Thèses : « Diseased red cell »

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Cytlak, Urszula Malgorzata. « Phosphatidylserine exposure in red blood cells from patients with sickle cell disease ». Thesis, University of Cambridge, 2015. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.708601.

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Barber, Latorya Arnold. « The Activity of Lipid Transport Proteins in Normal and Sickle Red Blood Cells ». University of Cincinnati / OhioLINK, 2009. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1243353188.

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Al, Balushi Halima. « Novel approach towards pathogenesis and treatment of sickle cell disease ». Thesis, University of Cambridge, 2019. https://www.repository.cam.ac.uk/handle/1810/288739.

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Sickle cell disease (SCD) is one of the most common genetic diseases worldwide. HbS polymerisation causes altered red blood cell (RBC) rheology and fragility, increase in blood viscosity with blockage of small blood vessels, and RBC membrane permeability changes. Excessive levels of cell-free Hb, high autoxidation of Hb, contribute to the production of reactive oxygen species (ROS) in SCD patients. In this work, oxidants showed direct and indirect effects on the main cation permeability pathways involved in dehydration of HbS/S RBCs - Psickle, the Gardos channel and the KCl cotransporter (KCC) - and thus on RBC volume causing polymersation. Psickle and Gardos channel activities showed significant correlation, consistent with the hypothesis that Ca2+ entry via Psickle causes activation of the Ca2+-dependent K+ channel. Treatment of SCD remains inadequate relying on the blood transfusion and supportive therapy depending on the organ affected. In the present study antioxidants and aromatic aldehydes showed some promising results towards future alternative treatments for SCD. Antioxidants showed inhibitory effects on the cation permeability pathways leading to inhibition of polymerisation and haemolysis and thus maintained RBC volume. Aromatic aldehydes interact with HbS and are usually given to increase oxygen affinity, thereby reducing its tendency to polymerise. GBT1118 had a marked inhibitory effect on all three cation permeability pathways. It reduced sickling, Psickle and Gardos activity. It inhibited KCC by affecting the regulatory protein phosphorylation cascade. It maintained RBC hydration, and stabilised RBCs. Historically Oman was the principal trading port of the Persian Gulf region, resulting in the complex mix of social and ethnic backgrounds. In 1989 a second mutation in the β chain of Hb, at position β121 was found in an Omani patient in addition to the usual HbS mutation at the β6 position, and termed HbS-Oman. At low percentage of HbS-Oman patients show severe SCD symptoms. Despite RBCs containing at most 25% HbS-Oman, there was high sickling percentage and K+ permeability showed many features similar to those seen in homozygous HbS/S patients. The presence of α thalassaemia was protective and represents an obvious potential prognostic marker for this rare SCD genotype. Overall, the present work contributes to elucidation of the pathogenesis of SCD, suggests approaches to the development of novel therapies and increases our understanding of a rare SCD genotype, HbS-Oman.

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Lizarralde, Iragorri Maria. « Impact of mechanical and oxidative stress on red blood cell properties in sickle cell disease ». Thesis, Sorbonne Paris Cité, 2018. http://www.theses.fr/2018USPCC324.

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Le globule rouge est une cellule simple avec l’une des fonctions les plus importantes de l’organisme : participer aux échanges gazeux et fournir l’oxygène aux tissus. C'est un disque biconcave hautement élastique grâce à un réseau de protéines du cytosquelette et de protéines membranaires spécifiques. La fonction, ainsi que la structure des globules rouges sont altérées dans plusieurs pathologies humaines telles que les hémoglobinopathies et les anomalies de membrane. La drépanocytose est une maladie héréditaire génétique caractérisée par une hémoglobine anormale qui polymérise en conditions hypoxiques provoquant la déformation des globules rouges circulants. La drépanocytose se caractérise également par une anémie hémolytique chronique et des crises vaso-occlusives douloureuses dues à l'obstruction des capillaires.Dans le but de mieux comprendre les mécanismes responsables des manifestations cliniques, nous avons étudié les propriétés mécaniques et adhésives des globules rouges de patients atteints de drépanocytose en évaluant 1) l'impact de contraintes mécaniques répétées sur la survie des globules rouges à l'aide d'un appareil microfluidique qui mime la microcirculation et 2) le rôle du stress oxydant dans l'activation des protéines d'adhérence érythroïde.Nous avons conçu une puce microfluidique et montré que le stress mécanique est un paramètre critique sous-jacent à l'hémolyse intravasculaire dans la drépanocytose et que des taux intracellulaires élevés d'hémoglobine fœtale préviennent cette lyse. Outre ces résultats, nous avons constaté que le traitement à l'hydroxyurée protège les globules rouges de la lyse lors d'un stress mécanique, même en l'absence d'expression de l'hémoglobine fœtale. D'autre part, nous avons étudié la structure et la fonction de la protéine d'adhérence érythroïde Lu/BCAM dans des conditions oxydantes en utilisant des approches biochimiques et d'imagerie. Nous avons observé que le stress oxydant active la fonction adhésive de Lu/BCAM par des modifications post-traductionnelles qui modifient sa distribution membranaire. Nous décrivons un nouveau mécanisme qui affecte les interactions en cis de Lu/BCAM à la surface cellulaire et qui pourraient expliquer l'adhérence anormale des globules rouges à la laminine en l'absence d'événements de phosphorylation.En conclusion, nous avons développé un modèle microfluidique reproduisant les dimensions physiologiques des microvaisseaux humains, permettant d’évaluer les caractéristiques cellulaires jusque-là inexplorées dans la drépanocytose. Nous montrons que le stress mécanique répété est en partie responsable de l'hémolyse chez les patients atteints d'anémie falciforme, ce qui pourrait contribuer aux niveaux élevés de stress oxydant en raison de l'hème libre dans la circulation. Nos travaux démontrent l'importance de la dimension mécanique dans l’obstruction des capillaires et la contribution critique du stress oxydant dans l'adhérence anormale des globules rouges dans cette maladie. Améliorer la déformabilité des globules rouges et cibler le stress oxydant pour inhiber l'adhérence des globules rouges serait une stratégie prometteuse pour cibler les principales caractéristiques de cette pathologie et alléger le fardeau de la maladie
The red blood cell is a simple cell with one of the most important functions in the organism, that is fulfilling the gas exchange function and delivering oxygen to the tissues. It is a highly elastic biconcave disk thanks to a network of specific skeletal and membrane proteins. The function and structure of the red cell are altered in several human pathologies like hemoglobinopathies and membrane disorders. Sickle cell disease is a genetic hereditary disorder characterized by abnormal hemoglobin that polymerizes under hypoxic conditions leading to the sickling and alteration of circulating red cells. The hallmarks of sickle cell disease are hemolytic anemia and painful vaso-occlusive crises due to the obstruction of fine capillaries.With the aim of better understanding the mechanisms behind these clinical manifestations we investigated the mechanical and adhesive properties of red blood cells from patients with sickle cell disease by assessing 1) the impact of repeated mechanical stress on red cell survival using a microfluidic device that mimic human microcirculation, and 2) the role of oxidative stress in the activation of erythroid adhesion proteins.We designed a microfluidic device that allowed us to show that mechanical stress is a critical parameter underlying intravascular hemolysis in sickle cell disease and that high intracellular levels of fetal hemoglobin protect against lysis. Furthermore, we found that treatment with hydroxyurea protects red blood cells from lysis upon mechanical stress even in the absence of fetal hemoglobin expression. On the other hand, we investigated the structure and function of the erythroid adhesion protein Lu/BCAM under oxidative conditions using biochemical and imaging approaches. We observed that oxidative stress activates the adhesive function of Lu/BCAM through post-translational modifications that alter its membrane distribution. We describe a novel mechanism that affects Lu/BCAM cis-interactions at the cell surface that might account for the abnormal adhesion of sickle red cells to laminin in the absence of phosphorylation events.In conclusion, we developed a microfluidic device replicating the physiological dimensions of human microvessels that allows assessing previously unexplored cellular characteristics in sickle cell disease. We show that repeated mechanical stress is partly responsible for hemolysis in patients with sickle cell disease, which might contribute to the high levels of oxidative stress because of free heme in the circulation. Our work demonstrates the importance of the mechanical dimension in the blockade of small capillaries and the critical contribution of oxidative stress in the abnormal adhesion of red cells in this disease. Improving red cell deformability and targeting oxidative stress to inhibit red cell adhesion would be promising strategies to target the main hallmarks of this pathology and alleviate the disease burden

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Simionato, Greta [Verfasser]. « The influence of hypoxia in erythropoiesis and morphology of red blood cells in sickle cell disease and hereditary spherocytosis. / Greta Simionato ». Saarbrücken : Saarländische Universitäts- und Landesbibliothek, 2020. http://d-nb.info/1221599666/34.

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Boa-Amponsem, Kwame Jr. « Genetics, humoral immunoresponsiveness, and disease resistance in chickens ». Diss., Virginia Tech, 1998. http://hdl.handle.net/10919/30580.

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Lines of White Leghorn chickens selected > 20 generations for (HA) and against (LA) antibody response to SRBC injected i.v. from 41 to 51 days of age, are now known to have diverged in primary antibody response to SRBC. Experiments described in this dissertation were designed to further evaluate the immune competence of these lines as influenced by age, diet, and a disease agent. A crossing experiment was also conducted to further describe the mode of inheritance of such competence. Humoral immunocompetence was evaluated by primary, memory, and maternal antibody responses to SRBC. Primary antibody response, measured 5, 10, and 20 days after inoculation with SRBC was greater in HA than LA chicks inoculated at 14, 21, and 28 days of age. In chicks injected at 7 days of age, a higher frequency of responders was observed for HA than LA chicks suggesting an earlier onset of immunocompetence in the high than low antibody line. Immunological memory antibody responses (secondary and tertiary) was studied in parallel experiments on two groups of chicks hatched at a 14-day interval. Chicks in both hatches were from the same matings of parental Lines HA and LA. Memory responses were evident in chicks at 14 days of age. Antibody responses to a second and third inoculation with SRBC were similar for both lines suggesting that genetic factors that influence primary and memory responses are not the same. The responses of LA chicks to repeat inoculations with SRBC were anamnestic whereas those of HA chicks initially inoculated at 28 days of age were not anamnestic. This study did not establish any major influence of nutrient density on either primary or memory immune responses even though the higher nutrient density diet improved growth performance. Assays in chicks indicated that maternal antibodies were transferred earlier into eggs laid by HA hens than in those of LA hens ( 7 to 9 days vs 10 to 12 days after inoculation) regardless of dosage administered. Response patterns whether assessed in terms of frequency of detection or magnitude of response showed divergence between the lines. Chicks of parental, reciprocal F , F , and backcrosses of 1 2 mating combinations of Lines HA and LA were injected with SRBC at 36 days of age. Contrasts between parental lines for antibody titers measured 5 and 12 days later showed higher antibody titers in HA than LA chicks. Sex-linked effects were evident because reciprocal contrasts for F crosses, individual heterosis, and 1 maternal heterosis were sex dependent. Response to marble spleen disease virus ( MSDV) measured 6 days after inoculation of chicks from parental, reciprocal F1, F2, and backcross matings of the lines, indicated that the mode of inheritance of spleen weight differed after infection. In the infected chicks, parental contrasts for absolute and relative spleen weights showed greater resistance to MSDV in LA than HA chicks. No other genetic effect was consistently important after infection.
Ph. D.

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Panknin, Christina Monika [Verfasser]. « Characterization of red blood cell functions in health and coronary artery disease / Christina Monika Panknin ». Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2017. http://d-nb.info/1136421815/34.

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Kucukal, Erdem. « BIOMIMETIC MICROFLUIDIC PLATFORMS FOR MONITORING CELLULAR INTERACTIONS IN MICROSCALE FLOW ». Case Western Reserve University School of Graduate Studies / OhioLINK, 2020. http://rave.ohiolink.edu/etdc/view?acc_num=case1576231265150031.

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Claveria, Pizarro Viviana Andrea. « Flow of healthy and sickle red blood cells in microcirculatory conditions : clustering process and self-margination phenomenon ». Thesis, Montpellier, 2017. http://www.theses.fr/2017MONTS081/document.

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J'ai caractérisé expérimentalement la formation de clusters au cours du passage de globules rouges (GRs) sains et drépanocytaires dans microcapillaires droites. L'effet de l'agrégation a été également étudié. J'ai montré que la formation des clusters dans des conditions physiologiques est due à la combinaison des interactions hydrodynamiques et des celles causées par les macromolécules du plasma. En effet, les interactions macromoléculaires ne sont pas complètement atténuées sous contraintes de cisaillement physiologiques et au contraire ils contribuent à la stabilité des clusters. En outre, j'ai découvert la présence d’une distribution bimodale en ce qui concerne les distances entre les cellules constituant les clusters hydrodynamiques.En plus, j'ai étudié expérimentalement le comportement collectif des globules rouges drépanocytaires oxygénés et leur distribution radiale le long de microcapillaires cylindriques avec un diamètre comparable à ces des veinules et des artérioles humaines. J'ai trouvé que les GRs montrent une distribution hétérogène en fonction de leur densité: les cellules plus légères ont tendance à rester prés du centre du canal, alors que la plupart des cellules denses (et aussi plus rigides) auto-marginent sous des conditions définies. L'agrégation semble d'inhiber l'auto-margination en fonction des patients et en particuliers des facteurs d’agrégation: le dextrane, par exemple, favorise l'auto-margination dans certains patients et il la diminue dans des autres. Le plasma montre de contraster l'auto-margination des GRs dans tous les sujets, en soulignant l'importance des protéines et des molécules adhésives du plasma dans les phénomènes d'agrégation. Finalement, j'ai observé que l'auto-margination se manifeste naturellement au cours de l’écoulement de globules rouges drépanocytaires
I experimentally characterized the clustering formation of healthy and sickle red blood cells (RBCs) flowing through straight micro-capillaries. The effect of aggregation was also investigated. I found that cluster formation under physiological conditions is most likely caused by a combination of hydrodynamic and macromolecule-induced interactions. Macromolecule-induced interactions are not fully overcome by shear stresses within the physiological range, and they contribute to cluster stability. Moreover, I found that a pronounced bimodal distribution of the cell-to-cell distances in the hydrodynamic clusters is produced.Additionally, I investigated experimentally the collective behavior of oxygenated sickle RBCs and their distribution along cylindrical micro-capillaries with diameters comparable to a human venule or arteriole. I have shown that there is a heterogeneous distribution of RBCs according to their density: low-density cells tend to stay closer to the center of the channel, while most dense cells (also more rigid) self-marginated under defined conditions. Aggregation seems to inhibit self-margination depending on the aggregative factor and patient: dextran allows self-margination in some patients and inhibits it in others. Plasma inhibits self-margination of cells in all cases, highlighting the importance of the plasma proteins and adhesive molecules in the aggregation phenomena

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El, Hoss Sara. « Novel insights into the role of fetal hemoglobin in spleen function, red cell survival and ineffective erythropoiesis in sickle cell disease ». Thesis, Université de Paris (2019-....), 2019. https://theses.md.univ-paris-diderot.fr/ELHOSS_Sara_va2_20190924.pdf.

