procedures and monitoring of GD patients are still based on the tedious evaluation of enzyme deficiency. Nevertheless, recent works suggest that these rheological disorders may be due to the accumulation of four sphingolipids, glucosylceramide, glucosylsphingosine, sphingosine and sphingosine-1-phosphate, which could be considered as relevant biomarkers. However, most of current determination methods of these sphingolipids require at least two liquid chromatographic runs, each with a time-consuming sample preparation step that does not facilitate a lipidomic approach. In addition, only glucosylceramide was quantified in RBC while the other three sphingolipids were quantified only in plasma. Thus, these biomarker candidates remain to be validated.In this PhD, we describe a simple and rapid UHPLC-MS/MS method of simultaneous determination of the 4 sphingolipids involved in GD in both plasma and RBC. The application of this method to RBC from GD patients, in collaboration with the Institut National de Transfusion Sanguine and Shire (USA), allowed us: 1- to validate one biomarker among the four proposed candidates and to show that the other three candidates are not specific; 2- to check the efficiency of the proposed ERT and 3- to confirm the initial hypothesis linking the RBC rheological abnormalities to its lipid composition.Also, a systematic study of the operating conditions allowed us to generalize the proposed method to the determination of not only all the sphingolipids present in RBC but also all phospholipids, which are the major constituents of its membrane. The application of the later method to the simultaneous quantification of thirty sphingolipids and phospholipids in normal and GD RBCs, allowed us to validate it and to unravel the involvement of other candidate biomarkers of GD, different from the 4 previous sphingolipids. Providing appropriate modifications, this method is intended to be used for the profiling of all lipid classes in plasma and RBC. This is our main objective in the medium-term.Finally, we evaluated other modern MS techniques such as high resolution (HRMS) and ion mobility (TWIMS and DIMS) in order to refine the investigation of new biomarker candidates, including the separation of lipid isomers that cannot be discriminated by conventional MS techniques. Indeed, in collaboration with the Laboratoire de Chimie Physique (LCP, CNRS UMR 8000), we here show the feasibility of this approach by achieving the separation of two isomers, by the DIMS technique: galactosylsphingosine 18:1 and glucosylsphingosine 18:1, which cannot be separated by conventional methods. We are currently pursuing these investigations in order to separate other isomers.

Cette thèse porte sur l’étude des interactions entre les globules rouges (GRs)et l’endothélium, la couche des cellules qui délimite les vaisseaux sanguins.Il a été démontré que l’endothélium et les GRs jouent des rôles actifs dans divers processus du système vasculaire, et leurs interactions produisent un signal bio chimique grâce à des moyens à la fois chimiques (molécules de signalisation) et mécaniques (taux de cisaillement sur les parois). D’abord,nous étudions le rôle des GRs, y compris dans des conditions pathologiques, dans la création de contraintes de cisaillement spatialement et temporellement dynamiques sur l’endothélium. Il a été montré que les contraintes de cisaillement constituaient un élément critique dans le déclenchement d’un signal bio mécanique depuis l’endothélium. Par ailleurs, étant donné qu’il a été montré que les parois des vaisseaux sanguins ondulent en raison des cellules endothéliales individuelles qui le composent, nous avons intégré à notre modélisation cette géométrie. On trouve que cette ondulation affecte la dynamique des GRs dans l’écoulement ainsi que le taux de cisaillement sur les parois. Nous étudions rapidement dans quelle mesure la déformabilité d’un GR affecte sa trajectoire dans un vaisseau ondulé. Pour cela, nous nous inspirons du processus de fonctionnement un appareil de déplacement latéral déterministe (DLD) qui utilise les variations de trajectoires des particules en fonction de leur taille pour les séparer dans l’écoulement. Nous étudions par ailleurs l’effet des suspensions de GRs sur les caractéristiques rhéologiques et les contraintes de cisaillement sur la paroi du vaisseau.Finalement, nous nous adressons à les interaction chimiques en développons un modèle numérique avec la méthode de Boltzmann sur réseaux-limite immergée (LB-IBM) pour résoudre la diffusion et l’advectiond’un soluté libéré par un particule en mouvement et déformable. L’oxygène et l’adénosine triphosphate (ATP) sont toutes les deux libérées par les GRs,se diffusent dans l'écoulement, et sont absorbées par l’endothélium. Ils représentent des facteurs de signalisation critiques pour les processus de l’inflammation et vasodilatation. Nous montrons que la morphologie des GRs affectera le temps de résidence et la dilution des espèces chimiques lorsqu’elles rentreront en contact avec la paroi du vaisseau. Ensemble, ces éléments nous conduisent vers la développement d’un modèle capable de simuler des processus vitaux du système vasculaire qui résultent d’événements locaux de composants individuels. / This thesis is devoted to the study of the interactions between red blood cells (RBCs) and the endothelium, the monolayer of cells lining blood vessels. The endothelium and RBCs have been shown to be active participants in various processes in the vascular system, and their interactions trigger biochemical signalling by mechanical (wall shear stress) and chemical (signalling molecules) means. We first investigate the role of RBCs, including pathological conditions, in creating time- and space-varying shear stress on the endothelium. Shear stress has been shown to be a critical element in biochemical signalling from the endothelium. In addition, as it has been shown that the endothelium is undulating due to the individual endothelial cells comprising it, we take this into account in our model of the geometry of the vessel wall. We find that this undulation affects the dynamics of the RBCs in the flow and the wall shear stress. We briefly explore how the deformability of a single RBC affects its trajectory in undulating channels, inspired by the idea behind deterministic lateral displacement devices (DLDs) which exploit the differing trajectories of particles based upon their sizes to separate them in flow. We also investigate the effect of suspensions of RBCs in undulating channels on rheological properties and wall shear stress. Finally, we address the chemical interactions by building a numerical model with the lattice Boltzmann-immersed boundary method (LB-IBM) to solve advection-diffusion of solute released from moving, deformable particles. Oxygen and adenosine triphosphate (ATP) are both released by RBCs and are advected and diffused in the flow and uptaken by the endothelium and serve as critical signalling factors in inflammation and vasodilation. We find that the morphology of RBCs will affect the residence time and dilution of the chemical species upon contact with the wall. Together, these elements lead us towards the development of a model capable of simulating vital processes in the vascular system which result from local interactions of individual components.

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