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La drépanocytose est une maladie génétique héréditaire récessive causée par la substitution d'un acide aminé dans la chaîne β-globine aboutissant à la production d'hémoglobine anormale (HbS). Dans des conditions hypoxiques, l’HbS polymérise entraînant une falciformation et une perte de déformabilité des globules rouges (GR). Au cours de la drépanocytose, le dysfonctionnement splénique entraîne des complications potentiellement mortelles, en particulier chez les jeunes enfants. Généralement, une asplénie fonctionnelle précède la survenue d’une asplénie anatomique avec cependant une grande variabilité inter-individuelle du fait de facteurs modulateurs génétiques et environnementaux. L’hémoglobine fœtale (HbF) est le principal modulateur de la gravité de la maladie, car la présence d’HbF inhibe la polymérisation d’HbS, retardant et prévenant ainsi les complications graves, améliorant la qualité de vie des patients et augmentant leur survie. Il existe une hétérogénéité assez bien caractérisée de la concentration et de la distribution cellulaire d'HbF dans les GR circulants, mais le rôle de l'HbF au cours de l'érythropoïèse est mal documenté.Dans le but de mieux comprendre le rôle de l’HbF dans la perte de fonctionnalité splénique, la survie des GR et l'érythropoïèse inefficace, nous avons étudié 1) l'histoire naturelle du dysfonctionnement splénique chez les enfants drépanocytaires, 2) l'expression cellulaire et la distribution de l'HbF comparativement chez les enfants, chez les patients naïfs et ceux traités par hydroxycarbamide (HC) et 3) le rôle de l'HbF au cours de l'érythropoïèse terminale.Nous avons d’abord développé une méthode de cytométrie en flux à haut débit pour mesurer la fonction de filtration splénique et avons montré que la perte de fonction splénique est présente très tôt dans la vie, dès 3 à 6 mois chez les enfants drépanocytaires, puis diminue avec l'âge. Nous avons également mis en évidence que les cellules irréversiblement falciformées (ISC) jouaient un rôle dans la survenue de la séquestration splénique aiguë, laquelle entraîne à son tour une perte supplémentaire de fonction. Dans la deuxième partie de ce travail, nous avons mis en place une approche originale pour déterminer la distribution d'HbF à l’échelle cellulaire. En utilisant une cohorte longitudinale de patients traités par HC (inducteur d'HbF), nous avons montré que celui-ci avait un impact positif global sur les GR, en augmentant non seulement le contenu en HbF, mais également le volume de tous les GR, indépendamment de leur contenu en HbF. Nous avons par ailleurs montré que les cellules riches en HbF (High-F) étaient un marqueur précis d’efficacité de l'HC. Dans la dernière partie de la thèse, nous avons démontré pour la première fois une érythropoïèse inefficace chez les patients drépanocytaires et avons révélé un nouveau rôle de l'HbF au cours de l'érythropoïèse terminale, celui de protéger les érythroblastes de l'apoptose.En conclusion, ce travail montre que l'HbF a un effet bénéfique supplémentaire sur la drépanocytose en conférant non seulement une survie préférentielle des cellules F dans la circulation, mais en diminuant également l'érythropoïèse inefficace. Ce résultat suggère que la persistance d’HbF dans la drépanocytose serait davantage la conséquence d’un enrichissement en F-érythroblastes au cours de la différenciation érythroïde terminale survenant peu après la naissance plutôt qu’un retard du switch des hémoglobines
Sickle cell disease (SCD) is caused by a single point mutation in the β-globin gene generating sickle hemoglobin (HbS). Hypoxia drives HbS polymerization that is responsible for red blood cell (RBC) sickling and reduced deformability. In SCD, splenic dysfunction results in life-threatening complications, particularly in early childhood. During the course of the disease, the spleen functionally declines and anatomically disappears, although with great individual variability depending on modulating genetic and environmental factors. The key modulator of disease severity is fetal hemoglobin (HbF), as the presence of HbF inhibits HbS polymerization, thus delaying and preventing severe complications, ameliorating patients’ quality of life and increasing survival. There is a rather well characterized hetero cellular concentration of HbF and distribution in circulating RBCs but the role of HbF during erythropoiesis, is poorly documented. With the aim of better understanding the role of HbF in spleen function, red cell survival and ineffective erythropoiesis we investigated 1) the natural history of spleen dysfunction in SCD children, 2) the cellular expression and distribution of HbF in SCD children, in untreated patients and patients treated with Hydroxycarbamide and 3) ineffective erythropoiesis and the role of HbF during terminal erythropoiesis.We developed a flow cytometry high-throughput method to measure splenic filtration function and showed that splenic loss of function is present very early in life at 3-6 months in SCD children and further declines with age. We also highlighted that irreversibly sickled cells (ISCs) are a potential contributor to acute splenic sequestration (ASS) which in turn results in further loss of splenic function. In the second part of this work, we set up an original approach to determine HbF distribution per cell. Using a longitudinal cohort of patients treated with hydroxycarbamide (HC - an inducer of HbF), we showed that HC has a global positive impact on RBCs, by not only increasing HbF content but also by increasing the volume of all RBCs independent of HbF. We moreover showed that High F-cells are a more precise marker of HC efficacy. In the last part of the thesis, we showed for the first time clear evidence of ineffective erythropoiesis in SCD and revealed a new role of HbF during terminal erythropoiesis protecting erythroblasts from apoptosis. In conclusion, this work shows that HbF has an additional beneficial effect in SCD by not only conferring a preferential survival of F-cells in the circulation but also by decreasing ineffective erythropoiesis. Importantly, it suggests that the delay in hemoglobin switch in SCD might be also due to an enrichment in F-erythroblasts during terminal erythroid differentiation occurring very early in infancy, shortly after birth

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Opi, Daniel Herbert. « Red blood cell polymorphisms and their associations with malaria and other nonmalaria related diseases in Kilifi, Kenya ». Thesis, Open University, 2013. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607150.

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The high morbidity and mortality associated with malaria has exerted considerable pressure on the human genome, selecting for polymorphisms thought to confer a survival advantage against severe and fatal malaria. The strongest protection is seen with HbAS and a+thalassaemia, offering up to 90% and 60% protection against severe malaria respectively. However, co-inheritance of these conditions, due to their geographical overlap, results in a negative interaction with their independent protection against malaria being lost. Through a series of in -vitro experiments, I show that this can be explained through the mechanism of cytoadherence. While Plasmodium Jalciparum infected HbAS and a+thalassaemia RBCs bound less well to endothelial receptors independently, this advantage was lost with co-inheritance of both conditions, a fact, however, not explained by changes in rosetting, PfEMPl or knob expression. The Sl2 and MCCb alleles of the Knops blood group system and blood group 0 of the ABO system occur at high frequencies in malaria endemic populations. However, sparse evidence is available on the protection they confer against malaria and biological mechanisms responsible. Using a series of epidemiological and clinical studies from Kilifi, I show that while Sl2 protects ag~inst cerebral and fatal malaria, MCCb increased susceptibility to both. MCCb is therefore under selection from nonmalaria related forces seen with its protection against lower respiratory tract infections and gastroenteritis. These associations with severe malaria could not be explained through the mechanisms of rosetting or cytoadherence. Non-O blood groups were associated with increased susceptibility to severe malarial anaemia and fatal malaria while protecting against several common non-malaria related diseases. While rosetting plays a role in these malaria related outcomes cytoadherence does not seem to be important. Finapy, I found that age, a+thalassaemia and the Knops blood group antigens . influence RBC CRi expression. Additionally, CRi deficiency is common in Kilifi, "associated with reduced rosetting.

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Peng, Xiao. « An inducible, conditional and targeted B cell ablation mouse model for studying B cell functionality in the pathogenesis of human diseases ». Master's thesis, Temple University Libraries, 2017. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/473128.

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Biomedical Sciences
M.S.
Primary objective of my MS thesis project is to characterize and develop a B cell ablation model for investigating the pathogenesis of human diseases such as hepatitis and systemic lupus erythematosus (SLE). Conditional and targeted cell ablation is a powerful approach for studying cellular functions in vivo. However, currently available cell ablation models still have some limitations and therefore limit their broader application in biomedical research. For example, two of the most common cell ablation methods currently employed utilize the herpes simplex virus 1 thymidine kinase (HSVtk) or diphtheria toxin (DT) receptor combined with their respective transgenic strategies. The ablation using HSVtk transgenic mice eliminates dividing cells, but does not affect non-dividing cells. In addition, because of its extremely high potency (a single molecule of DT-A, the active cleavage product of DT is sufficient to induce apoptosis), dose dependent responses are difficult to achieve and off-target effects are frequently observed. These facts highlight the unmet need to develop alternative methods of targeted cell ablation, which our model very successfully addresses. Our recently established approach using intermedilysin (ILY)-mediated cell ablation that is specific for human CD59 (hCD59) expressing cells, obviates these problems and provides an excellent and significantly improved alternative approach to the existing cell ablation methodologies. Intermedilysin (ILY), a toxin secreted by Streptococcus intermedius, exclusively binds to the human cell membrane protein CD59 (hCD59) but not to CD59 of any other species. Once bound, ILY rapidly and potently lyses the cells. Using genetic engineering, animal models can be created that express hCD59 in a spatially regulated manner. Administration of ILY will then selectively ablate only those cells in the animals that express hCD59 without any non-specific effect. To expand and facilitate the application of this newly generated model, we recently generated a Cre-inducible floxedSTOP-hCD59 transgenic mouse line (ihCD59), where specific hCD59 expression occurs following Cre-mediated recombination. By crossing ihCD59 mice with specific immune cell (T cells or macrophage) Cre transgenic lines, we obtained double transgenic mice expressing hCD59. ILY administration mediated specific cell ablation in these target cell populations in a dose dependent manner. Based on these results, I wanted to establish a new B cell ablation model for further studying B cell functionality in the pathogenesis of human diseases. CD19-Cre mice expressing the Cre-recombinase in B cell population were crossed with ihCD59 mice to generate the double positive transgenic mice (ihCD59+/-/CD19-Cre+/-). In Aim 1, I have demonstrated that hCD59 is specifically expressed in the B cell populations. In Aim 2, I have documented that 1) ILY has a large pharmacological window, and 2) ILY injection to ihCD59+/-/CD19-Cre+/- mice resulted in a rapid cell ablation of the B cell cells with off-target effect. Further, I have demonstrated that the specific ablation of B cells did not prevent the immune (Con A)-mediated hepatitis. In the future, I will apply this conditional B cell ablation model for investigating the functionality of B cells in the pathogenesis of human disease such as SLE.
Temple University--Theses

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Cuneo, Anthony. « THERAPEUTIC MECHANISMS OF INTERLEUKIN-19 FOR VASCULAR PROLIFERATIVE DISEASES ». Diss., Temple University Libraries, 2012. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/78032.

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Molecular and Cellular Physiology
Ph.D.
Cardiovascular disease is the leading cause of mortality in the western world. The pro-inflammatory and pro-proliferative etiology of vascular proliferative diseases is well characterized, while much less is known about the mechanisms of anti-inflammatory and anti-proliferative processes. Interleukin-19 (IL-19) is a newly described member of the IL-10 family of anti-inflammatory interleukins, and our group was the first to discover IL-19 expression in activated, synthetic, but not quiescent, contractile human vascular smooth muscle cells (hVSMC). We also found that IL-19 is anti-inflammatory and anti-proliferative for hVSMC. IL-19 is able to reduce the abundance of COX-2, IL-1β, IL-8, and Cyclin D1 transcripts which contain AU-rich elements (ARE) in their 3'-untranslated regions (3'-UTR). IL-19 is able to reduce the abundance of HuR, a stabilizing RNA-binding protein, which we feel provides a mechanism for these effects. The overall goal of this study is to elucidate IL-19's anti-inflammatory and anti-proliferative mechanism(s) in hVSMC in the context of vascular proliferative diseases. This goal has directed our overall hypothesis: IL-19's anti-proliferative and anti-inflammatory effects in hVSMC are mediated, at least in part, by modulation of HuR abundance and translocation, resulting in decreased stability of mRNA transcripts. HuR functions through a translocation mechanism, and IL-19 is able to reduce HuR cytoplasmic abundance. IL-19 also reduces HuR phosphorylation, which is a pre-requisite for HuR translocation, possibly through a PKCα-dependent mechanism. The stability of ARE-containing transcripts is reduced with IL-19 treatment, and reducing HuR expression by siRNA has the same inhibitory effect. VSMC are important mediators in the initiation of atherosclerosis. Oxidized low-density lipoprotein (ox-LDL) is able to induce IL-19 expression in these cells. VSMC are known to express scavenger receptors that take up ox-LDL. IL-19 is able to reduce the uptake of ox-LDL and the abundance of ox-LDL induced LOX-1 and CX-CL16 scavenger receptors. Interestingly, these scavenger receptors also have ARE in their 3'-UTR. IL-19 is able to reduce ox-LDL induced HuR cytoplasmic abundance. HuR knockdown by siRNA reduces the uptake of ox-LDL by hVSMC. These data suggest that IL-19 reduced scavenger receptor abundance may be due to decreased total and cytoplasmic HuR abundance. IL-19 reduces the abundance of ox-LDL induced COX-2 expression. Taken together, these results demonstrate that IL-19 down-regulates vital steps in vascular proliferative disease processes through an HuR-dependent mechanism.
Temple University--Theses

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Lamarre, Yann. « Implication de l’hémorhéologie dans la physiopathologie de la drépanocytose ». Thesis, Antilles-Guyane, 2013. http://www.theses.fr/2013AGUY0684/document.

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Nous avons étudié les marqueurs hémorhéologiques, hématologiques et biochimiques chez des sujets drépanocytaires homozygotes SS (HbS/HbS) et hétérozygotes composites SC (HbS/HbC) dans deux cohortes, pédiatriques et adultes, de patients drépanocytaires, et ce, à travers 7 complications récurrentes de la drépanocytose : 2 appartenant au profil hémolytique (l’ulcère de jambes et la glomérulopathie) et 5 appartenant au phénotype visqueux/vaso-occlusif (l’hypertension artérielle, le syndrome thoracique aigu (STA), la crise vaso-occlusive (CVO), la rétinopathie et l’ostéonécrose). Nous avons montré que : 1) une viscosité sanguine et une déformabilité érythrocytaire élevées sont des facteurs de risques de CVO chez les enfants homozygotes ; 2) Une viscosité sanguine élevée est associée à une hypertension artérielle systémique relative chez des adultes SS ; 3) les enfants SC présente une fonction vasculaire mieux préservée que les enfants SS pour faire face à une augmentation de la viscosité sanguine ; 4) les patients adultes SS avec une ostéonécrose présentent une déformabilité érythrocytaire plus élevée que les patients sans ostéonécrose ; 5) une viscosité sanguine élevée est associée à la présence d’une rétinopathie chez les adultes SC mais pas chez les SS ; 6) les patients adultes SS présentant une glomérulopathie ont un taux d’hémolyse élevé, une déformabilité érythrocytaire réduite et des agrégats érythrocytaires très robustes ; 7) les patients adultes SS avec des ulcères de jambes récurrents ont un taux d’hémolyse accru et une déformabilité érythrocytaire réduite. De plus, nos travaux confirment que l’-thalassémie module les propriétés de déformabilité érythrocytaire, mais montrent pour la première fois qu’elle module aussi les propriétés d’agrégation érythrocytaire, et notamment la force des agrégats érythrocytaires. En conclusion, ces travaux permettent de préciser le rôle de la rhéologie sanguine dans un certain nombre de complications de la drépanocytose et d’enrichir le modèle préexistant divisant les complications de la drépanocytose selon 2 phénotypes : hémolytique versus visqueux/vaso-occlusif. Nous montrons pour la première fois que le phénotype hémolytique est caractérisé aussi par des anomalies de la rhéologie du globule rouge : rigidité accrue et agrégats érythrocytaire robustes
Hemorheological, hemathological, and biochemical marquers of patients with sickle cell anemia (SS) and patients with sickle cell SC disease (SC) were studied in 2 cohorts: children and adults. We focused on 7 recurrent complications: 5 belonging to the viscosity/vaso-occlusion phenotype (systemic hypertension, acute chest syndrome (ACS), vaso-occlusive crisis (VOC), retinopathy and osteonecrosis) and 2 belonging to the hemolytic phenotype (leg ulcer and glomerulopathy). Our results show that 1) high viscosity is associated with increased risk for VOC in SS children; 2) blood viscosity is increased in SS adults with systemic relative hypertension; 3) SC children have preserved vascular function compared to SS children; 4) SS adults with osteonecrosis are characterized by higher red blood cell (RBC) deformability than SS adults without osteonecrosis; 5) high blood viscosity is associated with retinopathy in SC adults but not in SS adults; 6) SS adults affected by glomerulopathy have high hemolytic rate, low RBC deformability and increased RBC aggregates strenght; 7) SS adults with recurrent leg ulcers have high hemolytic rate and reduced RBC deformability. Moreover, our studies shows that alpha-thalassemia modulate RBC deformability and RBC aggregation properties. In conclusion, this work shows for the first time that the hemolytic phenotype is characterized by an abnormal RBC rheology which may play a role in several sickle cell complications

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15

Younce, Craig. « Zinc-Finger Protein MCPIP in Cell Death and Differentiation ». Doctoral diss., University of Central Florida, 2009. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/2279.

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Monocyte chemotactic protein-1 (MCP-1) plays a critical role in the development of cardiovascular diseases. How MCP-1 contributes to the development of heart disease is not understood. We present evidence that MCP-1 causes death in cardiac myoblasts, H9c2 by inducing oxidative stress, ER stress and autophagy via a novel Zn-finger protein, MCP-1 induced protein (MCPIP). MCPIP expression caused cell death and knockdown of MCPIP, attenuated MCP-1 induced cell death. Expression of MCPIP resulted in induction of iNOS and production of reactive oxygen (ROS). It caused induction of NADPH oxidase subunit phox47 and its translocation to the cytoplasmic membrane. Oxidative stress led to the induction of ER stress markers HSP40, PDI, GRP78 and IRE1α. ER stress lead to autophagy as indicated by beclin-1 induction, cleavage of LC3 to LCII and autophagolysosome formation. Here, MCPIP-induced processes lead to apoptosis as indicated by caspase 3 activation and TUNEL assay. This cell death involved caspase 2 and caspase 12 as specific inhibitors of these caspases prevented MCPIP-induced cell death. Inhibitors of oxidative stress inhibited ER stress, and cell death. Specific inhibitors of ER stress inhibited autophagy and cell death. Inhibition of autophagy inhibited cell death. Microarray analysis showed that MCPIP expression caused induction of a variety of genes known to be involved in cell death. MCPIP caused activation of JNK and p38 and induction of p53 and PUMA. These results collectively suggest that MCPIP induces ROS/RNS production that causes ER stress which leads to autophagy and apoptosis through caspase 2/12 and IRE1α –JNK/p38-p53-PUMA pathway. These results provide the first molecular insights into the mechanism by which elevated MCP-1 levels associated with chronic inflammation may contribute to the development of heart failure. A role for inflammation and MCP-1 in obesity and diabetes has been implicated. Adipogenesis is a key process involved in obesity and associated diseases such as type 2 diabetes. This process involves temporally regulated genes controlled by a set of transcription factors, C/EBPβ, C/EBPδ, C/EBPα, and PPARγ. Currently PPARγ is considered the master regulator of adipogenesis as no known factor can induce adipogenesis without PPARγ. We present evidence that a novel Zn-finger protein, MCPIP, can induce adipogenesis without PPARγ. Classical adipogenesis-inducing medium induces MCP-1 production and MCPIP expression in 3T3-L1 cells before the induction of the C/EBP family of transcription factors and PPARγ. Knockdown of MCPIP prevents their expression and adipogenesis. Treatment of 3T3-L1 cells with MCP-1 or forced expression of MCPIP induces expression of C/EBPβ, C/EBPδ, C/EBPα, PPARγ and adipogenesis without any other inducer. Forced expression of MCPIP induces adipogenesis in PPARγ-/- fibroblasts. Thus, MCPIP is a newly identified master controller that can induce adipogenesis without PPARγ. Heart failure is a major cause of death in diabetic patients. Hyperglycemia is a major factor associated with diabetes that causes cardiomyocyte apoptosis that leads to diabetic cardiomyopathy. Cardiomyoycte apoptosis is a key event involved in the pathophysiological progression of diabetic cardiomyopathy. We have recently found that in ischemic hearts, MCP-1 can induce the zinc-finger protein, MCP-1 induced protein (MCPIP) that causes cardiomyocyte apoptosis. Although there is evidence that inflammation may play a role in diabetic cardiomyopathy, the underlying mechanisms are poorly understood. In this study, we show that treatment of H9c2 cardiomyoblasts and Neonatal Rat Ventricular Myocytes (NRVM) with 28mmol/L glucose concentration results in the induction of both transcript and protein levels of MCP-1 and MCPIP. Inhibition of MCP-1 interaction with CCR2 via specific antibody or with the G-coupled receptor inhibitors propagermanium and pertussis toxin attenuated glucose-induced cell death. Knockdown of MCPIP with specific siRNA yielded similar results. Treatment of cells with 28mmol/L glucose resulted in increased ROS production and phox47 activation. Knockdown of MCPIP attenuated these effects. The increased ROS production observed in H9c2 cardiomyoblasts and NRVM’s resulted in increased ER stress proteins GRP78 and PDI. Knockdown of MCPIP attenuated expression of both GRP78 and PDI. Inhibition of ER stress with TUDC and 4’PBA prevented high glucose-induced cell death death. Treatment of cells with 28mmol/l glucose resulted in autophagy as determined by an increase in expression of beclin-1 and through increased cleavage of LC3I to LC3II. Knockdown of MCPIP attenuated expression of beclin-1 and prevented cleavage of LC3. Addition of the autophagy inhibitors 3’methyladenine and LY294002 attenuated high glucose-induced H9c2 cardiomyoblast death. We conclude that high glucose-induced H9c2 cardiomyoblast death is mediated via MCP-1 induction of MCPIP that results in ROS that leads to ER stress that causes autophagy and eventual apoptosis.
Ph.D.
Department of Biomolecular Science
Burnett College of Biomedical Sciences
Biomedical Sciences PhD

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16

Jacobsen, Christopher L. « Beta-Amyloid Inhibition of Alpha 7 Nicotinic Acetylcholine Receptors and Factors That Potentially Influence the AÎ²/nAChR Interaction ». BYU ScholarsArchive, 2013. https://scholarsarchive.byu.edu/etd/4179.

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Alzheimer's disease (AD) is a neurodegenerative disorder that manifests in the form of deficiencies in cognitive processes such as memory and learning. The pathological features of AD include hyperphosphorylated tau proteins that form neurofibrillary tangles as well as senile plaques composed primarily of the peptide β-amyloid (Aβ). When present in high concentrations in the brain, Aβ inhibits certain subtypes of neuronal nicotinic acetylcholine receptors (nAChRs) in the hippocampus. The effects of Aβ in the hippocampus have proven to be neurotoxic, resulting in reduced functionality of nAChRs and the subsequent death of neurons in the cholinergic pathway. The early stages of AD are characterized by reduction of nAChR density and by degeneration of the cholinergic neurons that provide input to the hippocampus. Because the hippocampus plays a critical role in memory formation and other cognitive processes, dysfunction in this brain region results in significant cognitive deficiencies. Understanding the interaction between Aβ and the structurally and functionally diverse nAChR subtypes and possible downstream effects in signaling cascades that might result from that interaction are important steps in comprehending AD pathogenesis. Comprehension of this interaction and factors that might influence it could lead to the development of pharmaceutical agents useful in the treatment of AD.

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17

Jan, Michael. « Novel Mechanisms Underlying Homocysteine-Suppressed Endothelial Cell Growth ». Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/264103.

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Pharmacology
Ph.D.
Cardiovascular disease (CVD) is the leading cause of death worldwide, and is projected to remain so for at least the next decade. Ever since its discovery in the urine and blood of children with inborn errors of metabolism, homocysteine (Hcy) at elevated plasma concentrations has been associated with CVD clinically and epidemiologically. Observational studies and meta-analyses have noted that changes in plasma Hcy by 5μM increase the odds ratio of developing coronary artery disease by 1.6-1.8 among other CVD. Clinical trials aimed at reducing plasma Hcy for benefit against development of subsequent cardiovascular events have had unconvincing results, but have moreover failed to address the mechanisms by which Hcy contributes to CVD. Recommendations from national agencies like the American Heart Association and the United States Preventive Services Task Force emphasize primordial prevention as a way to combat CVD. Reducing plasma Hcy as secondary and primary interventions does not fulfill this recommendation. In order to best understand the role of Hcy in CVD, an investigation into its mechanisms of action must be undertaken before measures of primordial prevention can be devised. Numerous experimental studies in the literature identify vascular endothelium as a target for the pathological effects of Hcy. Endothelial injury and impairment are contributory processes to atherosclerosis, and Hcy has been demonstrated to inhibit endothelial cell (EC) growth and proliferation through mechanisms involving cell cycle arrest, oxidative stress, and programmed cell death in vitro. Animal models have also confirmed that high levels of Hcy accelerate atherosclerotic plaque development and lead to impairment of vascular reendothelialization following injury. Hcy has been shown to have the opposite effect in vascular smooth muscle cells (SMC), causing their proliferation and again contributing to atherosclerosis. The cell-type specificity of Hcy remains to be understood, and among the aims of this research was to further characterize the effects of Hcy in EC. The overarching goal was discovery in order to direct future investigations of Hcy-mediated pathology. To begin, the first investigation considered the transcriptional and regulatory milieu in EC following exposure to Hcy. High-throughput screening using microarrays determined the effect of Hcy on 26,890 mRNA and 1,801 miRNA. Two different in vitro models of hyperhomocysteinemia (HHcy) were considered in this analysis. The first used a high dose of 500µ Hcy to mimic plasma concentrations of patients wherein the transsulfuration pathway of Hcy metabolism is impaired as in inborn cystathionine-ß-synthase deficiency. The other set of conditions used 50µ Hcy in the presence of adenosine to approximate impairment of the remethylation pathway of Hcy metabolism wherein s-adenosylhomocysteine accumulates, thus inhibiting s-adenosylmethionine formation and methylation reactions. These distinctions are important because most clinical trials do not distinguish between causes of HHcy, thereby ignoring the specific derangements underlying HHcy. mRNA and miRNA expression changes for both sets of treatment conditions identified CVD as a common network of Hcy-mediated pathology in EC. Moreover, methylation-specific conditions identified cell cycle modulation as a major contributory mechanism for this pathology, which agrees with recent findings in the literature. Analysis of significant mRNA changes and significant miRNA changes independently identified roles for Hcy in CVD and cell cycle regulation, thereby suggesting that miRNA may mediate the effects of Hcy in addition to gene expression changes alone. To investigate the role of Hcy in the cell cycle further, the next set of investigations considered the effect of Hcy under conditions approximating impaired remethylation in early cell cycle events. Previous studies have demonstrated that Hcy inhibits cyclin A transcription in EC via demethylation of its promoter. Conversely, Hcy induces cyclin A expression in SMC, again making the case for a cell type-specific mechanism in EC. Preceding cyclin A transcription and activation, canonical events in the early cell cycle include D-type cyclin activation, retinoblastoma protein (pRB) phosphorylation, and transcription factor E2F1 activation. In a series of in vitro experiments on EC, it was seen that Hcy inhibits expression of cyclin D2 and cyclin D3, but not cyclin D1. Next, pRB phosphorylation was seen to be decreased following treatment with Hcy. This also led to decreased E2F1 expression. However, this series of events could be reversed with E2F1 supplementation, allowing the cell cycle to proceed. As Hcy exerts a number of its effects via regulation of gene transcription, a final series of investigations aimed to predict potential targets of Hcy by examining patterns of transcription factor binding among known targets of Hcy regulation. Gene promoters of Hcy-modulated genes were analyzed in order to determine common transcription factors that potentially control their regulation. The locations of CpG-rich regions in promoters were identified to determine which regions would be most susceptible to regulation by DNA methylation. Next, high-throughput next-generation sequencing (NGS) and bisulfite NGS was performed for DNA from EC treated with Hcy in order to determine methylation changes after Hcy treatment. A number of potential transcription factors and their binding sites were identified as potential mediators of Hcy-mediated gene regulation. Taken together, these investigations represent an exploration of Hcy-mediated pathology in CVD, by focusing upon novel regulatory mechanisms in EC. Objective high-throughput arrays identified roles for Hcy in CVD and cell cycle pathways regulated by miRNA and gene expression, which were confirmed experimentally in vitro. These observations led to an investigation and identification of common transcription factors that potentially regulate Hcy-altered gene expression. This framework may be used to guide future investigations into the complex pathological network mediating the effects of Hcy in CVD. First, identification of a role for miRNA in mediating the effects of Hcy represents a novel regulatory mechanism, heretofore largely unexplored. Next, expanding the role of Hcy in EC cell cycle regulation to identify upstream mediators greatly adds to the published literature. Finally, noting that these changes center upon transcriptional and post-transcriptional regulation gives import to developing methods to characterize promoter and transcription factor regulation. The investigations presented herein and their results provide evidence that the future of Hcy research is vibrant, relevant, and not nearly surfeit.
Temple University--Theses

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18

Litwin, Nicole S. « Assessment of Red Blood Cell Membrane Fatty Acid Composition in Relation to Dietary Intake in Patients Undergoing Cardiac Catheterization ». Digital Commons @ East Tennessee State University, 2014. https://dc.etsu.edu/etd/2319.

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Red blood cells (RBC) have been shown to mediate plaque development seen in coronary artery disease (CAD). This study determined whether differences in RBC fatty acid (FA) composition were related to CAD risk. FAs were extracted from RBCs of 38 individuals who have undergone cardiac catheterization, 9 of whom had obstructive CAD, and analyzed via gas chromatography. Ferric reducing ability of plasma (FRAP) assay was used to determine oxidative stress. Food frequency questionnaires were used to correlate RBC omega-3 FA to daily intake of omega-3 FA. No correlation was found between RBC content and intake of omega-3 FA. FRAP values and RBC FA composition did not differ between the 2 groups with exception of the saturated FA, palmitic acid (p=0.018). These results suggest that RBC FA composition may differ between individuals with or at risk for CAD. Additional research is needed to validate this biomarker as a predictor of CAD.

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Oteng, Eugene K. « Discovery of a conserved Plasmodium antigen on the surface of malaria-infected red blood cells ». Thesis, University of Oxford, 2013. http://ora.ox.ac.uk/objects/uuid:592769fc-2628-4c6e-9a68-99060fb8c091.

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During its intraerythrocytic stages (IE), Plasmodium falciparum, the causative agent of the deadliest human malaria, remodels the host red cell membrane with a poorly defined assortment of parasite-encoded proteins that undergo antigenic variation. Despite the requirement for immunologic stealth, exported parasite proteins also mediate strain-independent functions such as endothelial sequestration that are critical for parasite survival and pathogenesis. This thesis explores the hypothesis that P. falciparum displays novel structurally conserved proteins on the IE surface and these proteins may serve as useful antigens for a broadly effective anti-malarial vaccine. In order to test this hypothesis, we developed an in vitro selection technique that sequentially incorporates unique P. falciparum isolates as the targets for Systematic Evolution of Ligands by EXponential enrichment (Serial-SELEX) to generate nucleic acid molecular probes, aptamers, capable of recognizing conserved cell surface determinants. Ten of 11 enriched aptamers were -parasite selective and three of these aptamers demonstrated strain-independent binding to P. falciparum. Aptamer recognition extended beyond the parasites used in Serial-SELEX to other laboratory and recent field isolates. Surprisingly the same three broadly binding aptamer selected against P. falciparum also recognized all laboratory-adapted and clinical isolates of P. vivax and P. knowlesi tested, strongly supporting our hypothesis that structurally conserved molecules are present on the surface IEs. Competition studies showed that the aptamers bound a single target which was confirmed as an IE membrane protein. Aptamer‐mediated affinity purification and tandem mass spectrometry enabled identification of the aptamer target as parasite-encoded protein. Discovery of a protein conserved between the major human malarias may have implications for vaccine development and validates the Serial‑SELEX technique as a powerful tool for antigen discovery.

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20

Kyriazis, George. « THE ENDOCYTIC PROTEIN NUMB REGULATES APP METABOLISM AND NOTCH SIGNALING : IMPLICATIONS FOR ALZHEIMER'S DISEASE ». Doctoral diss., University of Central Florida, 2008. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/3737.

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Increased production of amyloid beta (A-beta) peptide, via altered proteolytic cleavage of amyloid protein precursor (APP), and abnormalities in neuronal calcium homeostasis play central roles in the pathogenesis of Alzheimer's disease (AD). Notch1, a membrane receptor that controls cell fate decisions during development of the nervous system, has been linked to AD because it is a substrate for the gamma-secretase protein complex in which mutations cause early-onset inherited AD. Numb is an evolutionarily conserved endocytic adapter involved in the internalization of transmembrane receptors. Mammals produce four Numb isoforms that differ in two functional domains, a phosphotyrosine-binding domain (PTB) and a proline-rich region (PRR). Recent studies showed that the PTB domain of Numb interacts with the cytoplasmic tails of APP and Notch but the functional relevance of these interactions with respect to AD pathogenesis is not clear. In the current studies, we proposed to investigate the biological consequences of the interaction of the Numb proteins with APP and Notch in neural cells stably overexpressing each of the four human Numb proteins. In the first part of our studies, we found that expression of the Numb isoforms lacking the insert in the PTB (SPTB-Numb) caused the abnormal accumulation of cellular APP in the early endosomes, and increased the levels of C-terminal APP fragments and A-beta. By contrast, expression of the Numb isoforms with the insert in PTB (LPTB-Numb) leads to the depletion of cellular APP and coincides with significantly lower production of APP derivatives and A-beta. The contrasting effects of the Numb isoforms on APP metabolism were not attributed to differences in the expression of APP nor the activities of the various APP-processing secretases. In the second part of our studies, we found that expression of SPTB-Numb protein enhances neuronal vulnerability to serum deprivation-induced cell death by a mechanism involving the dysregulation of cellular calcium homeostasis. Neural cells expressing SPTB-Numb exhibited enhanced Notch activity, which markedly upregulated the expression of transient receptor potential canonical 6 (TRPC6) channels enhancing calcium entry in response to store depletion. We also found that serum deprivation increased TRPC6 expression, mediating the serum deprivation-induced death in neural cells. Interestingly, expression of LPTB-Numb protein suppressed serum deprivation-induced activation of Notch and the subsequent upregulation of TRPC6 and cell death. Finally, we showed that the Numb proteins differentially impact Notch activation by altering the endocytic trafficking and processing of Notch. Taken together, these studies demonstrate that aberrant expression of the Numb proteins may influence APP metabolism and Notch-mediated cellular responses to injury by altering their endocytic trafficking and processing.
Ph.D.
Department of Biomolecular Science
Burnett College of Biomedical Sciences
Biomedical Sciences PhD

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21

Vaja, V. « A HEPCIDIN INHIBITOR MOBILIZES IRON FOR INCORPORATION INTO RED BLOOD CELLS IN AN ADENINE-INDUCED KIDNEY DISEASE MODEL IN RATS ». Doctoral thesis, Università degli Studi di Milano, 2013. http://hdl.handle.net/2434/217464.

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Anemia is prevalent in patients with chronic kidney disease (CKD) and is primarily due to a complex interplay of relative erythropoietin deficiency, shortened red blood cell survival and abnormalities in iron homeostasis. A key feature in many patients with anemia of CKD is the limited iron availability for an efficient erythropoiesis despite adequate body iron stores. It is now well established that excess levels of the iron regulatory hormone hepcidin are responsible for downregulating the functional expression of the cellular iron exporter, ferroportin, thereby resulting in a blockade of iron absorption from the diet and iron retention in reticuloendothelial macrophage stores. Adenine treatment in rats has been proposed as an animal model of anemia of CKD with high hepcidin levels that mirrors the condition in patients. We developed a adenine-induced renal failure model in rats that simulates the renal failure and anemia condition in patients. We modified the Yokozawa et al. model by giving a diet supplemented with 0.75% adenine for 3 weeks followed by adenine free normal diet for another 3 weeks. We then tested whether the small molecule bone morphogenetic protein (BMP) inhibitor LDN-193189, which has previously been shown to lower hepcidin levels, was able to mobilize iron into the plasma and improve iron-restricted erythropoiesis in adenine-treated rats. The modified adenine model had a higher survival rate than previously reported models, while maintaining irreversible renal failure and anemia. We demonstrated that adenine rats had increased hepatic hepcidin mRNA levels, decreased serum iron concentration, increased spleen iron content, low hemoglobin levels and inappropriately low EPO levels relative to the degree of anemia, typical of the clinical condition in patients with anemia of CKD. LDN-193189 lowered hepatic hepcidin mRNA and mobilized stored iron into plasma in adenine-treated rats. Moreover, the iron was efficiently incorporated into hemoglobin in reticulocytes. However, LDN-193189 alone did not prevent anemia progression in our model. Lowering hepcidin improved iron availability, but did not improve anemia in an adenine-induced kidney disease model in rats. Co-administration of hepcidin lowering agents with erythropoiesis stimulating agents (ESAs) may be useful as a combination therapy to correct iron balance and thereby reduce the ESA dose needed to achieve target hemoglobin levels.

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Iampietro, Mary Catherine. « An Investigation of Episodic Memory Performance in Relation to Inflammation in Children with Sickle Cell Disease ». Diss., Temple University Libraries, 2014. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/301993.

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Psychology
Ph.D.
It is now well established that children with sickle cell disease (SCD) demonstrate cognitive deficits even in the absence of clinical stroke, but studies in children who have not experienced a stroke or other neurological event are lacking. Systemic processes that occur in SCD, like chronic inflammation and hypoxia, have been associated with hippocampal damage and episodic memory deficits in a range of clinical populations and animal models. However, studies examining episodic memory performance in children with SCD and in relation to systemic processes are largely absent. The present study addressed these gaps in young children with SCD (Mage = 7.37 years, SD = 1.51) who had not experienced a clinical stroke. Participants (N = 31) completed various memory measures as part of a larger neuropsychological protocol and participated in routine clinical blood draws. Latent class analysis (LCA) was used to empirically define SCD groups based on measures of specific visual memory processes, and results revealed two distinct visual memory groups, characterized by (1) visual memory deficits, specifically in delayed recognition abilities, and (2) intact visual memory. Follow-up analyses revealed that the two classes did not significantly differ on verbal memory performance. The relation between memory processes and both biomarkers of inflammation and adaptive functioning also were examined with variable-centered analyses. Results showed only one significant relation between C-reactive protein (CRP) and a measure of verbal delayed recognition. In sum, young children with SCD demonstrate variable episodic memory performance, with most notable deficits in visual delayed recognition. Higher levels of CRP, a biomarker of inflammation, were associated with poorer verbal delayed recognition. The results indicate that young children with SCD experience deficits in memory, even in the absence of a neurological event, and specific memory processes should be assessed in these children to guide targeted interventions.
Temple University--Theses

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Newman, Tiffanny Nicole. « ROLE OF TULA-FAMILY PROTEINS IN T CELL DRIVEN RESPONSES ». Diss., Temple University Libraries, 2011. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/210733.

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Microbiology and Immunology
Ph.D.
The TULA-family consists of two proteins implicated in cellular regulation. TULA-1 is expressed in T-cells and is involved in apoptosis. TULA-2 is a ubiquitously expressed phosphatase that suppresses receptor-mediated signaling. T cells from mice lacking TULA-1 and 2 (double knockout, or dKO) are hypersensitive to TCR stimulation. This may be due to these proteins having a similar function working synergistically or dissimilar functions having a convergent effect. To understand functional interaction of these proteins we have characterized TULA-family knockout mice without and during an immune challenge. We show that CD4+ T cells of dKO mice have a characteristic CD45RB distribution, and that within the CD45RBlow subset effector/memory T cells are expanded only in dKO, but not in single knockouts (sKO) of either TULA-1 or TULA-2. However, CD4+ T cells of sKO and wild-type (WT) mice respond differently to TCR stimulation as seen using signaling and responses in vitro. To evaluate consequences of TULA deficiency in vivo, we utilized two mouse models of inflammatory bowel disease: TNBS-induced colitis and colitis induced by the adoptive transfer of CD45RBhigh CD4+ T cells. Studies utilizing TNBS indicate that deficiency of any TULA-family protein exacerbates TNBS-induced colitis. Likewise, dKO CD45RBhigh CD4+ T cells were significantly more colitogenic than cells from WT mice in the transfer model. Taken together, our data indicate that TULA-family proteins are key to the physiological regulation of T-cell reactivity that drives intestinal inflammation.
Temple University--Theses

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Chatila, Wissam M. F. « MicroRNA Expression in Regulatory T Cells in Chronic Obstructive Pulmonary Disease ». Diss., Temple University Libraries, 2015. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/333572.

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Microbiology and Immunology
Ph.D.
COPD is characterized by an abnormal regulatory T cell (Treg) response with a shift towards a Th1 and Th17 cell responses. However, it is unclear if the function of Treg cells is impaired by smoking and in COPD. In addition, the miRNA profile of Treg cells in COPD is unknown and whether miRNA deregulation contributes to COPD immunopathogenesis. We set the objective to study Treg cell function isolated from peripheral blood of patients with COPD versus controls and to compare their miRNA profiles. We also were interested in exploring the function of some of the differentially expressed Treg cell miRNAs. We assessed the Treg cell function by observing their suppressive activity on autologous effector T cells and analyzed their miRNA expression initially by microarray analysis then conducted real time RT-PCR validation for selected miRNAs. In Silico target gene analysis for the validated miRNAs suggested that miR-199-5p is particularly relevant to Treg cell physiology so its function was investigated further using CCD-986Sk and MOLT-4 cells. We found no difference in Treg cell function between COPD and controls but we were able to identify 6 and 96 miRNAs that were differentially expressed in COPD versus control Treg cells. We confirmed that miR-199a-5p was repressed by approximately 4 fold in Treg cells of COPD patients compared to cells in healthy smokers. Importantly, miR-199a-5p had significant overrepresentation of its target genes in the Treg cell transcriptome, with many targets associated with the TGF-b activation pathway. We also confirmed the function of miR-199a5p in an in-vitro loss-of-function cell model running TaqMan® arrays of the Human TGF-b Pathway. These findings suggest that the abnormal repression of miR-199a-5p in patients with COPD compared to unaffected smokers may be involved in modulating the adaptive immune balance in favor of a Th1 and Th17 response.
Temple University--Theses

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Luongo, Timothy Scott. « The Role of Mitochondrial Calcium Exchange in Cardiac Physiology and Disease ». Diss., Temple University Libraries, 2017. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/437718.

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Biomedical Sciences
Ph.D.
The high metabolic demand of the heart makes it essential that an efficient and tightly controlled system be in place to regulate energy production. Contractility is mediated by a variable flux in intracellular calcium (iCa2+), which is proposed to be integrated into mitochondria to regulate cardiac energetics. Moreover, mitochondrial Ca2+ (mCa2+)-overload is known to activate the mitochondrial permeability transition pore (MPTP) and induce cell death. However, the true function of cardiac mCa2+ in physiology remains unknown. Recent studies have reported that the Mcu gene encodes the channel-forming portion of the mitochondrial calcium uniporter (MCU) and is required for mCa2+ uptake (Baughman et al., 2011; De Stefani, Raffaello, Teardo, Szabo, & Rizzuto, 2011). To examine the role of mCa2+ in the heart, we generated a conditional, cardiac-specific knockout model and deleted Mcu in adult mice (Mcu-cKO). Loss of Mcu protected against myocardial ischemia-reperfusion (IR) (40 min occlusion of the left coronary artery (LCA) followed by 24h reperfusion) injury by preventing the activation of the MPTP. We observed a 45% reduction in infarct size per area-at-risk and a 65% reduction in cardiac troponin-I serum levels from 24h post-IR. In addition, while we found no baseline phenotype or change in baseline mCa2+ content, Mcu-cKO mice lacked contractile responsiveness to β-adrenergic receptor stimulation (isoproterenol infusion) as assessed by invasive hemodynamics, and, in parallel, were unable to activate mitochondrial dehydrogenases, thereby decreasing tricarboxylic acid (TCA) cycle flux and cardiac NADH. We found that Mcu-cKO mice had a 3-fold increase in pyruvate dehydrogenase (PDH) phosphorylation and a 50% decrease in PDH activity post-isoproterenol infusion. Further experimental analyses in isolated adult cardiomyocytes confirmed a lack of energetic responsiveness to acute sympathetic stress (isoproterenol failure to mediate an increase in oxidative phosphorylation capacity) supporting the hypothesis that the physiological function of the MCU in the heart is to modulate Ca2+-dependent metabolism during the ‘fight or flight’ response. However, questions still remain on how basal mCa2+ levels are regulated and if it contributes to cardiac disease. The mitochondrial sodium/calcium exchanger (mNCX) is hypothesized as the primary mechanism of mCa2+ efflux, but to date no study has confirmed its identity or function in an in vivo system (Palty et al., 2010). To investigate the role of mNCX in the heart, we generated mutant mice with loxP sites flanking exons 5-7 of the candidate gene, Slc8b1, and crossed them with a tamoxifen-inducible, cardiomyocyte-specific, αMHC-Cre mouse to delete mNCX in the adult heart (mNCX-cKO). Biophysical study of cardiomyocytes isolated from mNCX-cKO mice revealed a significant reduction in mCa2+ efflux rate. Tamoxifen-induced deletion of Slc8b1 in adult hearts caused sudden death with less than 15% of mice surviving after 10 days. Echocardiographic evaluation of mNCX-cKO hearts 3d post-tamoxifen revealed significant left ventricular (LV) remodeling, characterized by significant dilation and a substantial decrease in function. In addition, mNCX-cKO hearts exhibited increased reactive oxygen species generation when assessed by DHE imaging of live myocardial tissue and mitoSOX Red imaging in isolated adult cardiomyocytes. Using an Evan’s blue dye exclusion technique, we found that mNCX-cKO hearts displayed significant sarcolemmal rupture (~8% of all myocytes at a single time point 3d post-tamoxifen), indicative of cellular necrosis. To rescue the sudden death phenotype and acute loss of cells, we crossed our mNCX-cKO mice with the cyclophilin d (a mediator of MPTP-opening) knockout mice. mNCX-cKO x CypD-KO mice had a significant improvement in survival and LV-function. In addition, loss of MPTP activation also rescued mitochondrial pathology on the subcellular level. Since deletion of mNCX was detrimental on cardiac function, we thought that increasing mNCX could protect cardiomyocytes by reducing mCa2+-overload during cardiac disease. To test this, we generated a conditional, cardiac-specific mNCX overexpression mouse model (mNCX-Tg) to assess if increasing mCa2+ efflux would prevent cardiac injury in multiple pathological surgical models. mNCX-Tg and controls were subjected to in vivo IR injury followed by 24h reperfusion and myocardial infarction (MI) (permanent LCA ligation). mNCX-Tg mice displayed reduced cell death (a 43% reduction in infarct size 24h post-IR and a 33% reduction in scar size 4w post-MI), preserved LV function, a reduction in ROS generation, and a decrease in numerous HF indices. For the first time, we showed that mNCX is essential for maintenance of the mCa2+ microdomain in cardiomyocytes and that mNCX represents a novel therapeutic target in HF.
Temple University--Theses

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Jaffal, Jad M. « Isolation of potential protein targets of MS-818 using affinity chromatography ». Honors in the Major Thesis, University of Central Florida, 2010. http://digital.library.ucf.edu/cdm/ref/collection/ETH/id/1428.

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This item is only available in print in the UCF Libraries. If this is your Honors Thesis, you can help us make it available online for use by researchers around the world by following the instructions on the distribution consent form at http://library.ucf.edu/Systems/DigitalInitiatives/DigitalCollections/InternetDistributionConsentAgreementForm.pdf You may also contact the project coordinator, Kerri Bottorff, at kerri.bottorff@ucf.edu for more information.
Bachelors
Medicine
Molecular and Microbiology

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Porro, B. « EVALUATION OF L-ARGININE/NITRIC OXIDE METABOLIC PATHWAY IN ERYTHROCYTES IN RELATION WITH OXIDATIVE STRESS : FOCUS ON DIFFERENT CARDIOVASCULAR DISEASES ». Doctoral thesis, Università degli Studi di Milano, 2014. http://hdl.handle.net/2434/247161.

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Background: A decreased nitric oxide (NO) bioavailability and an increased oxidative stress play a pivotal role in different cardiovascular pathologies. Recent studies have shown that red blood cells (RBCs) participate in NO formation in the bloodstream. Aim: The aim of this study was to assess the L-arginine (Arg)/NO pathway and the oxidative stress status in RBCs and in plasma of patients with microvascular angina (MVA), investigating similarities and differences with respect to coronary artery disease (CAD) patients or healthy controls (Ctrl). Materials and Methods: Analytes involved in Arg/NO pathway and the ratio between the oxidized and the reduced forms of glutathione, as index of oxidative stress, were measured by liquid-chromatography tandem mass spectrometry (LC-MS/MS). The arginase and the NO synthase (NOS) expression were assessed by immunofluorescence staining. NOS activity was evaluated by ex-vivo experiments through the conversion of L-[15N2]arginine to L-[15N]citrulline. Results: Both MVA and CAD patients showed alterations in the ability of RBCs to produce NO, based on an increase of NO synthesis inhibitors, parallel to that found in plasma, a reduction of NOS expression and activity and an increased arginase expression. When summary scores of NO synthesis and of oxidative stress were computed, both patient groups were associated with a positive oxidative score and a negative NO score, with the CAD group located in a more extreme position with respect to Ctrl. Conclusions: This finding points out to an impairment of the capacity of RBCs to produce NO in pathological conditions characterized by alteration at the microvascular bed with/without no significant coronary stenosis.

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Manokaran, Thirumakal [Verfasser], Miriam [Gutachter] Cortese-Krott et Inge [Gutachter] Bauer. « Nitric oxide-dependent red cell sGC activity and sGC/cGMP pathway in coronary artery disease patients and healthy controls / Thirumakal Manokaran ; Gutachter : Miriam Cortese-Krott, Inge Bauer ». Düsseldorf : Universitäts- und Landesbibliothek der Heinrich-Heine-Universität Düsseldorf, 2019. http://d-nb.info/1189901757/34.

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Yue, Yujia. « INFLAMMATION ALTERS ENDOTHELIAL PROGENITOR CELL-DERIVED EXOSOME CONTENTS AND THERAPEUTIC EFFECT ON MYOCARDIAL REPAIR ». Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/559194.

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Biomedical Sciences
Ph.D.
Cardiovascular disease remains the leading cause of morbidity and mortality worldwide and Myocardial Infarction (MI) and subsequent heart failure remains the leading cause for death. Despite the improvement in prognosis and treatment of acute MI patients, the underlying causes including loss of cardiomyocytes and microvasculature remain potential risk and lack proper and efficient solutions. Stem cell-based therapies for repair and regeneration have evolved and have been applied in clinical trials. Different types of stem cells, including Endothelial progenitor cell (EPC), Mesenchymal Stem Cell (MSC), induced Pluripotent Stem Cell (iPSC) and cardiac progenitor cells etc. have been used for potential long term recovery and cardiac regeneration. However, results from the clinical trials have been largely disappointing and improvement in cardiac functions have been modest likely due to the limitations of cell therapy including low integration in myocardium, poor survival, cellular dysfunction and limited differentiation ability. It is therefore necessary and urgent to develop cell free alternatives as next generation regenerative therapies. There is a consensus that the beneficial effect of stem cell therapy is largely due to paracrine effects. Exosomes have recently emerged as important functional units mediating stem cell paracrine effects. Exosomes are the family of extracellular vesicles (EV) which are 30-150nm in size, secreted by almost all types of cells and responsible for cell-cell communication via delivering their cargo including RNAs and proteins to host cells. Studies from our and other labs have shown that exosomes mimic parental stem cell in improving post-MI functions. The essential feature of exosome is decided by their cargo including RNA and protein, which are subject to dynamic changes depending on the environment of parental cells. Our studies were focused on Endothelial Progenitor Cell (EPC)-derived exosomes. EPCs are generated in bone marrow, and home to the site of tissue injury and orchestrate neovascularization and tissue repair. Patients with ischemic heart disease, are usually accompanied with comorbidities such as systemic inflammation, aging, diabetes, etc. which are known to compromise EPC functions. We hypothesized that EPCs under inflammatory stress produce dysfunctional exosomes with altered RNA and protein content, leading to impaired cardiac reparative properties. We chose interleukin-10 knockout (IL-10KO) mice as a model of systemic inflammation. EPCs were isolated from IL-10KO and wild-type (WT) mice, and their exosomes (Exo) were compared for their reparative properties both in vitro and in vivo. Our in vitro studies showed WT-EPC-Exo treatment attenuated recipient cell apoptosis, enhanced cell mobilization and tube formation, whereas IL-10KO-EPC-Exo were functionally deficient or even had detrimental effects. We used MI mouse model to compare the in vivo function of two groups of exosomes and found WT-EPC-Exo treatment significantly improved left ventricular (LV) cardiac function, inhibited cell death, promoted angiogenesis and attenuated cardiac remodeling; while these cardioprotective effects were lost in IL-10KO-EPC-Exo treated group. Both in vitro and in vivo studies proved that even the same progenitor cell type (EPCs), under inflammatory stimulus (IL-10KO), secretes exosomes with different reparative properties. Next, we explored whether the observed difference in exosome function is caused by altered exosome content. Using Next Generation RNA Sequencing (NGS RNAseq) and mass spectrometry we found RNA and protein expression patterns were drastically different in wild type and IL-10 knockout EPC derived exosomes. This evidence leads to the conclusion that alteration in exosome content is fundamental for exosome function. We picked two candidates that are highly enriched in IL-10KO-EPC-Exo for further study, miR-375 and Integrin-Linked Kinase (ILK). We treated IL-10KO-EPC with anti-miR against miR-375 and siRNA against ILK separately, and successfully decreased the expression of miR-375 and ILK in both EPCs and EPC derived exosomes. Then we explored the function of those miR and protein ‘modified exosomes’ with similar in vitro and in vivo experiments as previously described. Compared to IL-10KO-EPC-Exo, miR-375 knockdown exosomes showed enhanced angiogenesis and inhibited cell apoptosis, while ILK knockdown in exosomes rescued functions in both in vitro and in vivo experiments. These results suggested the possibility that exosome manipulation of identified factors may partially rescue their reparative functionality. In summary, our studies revealed that stem cell derived exosomes are capable for independent cardiac repair in ischemic heart disease, however, parental stem cells under pathological stimulus secrete dysfunctional exosomes with altered RNA and protein content. Exosome function can be rescued or enhanced through RNA and protein content modification.
Temple University--Theses

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Sommerville, Laura Jean. « The Role of Allograft Inflammatory Factor-1 in Vascular Smooth Muscle Cell Activation and Development of Vascular Proliferative Disease ». Diss., Temple University Libraries, 2010. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/76756.

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Molecular and Cellular Physiology
Ph.D.
The underlying cause of all vascular proliferative diseases is injury-induced activation of vascular endothelium and vascular smooth muscle cells (VSMC). Activated VSMC proliferate, than migrate from the arterial media to the intima, contributing to neointima formation. Activated immune cells, vascular cells, and their endogenous regulators mediate this complex process. One integral regulator of VSMC activation is allograft inflammatory factor-1 (AIF-1). AIF-1 is a cytoplasmic scaffold protein, expressed constitutively in lymphoid cells and induced in VSMC by injury. Stable over expression of AIF-1 increases VSMC proliferation and migration in vitro, causes increased injury-induced neointima formation, and increases Rac1 and p38 MAP Kinase activity. Recent studies show a correlation between VSMC expression of AIF-1 and atherosclerosis development. We hypothesize that VSMC over expression of AIF-1 contributes to atherosclerosis development by increasing activity of inflammatory signaling molecules, and that inhibiting VSMC AIF-1 expression will decrease injury-induced neointima formation. Rat carotid arteries transfected with AIF-1 si RNA adenovirus after balloon angioplasty developed significantly less neointima compared to controls. AIF-1 si RNA transfected VSMC proliferated significantly less than AIF-1 or GFP transfected VSMC, while AIF-1 si RNA transfection did not attenuate AIF-1-mediated migration. p38 inhibition showed that AIF-1-mediated proliferation is dependent on p38 activation while AIF-1-mediated migration is not. AIF-1 transgenic mice fed a high fat diet showed significantly more atherosclerotic lesions than WT littermates. Boyden Chamber assays showed OxLDL treatment increases VSMC migration but does not effect AIF-1-mediated migration. Expression of migration and inflammatory responsive genes in AIF-1 and XGal transfected VSMC after OxLDL treatment at various time points were examined. MMP-2 and -9 expression did not change. ICAM-1 and VCAM-1 expression increased in both groups. AIF-1 VSMC showed significantly higher ICAM-1 expression at baseline and early time points and elevated, but not significantly higher VCAM-1 expression at early time points. Western blots showed increased activation of NF-kB in AIF-1 transfected VSMC at baseline and 30 minutes after OxLDL stimulation compared to XGal transfected VSMC. Expression of the scavenger receptor receptors CD36 and SRA(I) expression increased after lipid treatment in AIF-1 and XGal transfected groups. AIF-1 VSMC showed sustained expression of both receptors after 16 hours of treatment compared to XGal VSMC, which showed decreased expression at that time point. CXCL16/PSOX expression increased with treatment, but differences in expression patterns were not seen between cell groups. Analysis showed significantly more OxLDL was taken up by AIF-1 VSMC compared to XGal VSMC. These data show that AIF-1 expression in VSMC is tightly linked to the vascular response to injury and development of vascular disease. Although AIF-1-mediated migration is not p38 dependent, AIF-1 may contribute to increased VSMC migration in part by upregulating NF- kB downstream effectors through increased NF-kB activity. AIF-1 may also speed the progression of atherosclerosis by increasing scavenger receptor expression and thereby increasing OxLDL uptake and foam cell formation. Although more study is required to fully elucidate the molecular mechanisms leading to AIF-1 mediated VSMC activation, these data have further established AIF-1 as an integral regulator of the VSMC response to injury.
Temple University--Theses

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Bizumukama, Leonidas. « Contribution à l'étude du mécanisme d'action anti-drépanocytaire du cromoglycate disodique ». Doctoral thesis, Universite Libre de Bruxelles, 2011. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/209805.

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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.

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.

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.

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.

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.

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.

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.

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.

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.

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

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Herman, Allison. « RNA-binding proteins mediate anti-inflammatory regulation of vascular disease ». Diss., Temple University Libraries, 2019. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/554883.

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Biomedical Sciences
Ph.D.
This work identifies the Fragile X-related protein (FXR1) as a reciprocal regulator of HuR target transcripts in vascular smooth muscle cells (VSMC). FXR1 was identified as an HuR interacting protein by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The-HuR-FXR1 interaction is abrogated in RNase-treated extracts, indicating that their association is tethered by mRNAs. FXR1 expression is induced in diseased, but not normal arteries. SiRNA knock down of FXR1 increases abundance and stability of inflammatory mRNAs, while overexpression of FXR1 reduces their abundance and stability. RNA-EMSA and RIP demonstrate that FXR1 directly interacts with an ARE and a previously uncharacterized element in the 3’UTR of TNFa. FXR1 expression is increased in VSMC challenged with the anti-inflammatory cytokine IL-19, and FXR1 is required for IL-19 reduction of HuR. This suggests FXR1 is an anti-inflammation responsive, HuR counter-regulatory protein that reduces abundance of pro-inflammatory transcripts. Additionally, we observed significantly increased poly-A-Binding protein (PABP) expression localizing to discrete punctate structures in both vascular smooth muscle (VSMC) and endothelial cells (EC) of the aortic arch of Ldlr-/- mice, as compared to WT controls. EIF2α phosphorylation, requisite for SG formation, was also induced by clotrimazole and oxLDL in these cells. Interestingly, VSMCs pre-treated with anti-inflammatory cytokine IL-19 followed by clotrimazole significantly reduced the formation of SGs and eIF2a phosphorylation, suggesting a relationship between inflammation and SG formation in vascular cells. Reduction of SG component G3BP1 by siRNA knockdown significantly reduced stress granule formation and inflammatory gene abundance in hVSMC. Microtubule inhibitors reduced SG formation in hVSMC. These results support the hypothesis that SG formation in atherosclerosis is driven by inflammation, SG may mediate the cellular response to inflammation, and that anti-inflammatory treatment may lessen atherosclerosis progression and plaque formation by reduction of SGs.
Temple University--Theses

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Moussaed, Mireille. « Étude de Reg-1α dans les processus neurodégénératifs associés aux tauopathies ». Thesis, Paris, EPHE, 2016. http://www.theses.fr/2016EPHE3048.

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Reg-1α est une protéine essentielle du système digestif impliquée dans des fonctions de prolifération, différenciation et régénération. Elle est exprimée et sécrétée par les neurones du système nerveux central où elle stimule la croissance neuritique et régule la différenciation et la migration des cellules précurseurs neurales via son récepteur EXTL3 et la voie GSK-3β. Par ailleurs, Reg-1α est surexprimée dans les cerveaux de patients Alzheimer et nos études préliminaires montrent qu’elle est associée à Tau hyperphosphorylée. Nous avons étudié dans ce contexte (1) le rôle de Reg-1α dans les processus neurodégénératifs liés à Tau et les voies de signalisation impliquées et (2) sa localisation dans le cerveau de souris transgéniques exprimant Tau mutée P301L/R406W. Nous avons montré que la surexpression de Reg-1α dans des neurones différenciés ne modifie pas significativement la voie Akt/GSK-3β/P-Tau mais induit la formation d’amas de Tau anormalement phosphorylée et une perturbation modérée du transport axonal. Par contre, sur des neurones surexprimant TauP301L, Reg-1α stimule la phosphorylation de Tau via Akt/GSK-3β entrainant la formation accentuée de renflements neuritiques et la perturbation sévère du transport axonal. In vivo, Reg-1α augmente avec l’âge dans le cerveau des souris contrôles et son expression est plus importante dès 5 mois chez les souris PLB2 Tau comparée aux souris sauvages. De plus, Reg-1α est associée à l’accumulation de Tau phosphorylée en S202 chez les souris transgéniques. Reg-1α est une protéine pouvant moduler l’hyperphosphorylation de Tau et le transport axonal et donc pourrait jouer un rôle dans le développement des tauopathies
Reg-1α is an essential protein of the digestive system involved in proliferation, differentiation and regeneration functions. It is also expressed and secreted by neurons of the central nervous system where it stimulates neurite outgrowth and regulates differentiation and migration of neural precursor cells via its receptor EXTL3 and GSK-3β pathway. Moreover, Reg-1α is overexpressed in the brain of Alzheimer's patients and our preliminary studies show that it is associated with hyperphosphorylated Tau. We studied in this context (1) the role of Reg-1α in neurodegenerative processes associated with Tau and the involved signaling pathways and (2) its location in the brain of transgenic mice expressing mutated Tau P301L/R406W (PLB2 mice). We showed that overexpression of Reg-1α in differentiated neurons does not significantly modify the Akt/GSK-3β/P-tau pathway. However it induces the formation of neuritic swellings associated with abnormal phosphorylated Tau and leads to the disruption of axonal transport. Furthermore, in neurons overexpressing TauP301L, Reg-1α overexpression stimulates Tau phosphorylation via Akt/GSK-3β regulation and results in the increase of neuritic bulges and severe disruption of axonal transport. In vivo, Reg-1α expression increases with age in brains of control mice and is already higher in 5 month-old PLB2 Tau mice compared with age-matched controls. Cellular localization showed that Reg-1α is associated with the accumulation of phosphorylated Tau S202 in PLB2 mice. Finally, Reg-1α can regulate Tau hyperphosphorylation and axonal transport and consequentely could be involved in Tauopathy development

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Dysart, Anna, W. Andrew Clark, Jo-Ann Marrs, Jonathan M. Peterson, Michelle Eileen Johnson et Arsham Alamian. « Evaluation of Dietary Intake and Red Blood Cell Membrane Fatty Acid Profile on the Incidence of Metabolic Syndrome in Hispanic Children from 2 to 10 Years of Age ». Digital Commons @ East Tennessee State University, 2017. https://dc.etsu.edu/etsu-works/1388.

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Balakrishnan, Meenakshi Puthucode. « Studies on a novel human cardiospecific transcription factor and its involvement in Omi/HtrA2 mediated cell death ». Doctoral diss., University of Central Florida, 2010. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4649.

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Omi/HtrA2 is a mitochondrial serine protease that is known to translocate to the cytoplasm upon induction of apoptosis and to activate caspase-dependent and caspase-independent cell death. The molecular mechanism of Omi/HtrA2's function is not clear but involves degradation of specific substrates. These substrates include cytoplasmic, mitochondrial, as well as nuclear proteins. We have pubmedisolated a new Omi/HtrA2 interactor, the THAP5 protein. THAP5 is a fifth member of a large family of transcription factors that are involved in cell proliferation, apoptosis, cell cycle control, chromosome segregation, chromatin modification and transcriptional regulation. THAP5 is an approximately 50kDa nuclear protein, with a restricted pattern of expression. Furthermore, there is no mouse or rat homolog for this protein. THAP5 mRNA is highly expressed in the human heart but some expression is also seen in the brain and skeletal muscle. The normal function of THAP5 in the heart or heart disease is unknown. THAP5 protein level is significantly reduced in the myocardial infarction (MI) area in the heart of patients with coronary artery disease (CAD). This part of the heart sustains most of the cellular damage and apoptosis. Our data clearly show that THAP5 is a specific substrate of the proapoptotic Omi/HtrA2 protease and is cleaved and removed during cell death. The molecular mechanism of THAP5's function is unclear. THAP5 can bind to a specific DNA sequence and repress transcription of a reporter gene. Our work suggests that THAP5 is a tissue specific transcriptional repressor that plays an important role in the normal function of the human heart as well as in the development of heart disease.
ID: 029050522; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (Ph.D.)--University of Central Florida, 2010.; Includes bibliographical references (p. 68-79).
Ph.D.
Doctorate
Burnett School of Biomedical Sciences
Medicine

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Toral, Rizo Victor Hugo 1977. « Hodgkin / Reed-Sternberg-like cells in diffuse large B cell lymphoma of the oral cavity = histopathological, immunohistochemistry and in situ hybridization study = Células de Hodgkin/Reed-Sternberg-like em linfoma difuso de grandes células B de boca : estudo histopatológico, imunoistoquímico e de hibridização in situ ». [s.n.], 2013. http://repositorio.unicamp.br/jspui/handle/REPOSIP/288358.

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Orientador: Oslei Paes de Almeida
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O linfoma difuso de grandes células B (LDGCB) é o linfoma da cavidade bucal mais comum. Alguns dos LDGCB podem apresentar células grandes morfologicamente similares às células Hodgkin e Reed/Sternberg (HRS) dos linfomas de Hodgkin clássico (LHC). O objetivo deste estudo foi comparar os LDGCB bucal que apresentem células HRS-like (LDGCB-HRS) com o linfoma de Hodgkin primário nodal, considerando os aspectos histológicos e imunoistoquímicos (IQs), angiogênese, índice de mastócitos e células dendríticas (CD), por meio de um amplo painel IQ. Quize casos foram estudados, nos quais sete eram LDGCB-HRS like e oito eram LHC nodal. Para a análise dos aspectos histológicos e IQs foram utilizados os seguintes anticorpos: CD3, CD15, CD20, CD30, CD43, LCA, CD45RO, CD79a, CD83, EMA, MUM-1, PAX-5, perforina, granzyme B, FASN, Ki-67, LMP-1; e EBER1/2. Já para a análise da angiogênese foram utilizados os anticorpos CD34, CD31, D2-40, CD105, vWF e VEGF; e para o índice de mastócitos utilizou-se o mast cell triptase. Finalmente, para avaliar a expressão IQ das CD os anticorpos CD1a, CD83, CD123, CD207, S-100 e FXIIIa foram utilizados. Todas as lâminas foram escaneadas e as células HRS-like, mastócitos e CD imunopositivas foram analisados, assim como os parâmetros morfométricos da angiogênese. Os resultados mostraram que a imunoexpressão foi postiva em 100% de casos de LHC e em 57% dos casos de LDGCB de boca, enquanto que LCA, CD20 e CD79a foram exclusivos para todos os LDGCB, e apenas CD15 foi exclusivo para os LHC. Angiogênese e o índice de mastócitos estavam aumentados em ambas as lesões, e entre elas, o LHC obteve maiores valores que o LDGCB da cavidade bucal em todos os anticorpos analisados. Por fim, o índice de CDs foram estatisticamente significante entre os grupos, exceto para CD83, que não mostrou nenhuma diferença estatística. A distribuição de CD foi reconhecida principalmente na área tumoral e ao redor das células neoplásicas em ambas as entidades. Foi possível concluir que os LDGCB com células HRS-like da cavidade bucal devem ser incluídos no diagnóstico diferencial de LHC da cavidade bucal. Quando da avaliação destes casos, a analise morfológica detalhada assim como o uso de um amplo painel de IQ são recomendados para realizar o diagnóstico correto. A angiogênese é essencial para o desenvolvimento de LDGCB da cavidade bucal, e quaisquer dos anticorpos CD34, CD31 e vWF podem ser utilizados para avaliar os parâmetros morfométricos. A presença significativa de CD nestes linfomas provavelmente desempenha um papel patologicamente relevante nos linfomas. Nossos resultados sugerem que o aumento no número de CD parece ser um fator contribuinte para a resposta imune estimulada pelo crescimento tumoral
Abstract: Diffuse large B-cell lymphoma (DLBCL) is the most common oral lymphoma. Some DLBCLs can present large cells morphologically similar to Hodgkin and Reed-Sternberg (HRS) cells of classical Hodgkin lymphoma (cHL). The objective of this study was to compare oral DLBCL presenting HRS-like cells (DLBCL-HRS like) with primary nodal cHL, considering the following aspects: histological and immunohistochemical (IHC), angiogenesis, index of mast cells and dendritic cells (DCs); through a broad immunohistochemical panel. Fifteen cases were studied, of which, seven were DLBCL-HRS like and eight were nodal cHL. For histological and IHC aspects, immunoexpression of CD3, CD15, CD20, CD30, CD43, LCA, CD45RO, CD79a, CD83, EMA, MUM-1, PAX-5, perforin, granzyme B, FASN, Ki-67, LMP-1; and EBER1/2, were assessed. As for angiogenesis analysis, the antibodies used were CD34, CD31, D2-40, CD105, vWF and VEGF; and for the index of mast cell were used the mast cell tryptase. Finally, for IHC expression of DCs, the antibodies used were CD1a, CD83, CD123, CD207, S-100, and FXIIIa. All slides were scanned and positive immunoreactive cells HRS-like, mast cell and DCs were analyzed, as well as morphometric parameters of angiogenesis. The results showed that the immunoexpression of CD30 was 100% positive in cHL and 57% in oral DLBCL HRS-like, while LCA, CD20 and CD79a were exclusive for all oral DLBCL, and only CD15 was exclusive for cHL. Angiogenesis and mast cell index values were increased in both lesions and between them, cHL was greater than oral DLBCL with all antibodies studied. Finally, DC subsets were statistically significant between groups, except CD83, which did not show statistical significance. The distribution of DCs was mainly in the tumor area, around neoplastic cells in both entities. It was possible to conclude that DLBCL-HRS should be included in the differential diagnosis of oral cHL. When evaluating these cases, a detailed morphologic and a broad IHC analyses for the correct diagnosis are recommended. Angiogenesis is essential to the development of DLBCL of the oral cavity and any of the antibodies CD34, CD31 and vWF could be used to evaluate morphometric parameters. The presence of significantly higher numbers of DCs in these lymphomas could suggest that these cells are likely to play a pathological relevant role in lymphomas. Our findings suggest that increased number of DCs in lymphomas appears to be a factor contributing to the immune response against tumor growth
Doutorado
Patologia
Doutor em Estomatopatologia

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Ferreira, Mônica Calil Borges. « Doença falciforme : um olhar sobre a assistência prestada na rede pública estadual – Hemocentro Regional de Juiz de Fora ». Universidade Federal de Juiz de Fora, 2012. https://repositorio.ufjf.br/jspui/handle/ufjf/1748.

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As hemoglobinopatias constituem o distúrbio genético de maior frequência nos seres humanos, sendo a doença falciforme (DF), com destaque para a anemia falciforme, a de maior impacto clínico, social e epidemiológico. Devido às características raciais do Brasil essas desordens genéticas passaram a representar um grave problema de saúde pública. Minas Gerais por meio da Fundação Centro de Hematologia e Hemoterapia (Hemominas) é pioneira na implantação de uma política de atenção aos portadores de DF, sendo que, desde 1998 a doença foi incluída na triagem neonatal (TN), enquanto que no Programa Nacional tal vinculação só ocorreu a partir de 2001. No Brasil, dos seus 27 estados apenas 18 realizam a TN para a DF. A implantação de uma triagem precoce para hemoglobinopatias não garante por si só o sucesso do Programa, pois é necessário acompanhar constantemente a rede de atenção a DF, visando avaliar e promover melhorias desde a atenção básica à saúde, com o “teste do pezinho”, até o tratamento em serviços de maior complexidade. Trata-se de um estudo quantitativo que se propôs a avaliar o espaço cronológico entre as etapas da TN, assim como a frequência e caracterização socioeconômica dos casos de portadores de DF matriculados no Hemocentro Regional de Juiz de Fora (HRJF) - Hemominas, durante o período de 1998 a 2007. No período proposto, foram triados em Minas Gerais 2.549.097 recém-nascidos, sendo que, 210.696 nascidos nas 34 cidades que referenciam o HRJF como centro de tratamento da DF. As cidades que melhor representam a incidência estadual de DF são Juiz de Fora e Ubá. Das crianças estudadas com DF não houve diferença significativa entre os gêneros. Em relação ao perfil hematológico dos acompanhados pelo HRJF (n=109) a HbSS esteve presente em 42,2% pacientes, a HbFC em 27,5%, a HbFS em 23,8% e a HbS/B-talassemia em 6,4%, sendo o percentual de meninos HbSS de 48,2% para 35,8% meninas HbSS. A maioria das famílias relatou viver com renda familiar menor que um salário mínimo por mês (37%). Em relação a fonte de renda foi identificado que o pai trabalha com carteira assinada em 44,9% e as mães em apenas 18,3%. Em 7,33% das famílias o pai está desempregado e as mães em 32,1%, fato que reforça a vulnerabilidade social das crianças portadoras de DF. Outro aspecto importante é a presença da DF em mais de um filho na mesma família, constatando a presença de 56% dos irmãos com a doença, sendo que deste, em 41% o diagnóstico é de anemia falciforme. Quanto ao traço falciforme, 36,7% possuem ao menos mais um filho com traço falciforme e 6,4% desconhece a presença do traço entre os irmãos da criança entrevistada, o que demonstra a necessidade de orientação aos pais quanto ao planejamento familiar. O espaço cronológico entre a coleta de sangue e o cadastro no HRJF foi de 17 dias, período este considerado ideal. Como produto geral da pesquisa, obteve-se um maior conhecimento dos programas integrais de atenção à DF implementados pelo HRJF propiciando uma compreensão mais ampla da situação da DF no nosso Estado na tentativa de favorecer num futuro bem próximo o planejamento de políticas públicas e outras ações que possam contribuir para reduzir a morbimortalidade e melhorar a qualidade de vida do doente falciforme. Além disso, como o Programa Nacional de TN está em alguns estados brasileiros em fase inicial de implantação, em muito contribuiria para esta iniciativa uma ampla divulgação dos estudos, para que medidas de prevenção e controle sejam melhor implementadas.
Hemoglobinopathies are the most frequent genetic disease in humans, and sickle cell disease (SCD), especially for sickle cell anemia, the most clinical impact, social and epidemiological. Due to the racial characteristics of Brazil these genetic disorders now represent a serious public health problem. Minas Gerais through the Foundation Center of Hematology (Hemominas) is pioneer in implementing a policy of care for patients with SCD, and since 1998 the disease was included in newborn screening (NS), while in this National Program Binding occurred only after 2001. In Brazil, the 27 states only 18 do the NS to perform the SCD. The implementation of an early screening for hemoglobinopathies is not in itself guarantee the success of the program, it is necessary to constantly monitor the care net SCD, to evaluate and promote improvement since the primary health care, with the "Guthrie test" to the treatment services of greater complexity. This is a quantitative study aimed to evaluate the space between the chronological stages of NS, as well as the frequency and socioeconomic characteristics of the cases of patients with SCD enrolled in Regional Blood Center of Juiz de Fora (RBCJF) - Hemominas during the period 1998 to 2007. The proposed period, were screened in Minas Gerais 2,549,097 newborns, and that 210,696 newborns in 34 cities that reference the RBCJF as a center for treatment of SCD. The cities that best represent the incidence of SCD are state Juiz de Fora and Uba. From these children with SCD did not differ between genders. Regarding the hematological profile of RBCJF accompanied by (n = 109) to HbSS was present in 42.2% patients, HBFCs by 27.5% to 23.8% and HbFS HbS / B thalassemia in 6.4 %, the percentage of boys HbSS 48.2% to 35.8% HbSS girls. Most families reported living with family income less than one minimum wage per month (37%). Regarding the source of income was identified as the father works with a formal contract in 44.9% and mothers in only 18.3%. In 7.33% of families the father is unemployed and mothers in 32.1%, a fact that reinforces the social vulnerability of children with SCD. Another important aspect is the presence of SCD in more than one child in the family, noting the presence of 56% of the siblings with the disease, and this, in 41% the diagnosis is sickle cell anemia. As for the sickle cell trait, 36.7% have at least one child with sickle cell trait and 6.4% were unaware of the presence of the trait among the siblings of children interviewed, which demonstrates the need for guidance to parents about family planning. The space between the chronological collection of blood and register for RCBJF was 17 days, a period considered ideal. As a product of the research, we obtained a greater knowledge of comprehensive attention to SCD RCBJF implemented by providing a broader understanding of the situation in our state of the SCD in trying to promote in the near future planning policies and other actions that may help reduce morbidity and improve quality of life of sickle cell patients. Moreover, as the National Program for NS is in some Brazilian states in the initial deployment, greatly contribute to this initiative a wide dissemination of studies, so that prevention and control measures are best implemented.

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Xu, Tieying. « Analyse des propriétés en déformabilité de globules rouges par impédancemétrie au sein d’une puce microfluidique : application à la drépanocytose ». Thesis, université Paris-Saclay, 2020. http://www.theses.fr/2020UPASN010.

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En raison d’une mutation génétique, l'hémoglobine contenue dans les globules rouges (GRs) se polymérise sous conditions de désoxydation, conduisant à une modification des propriétés en déformabilité des GRs. Ce travail de thèse propose une investigation par des mesures électriques de cinétique de passage comme nouveau moyen pour une caractérisation plus rapide et plus poussée de la déformabilité des GRs. Il s’appuie sur une puce microfluidique réutilisable contenant un réseau de capillaires mimétique de la circulation sanguine au sein des organes et intégrant des électrodes de mesure électrique. La discrimination entre des globules rouges normaux, des globules rouges chauffés, des globules rouges sphérocytaires et des globules rouges drépanocytaires constitue le cœur de cette thèse.Pour la mise en œuvre de ce projet, une analyse analytique utilisant un modèle numérique a été utilisée pour estimer le comportement fluidique et électrique de la puce microfluidique. La microfabrication a été utilisée pour obtenir la puce microfluidique. Le packaging utilise un PDMS recouvert de parylène permettant un assemblage réversible et simplifiant la mise en œuvre du système.Plus de 2000 globules rouges ont été analysés pour la comparaison entre des globules rouges normaux ou pathologiques (drépanocytaires ou atteints par la sphérocytose héréditaire). Une discrimination entre les globules rouges normaux et anormaux a été observée en comparant ces paramètres. Une corrélation entre les caractérisationsélectriques mesurées et les propriétés mécaniques des globules rouges est ainsi obtenue
Due to the genetic disorder, the hemoglobin in red blood cell (RBC) polymerizes under deoxidation conditions, leading to the variation of RBC deformability. Statistical analyses of the variation of RBC deformability are usually performed with an optical microscope, by observing RBC shape and behaviour under flow rate. However, this method lacks productiveness. This thesis studies an electrical investigation of flow kinetics across the mimetic capillary. It offers a new original way for faster and further characterization of the alteration of red blood cell (RBC) deformability. It is based on a reusable microfluidic chip which contains a mimicking capillaries network with embedded electrodes. Discrimination between normal RBCs, heated RBCs, hereditary spherocytosis RBCs and sickle cells has been achieved.During this project, an analytical approach and a finite element analysis were used to estimate the fluidic and electric behaviour of the cell flowing in the microfluidic device. Specific microfabrication was required to obtain the reusable microfluidic PDMS chip and electrodes. The packaging uses a parylene-coated PDMS allowing a reversible assembly and simplifying the implementation of the system.More than 2000 red blood cells were analysed for comparison between normal and pathological red blood cells (sickle cell disease or hereditary spherocytosis). The results mainly exploit the transit time and the amplitude of the current blockage when RBCs transit within the capillary. Discrimination between normal and abnormal RBCs was observed with these data. A correlation between the measured electrical characterizations and the mechanical properties of the red blood cells is thus obtained

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Nwaneshiudu, Adaobi I. « The Role of Gamma-Delta TCR+ T-cells in the Pathogenesis of Systemic Sclerosis ». Diss., Temple University Libraries, 2008. http://cdm16002.contentdm.oclc.org/cdm/ref/collection/p245801coll10/id/11843.

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Microbiology and Immunology
Ph.D.
The human gamma-delta (gd) TCR+ T-cell subset may undergo specific antigen-driven activation and clonal expansion, in the context of systemic sclerosis (SSc) pathogenesis. The purpose of this study was; 1) To determine whether gd TCR+ T-cells are clonally expanded in skin biopsies and peripheral blood from patients with SSc; and 2) To develop approaches for identification of the antigens recognized by these clonally-expanded gd TCR+ T-cells. Total RNA was isolated from the skin biopsies and peripheral blood of patients with SSc (n=8). After cDNA synthesis, the g- and d-chain TCR transcripts were amplified by PCR, cloned and sequenced for analysis. Full length copies of the TCR transcripts were constructed, expressed in a TCR-negative Jurkat T-cell line using retroviral gene transduction, and verified by RT-PCR and flow cytometry for gd TCR expression. Putative antigen recognition, by the transduced gd TCR+ Jurkat T-cell lines, was assessed via; 1) Measuring intracellular calcium flux in the transduced cells after stimulation with putative SSc antigens, including DNA topoisomerase I, centromere proteins A and B, hsp 27, hsp 90 and the viral lysate of human cytomegalovirus; and 2) Cytotoxicity against human endothelial cell lines (HUVEC and HLMVEC) via measurement of lactate dehydrogenase release from the targets. We report the presence of substantial, statistically-significant, proportions of identical g- and d-chain transcripts in skin biopsies and PBMC of patients with SSc, demonstrating the presence of antigen-driven clonal expansions. Jurkat T-cells, transduced with the clonally-expanded gd TCR transcripts from a patient, showed no evidence of cytotoxicity against the human endothelial cell lines, or calcium flux in response to stimulation with the putative SSc antigens assessed. In conclusion, extensive clonal expansions of g- and d-chain TCR transcripts were identified in skin biopsies and peripheral blood of patients with SSc, demonstrating the presence of oligoclonal populations of gd TCR+ T-cells in these patients. These gd TCR+ T-cells have undergone proliferation and clonal expansion in vivo in response to as yet unidentified antigens. Furthermore, an approach has been developed for the identification of the antigens recognized by the clonally-expanded gd TCR transcripts, which can be expanded to additional patients with SSc.
Temple University--Theses

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Kohli, Neha. « Amelioration of Amyloid Burden in Advanced Human and Mouse Alzheimer's Disease Brains by Oral Delivery of Myelin Basic Protein Bioencapsulated in Plant Cells ». Master's thesis, University of Central Florida, 2012. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/5380.

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One of the pathological hallmarks of Alzheimer's disease (AD) is the amyloid plaque deposition in aging brains by aggregation of amyloid-? (A?) peptides. In this study, the effect of chloroplast derived myelin basic protein (MBP) fused with cholera toxin subunit B (CTB) was investigated in advanced diseased stage of human and mouse AD brains. The CTB-fusion protein in chloroplasts facilitates transmucosal delivery in the gut by the natural binding ability of CTB pentameric form with GM1 receptors on the intestinal epithelium. Further, bioencapsulation of the MBP within plant cells confers protection from enzymes and acids in the digestive system. Here, 12-14 months old triple transgenic AD mice were fed with CTB-MBP bioencapsulated in the plant cells for 3 months. A reduction of 67.3% and 33.3% amyloid levels in hippocampal and cortical regions, respectively were observed by immunostaining of brain sections with anti- A? antibody. Similarly, 70% decrease in plaque number and 40% reduction of plaque intensity was observed through thioflavin S (ThS) staining that specifically stains amyloid in the AD brain. Furthermore, ex vivo 3xTg AD mice brain sections showed up to 45% reduction of ThS stained amyloid levels when incubated with enriched CTB-MBP in a concentration dependent manner. Similarly, incubation of enriched CTB-MBP with ex vivo postmortem human brain tissue sections with advanced stage of AD resulted up to 47% decrease of ThS stained amyloid plaque intensity. Lastly, lyophilization of plant material facilitates dehydration and long term storage of capsules at room temperature, in addition to increasing CTB-MBP concentration by 17 fold. These observations offer a low cost solution for treatment of even advanced stages of the AD by facilitating delivery of therapeutic proteins to central nervous system to address other neurodegenerative disease.
M.S.
Masters
Molecular Biology and Microbiology
Medicine
Biotechnology

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Eggart, Benjamin [Verfasser], et Thomas [Akademischer Betreuer] Franke. « Deformation and Shape Transition Studies of Single Mammalian Red Blood Cells in View of the Effects of Diseases or Chemical Modifications by Means of Microfluidic Devices / Benjamin Eggart. Betreuer : Thomas Franke ». Augsburg : Universität Augsburg, 2015. http://d-nb.info/107770593X/34.

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Neel, Sarah Elizabeth. « Transplantation of iPS cells reduces apoptosis and fibrosis and improves cardiac function in streptozotocin-induced diabetic rats ». Master's thesis, University of Central Florida, 2010. http://digital.library.ucf.edu/cdm/ref/collection/ETD/id/4686.

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Background: Streptozotocin (STZ) induced diabetes leads to various complications including cardiomyopathy. Recent data suggests transplanted bone marrow stem cells improve cardiac function in diabetic cardiomyopathy. However, whether modified ES, iPS cells, or factors released from these cells can inhibit apoptosis and fibrosis remains completely unknown. The present study was designed to determine the effects of transplanted ES cells overexpressing pancreatic transcription factor 1 a (Ptf1a), a pro-pancreatic endodermal transcription factor, iPS cells, or their respective conditioned media (CM) on diabetic cardiomyopathy. Methods: Experimental diabetes was induced in male Sprague Dawley rats (8-10 weeks old) by intraperitoneal STZ injections (65 mg/kg body weight for 2 consecutive days). Animals were divided into six experimental groups including control, treated with sodium citrate buffer IP, STZ, STZ + ES-Ptf1a cells, STZ + iPS cells, STZ + ES-Ptf1a CM and STZ + iPS CM. Following STZ injections, appropriate cells (1 X 106/mL/injection/day) or CM (2 mL injection/day) were given intravenously for 3 consecutive days. Animals were sacrificed and hearts were harvested at day 28. Histology, TUNEL staining, and Caspase-3 activity were used to assess apoptosis and fibrosis. ERK1/2 phosphorylation was quantified using ELISAs. M-mode echocardiography fractional shortening was used to assess cardiac function. Results: Animals transplanted with ES cells, iPS cells, or both CMs showed a significant (pless than]0.05) reduction in interstitial fibrosis, and apoptosis compared with STZ group. ERK expression was not significantly different compared with STZ. Echocardiography showed a significant (pless than]0.05) improvement in fractional shortening in cell and media transplanted groups compared with STZ. Conclusions: Our data suggest that ES cells, iPS cells, and/or CMs inhibit apoptosis, reduce fibrosis, and improve cardiac function in STZ-treated diabetic rats.
ID: 029049879; System requirements: World Wide Web browser and PDF reader.; Mode of access: World Wide Web.; Thesis (M.S.)--University of Central Florida, 2010.; Includes bibliographical references (p. 33-40).
M.S.
Masters
Burnett School of Biomedical Sciences
Medicine

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Monedero, Alonso David. « Characterization of cationic conductances of human erythrocytes and their involvement in health and disease ». Electronic Thesis or Diss., Sorbonne université, 2019. http://www.theses.fr/2019SORUS554.

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La membrane des globules rouges est dotée de plusieurs canaux ioniques. Normalement silencieux, ils peuvent dissiper rapidement les gradients ioniques une fois activés. Lors de cette étude, l'utilisation du NS3623 à des concentrations supérieures à celles requises pour l'inhibition des voies de conductances anioniques montre que ce composé active les canaux cationiques non sélectifs permettant ainsi leur étude y compris en conditions hyperpolarisantes. Le suivi en temps réel du potentiel membranaire à l'aide de l'ionophore à protons CCCP permet d’observer directement l'activité des canaux ioniques lorsque leur ouverture modifie le potentiel membranaire. Cette méthode a été utilisée pour décrire l'homéostasie cationique dysfonctionnelle dans des cellules de patients affectés par différentes mutations sur les canaux Gárdos ou Piezo1. Elle pourrait constituer un outil de diagnostic alternatif. L'activité des canaux ioniques a été caractérisée tout au long de la période de stockage réglementaire des globules rouges stockés à 4 °C (42 jours), afin de mieux comprendre les lésions de stockage. Il a été démontré que l’activité du NSC augmentait avec le temps, devenant spectaculaire la dernière semaine de stockage. En conclusion, les canaux cationiques non sélectifs jouent un rôle dans l'homéostasie des globules rouges matures. Ils contribuent ou peuvent constituer l'origine de la fuite de cations. Ils sont à l'origine de maladies en cas de dysfonctionnement et la compréhension de leur fonctionnement dans ces conditions peut fournir des stratégies thérapeutiques. Enfin, ils sont impliqués dans les lésions de stockage compromettant par leur activité l'efficacité transfusionnelle
Red cell membranes are endowed with several ion channels. Normally silent, they will rapidly dissipate ionic gradients once activated. I present a pharmacological means (NS3623) for the enhancement of NSC channels in hyperpolarizing conditions with concomitant chloride conductance inhibition in freshly drawn healthy mature RBCs. Membrane potential estimation aided by proton ionophore CCCP allows the recording of membrane potential changes in real time, enabling the observation of ion channel activity as their opening alters the membrane potential. This method was used to describe dysfunctional cation homeostasis in hereditary anemia using patient cells affected by different mutations on Gárdos or Piezo1 channels. The technique is fast, reliable and inexpensive providing an alternative diagnostic tool with the added advantage of producing ion channel activity information. Ion channel activity was characterized throughout 42-day storage period of RBCs stored at 4 C in CPD-SAGM according to French regulations to address the issue of storage lesions, which reduce transfusion efficacy. NSC activity was shown to increase over time during storage and dramatic ion channel activity was observed during the last week. Consequently, NSC activity may jeopardize cell volume and morphology upon reinfusion. In conclusion, Non-Selective Cation channels play an important role in mature RBCs. They contribute or may constitute the origin of cation leak. They cause disease when malfunctioning and insight into their operation in these conditions may supply with therapeutic strategies. They are involved in the storage lesion, and may account for RBCs demise once back in the circulation

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Lee, David. « Age-Related Differences in In-vitro Sensitivity to Inhibition of Human Red Blood Cell Acetylcholinesterase and Plasma Butyrylcholinesterase by the Cholinesterase Inhibitors Physostigmine (PHYS), Pyridostigmine (PYR), Donepezil (DON) and Galantamine (GAL) ». VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1937.

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Alzheimer’s disease (AD) is a chronic, progressive neurodegenerative disorder, characterized clinically by a progressive loss of memory, cognitive function, ability to care for oneself and psychiatric symptoms. First-line agents for the treatment of AD are ChE inhibitors (DON, GAL), whose modest clinical efficacy and the high incidence of dose-limiting toxicities limit their clinical utility. In addition to AD, ChE inhibitors (PYR) are used for other medical conditions, such as myasthenia gravis (MG). Furthermore, ChE inhibitors (PYR) are used by military personnel prophylactically if impending exposure to chemical warfare agents, e.g., soman, is suspected. The purpose of this research project was to understand the effect of age on the in-vitro sensitivity of ChE inhibitors in human RBCs and plasma. Understanding possible covariates, such as age and gender, may assist in optimizing dosing regimens of ChE inhibitors and/or developing newer ChE inhibitors with better adverse effect profiles. Plasma PHYS concentrations were measured by a validated HPLC-FD method. RBC AChE activity and plasma BuChE activity were measured by a modified Ellman’s colorimetric method using the model substrates, acetylthiocholine and butyrylthiocholine, respectively. The kinetics of RBC and plasma ChE activity followed Michaelis-Menten kinetics. Acetylthiocholine was found to be a nonselective substrate (RBC AChE Km = 73 μM; plasma BuChE Km = 117 μM); while butyrylthiocholine was a selective substrate for plasma BuChE (RBC AChE Km = 130,000 μM; plasma BuChE Km = 72 μM). For the following studies, RBC AChE activity was measured using acetylthiocholine as the substrate and plasma BuChE activity was measured using butyrylthiocholine as the substrate. This research project was performed in two parts: First, mechanistic studies of PHYS, PYR, DON and GAL, explored and determined the mechanism of in-vitro inhibition of RBC AChE and plasma BuChE inhibition, as well as the in-vitro degradation of PHYS in human whole blood, plasma and RBC. PHYS was rapidly degraded in human whole blood, RBC and plasma and followed Michaelis-Menten kinetics but its degradation clearance - scaled to whole blood clearance - was only predicted to account for 4-6% (i.e., 195-261 ml/min) of the reported total body clearance for PHYS (4500 ml/min). RBCs were responsible for 60% of the whole blood clearance while plasma accounted for 40% of the whole blood clearance. Inhibition results indicated that both PHYS and PYR were nonselective and rapid suicide ChE inactivators. PYR inactivated RBC AChE more rapidly at low concentrations and inactivated plasma BuChE more rapidly at high concentrations, but inactivated both more rapidly than PHYS. PHYS was a more potent inactivator than PYR with a Ki for RBC AChE of 0.011 μM and 0.063 μM, respectively, and 0.023 μM and 0.036 μM, respectively for plasma BuChE. DON was found to be a noncompetitive inhibitor for RBC AChE (Ki,noncomp = 114 μM), but a competitive inhibitor for plasma BuChE (Ki,comp = 213 μM). GAL was found to be a competitive inhibitor for both RBC AChE (Ki,comp = 66 μM) and plasma BuChE (Ki,comp = 358 μM). The second part involved a clinical study with ten young and nine elderly healthy subjects, balanced for gender, who donated blood for an in-vitro study in order to assess any age- and gender-related differences in in-vitro sensitivity to RBC AChE and plasma BuChE inhibition to all four ChE inhibitors. Elderly adults were found to be 2-3-fold less sensitive compared to the young adults for PHYS (BuChE Ki,pss; 0.010 and 0.015 μM, young and elderly, respectively) and PYR (AChE Ki,pss; 0.12 and 0.25 μM, young and elderly, respectively) only, while neither DON nor GAL showed any age-related differences in sensitivity. The observed differences for PHYS and PYR may be due to kinetic differences in ChE inactivation between young and aged adults, rather then a difference in binding affinities/potencies. These carbamate ChE inhibitors, presumably, have a slower decarbamoylation rate in younger adults than elderly adults, which leads to the observed difference in in-vitro sensitivity. The above in-vitro results were consistent with results of a meta-analysis: In a study by Knapp et al. (1991), young males (n=6), receiving 18 mg, 24 mg and 30 mg PHYS tablets, showed similar ex-vivo plasma BuChE sensitivity to (28 %/(ng/ml)) as the in-vitro sensitivity for young males in the current study (33 %/(ng/ml)). On the other hand, in the study by Men (2004), elderly males (n=8) and females (n=8), receiving 6.7 μg/kg PHYS as 30-minute infusion, showed similar ex-vivo RBC AChE sensitivity (12 %/(ng/ml)) as the in-vitro sensitivity for elderly subjects in the current study (9.7 %/(ng/ml)). This suggests that in-vitro measurement of ChE sensitivity is predictive of ex-vivo sensitivity in clinical studies. The study results suggest that elderly adults may require a 2-3-fold higher blood concentration than young adults to achieve the same ChE inhibition. This may explain why for epistigmine, an investigational carbamate ChE inhibitor for the treatment of AD, the maximum tolerated dose observed in young adults (40 mg single dose) was lower than for older adults (90 mg/day). Higher sensitivity in young adults prevented further dose escalation, while all elderly subjects tolerated higher doses. This research may have implications for other diseases and conditions, most notably MG and as a prophylaxis of nerve gases poisoning. As patients with MG age, they may become less sensitive to PYR, the most common symptomatic treatment for MG, and an increase in dose may be required. Further, older military personnel assigned to receive PYR, may require increased doses to achieve the targeted 10% RBC AChE inhibition, necessary to protect against nerve gas poisoning.

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Chipeaux, Caroline. « Recherche et validation de biomarqueurs lipidiques du globule rouge par chromatographie en phase liquide couplée à la spectrométrie de masse. Application au diagnostic et au suivi thérapeutique de la maladie de Gaucher ». Thesis, Université Paris-Saclay (ComUE), 2019. http://www.theses.fr/2019SACLS419.

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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

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PILLAI, Vinoshene. « Intravital two photon clcium imaging of glioblastoma mouse models ». Doctoral thesis, Scuola Normale Superiore, 2021. http://hdl.handle.net/11384/109211.

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Tavares, Lucas Alves. « O envolvimento da proteína adaptadora 1 (AP-1) no mecanismo de regulação negativa do receptor CD4 por Nef de HIV-1 ». Universidade de São Paulo, 2016. http://www.teses.usp.br/teses/disponiveis/17/17136/tde-06012017-113215/.

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O Vírus da Imunodeficiência Humana (HIV) é o agente etiológico da Síndrome da Imunodeficiência Adquirida (AIDS). A AIDS é uma doença de distribuição mundial, e estima-se que existam atualmente pelo menos 36,9 milhões de pessoas infectadas com o vírus. Durante o seu ciclo replicativo, o HIV promove diversas alterações na fisiologia da célula hospedeira a fim de promover sua sobrevivência e potencializar a replicação. A rápida progressão da infecção pelo HIV-1 em humanos e em modelos animais está intimamente ligada à função da proteína acessória Nef. Dentre as diversas ações de Nef está a regulação negativa de proteínas importantes na resposta imunológica, como o receptor CD4. Sabe-se que esta ação resulta da indução da degradação de CD4 em lisossomos, mas os mecanismos moleculares envolvidos ainda são totalmente elucidados. Nef forma um complexo tripartite com a cauda citosólica de CD4 e a proteína adaptadora 2 (AP-2), em vesículas revestidas por clatrina nascentes, induzindo a internalização e degradação lisossomal de CD4. Pesquisas anteriores demonstraram que o direcionamento de CD4 aos lisossomos por Nef envolve a entrada do receptor na via dos corpos multivesiculares (MVBs), por um mecanismo atípico, pois, embora não necessite da ubiquitinação de carga, depende da ação de proteínas que compõem os ESCRTs (Endosomal Sorting Complexes Required for Transport) e da ação de Alix, uma proteína acessória da maquinaria ESCRT. Já foi reportado que Nef interage com subunidades dos complexos AP-1, AP-2, AP-3 e Nef não parece interagir com subunidades de AP-4 e AP-5. Entretanto, o papel da interação de Nef com AP-1 e AP-3 na regulação negativa de CD4 ainda não está totalmente elucidado. Ademais, AP-1, AP-2 e AP-3 são potencialmente heterogêneos devido à existência de isoformas múltiplas das subunidades codificadas por diferentes genes. Todavia, existem poucos estudos para demonstrar se as diferentes combinações de isoformas dos APs são formadas e se possuem propriedades funcionais distintas. O presente trabalho procurou identificar e caracterizar fatores celulares envolvidos na regulação do tráfego intracelular de proteínas no processo de regulação negativa de CD4 induzido por Nef. Mais especificamente, este estudo buscou caracterizar a participação do complexo AP-1 na modulação negativa de CD4 por Nef de HIV-1, através do estudo funcional das duas isoformas de ?-adaptina, subunidades de AP-1. Utilizando a técnica de Pull-down demonstramos que Nef é capaz de interagir com ?2. Além disso, nossos dados de Imunoblot indicaram que a proteína ?2-adaptina, e não ?1-adaptina, é necessária no processo de degradação lisossomal de CD4 por Nef e que esta participação é conservada para degradação de CD4 por Nef de diferentes cepas virais. Ademais, por citometria de fluxo, o silenciamento de ?2, e não de ?1, compromete a diminuição dos níveis de CD4 por Nef da membrana plasmática. A análise por imunofluorêsncia indireta também revelou que a diminuição dos níveis de ?2 impede a redistribuição de CD4 por Nef para regiões perinucleares, acarretando no acúmulo de CD4, retirados por Nef da membrana plasmática, em endossomos primários. A depleção de ?1A, outra subunidade de AP-1, acarretou na diminuição dos níveis celulares de ?2 e ?1, bem como, no comprometimento da eficiente degradação de CD4 por Nef. Além disso, foi possível observar que, ao perturbar a maquinaria ESCRT via super-expressão de HRS (uma subunidade do complexo ESCRT-0), ocorreu um acumulo de ?2 em endossomos dilatados contendo HRS-GFP, nos quais também detectou-se CD4 que foi internalizado por Nef. Em conjunto, os resultados indicam que ?2-adaptina é uma importante molécula para o direcionamento de CD4 por Nef para a via ESCRT/MVB, mostrando ser uma proteína relevante no sistema endo-lisossomal. Ademais, os resultados indicaram que as isoformas ?-adaptinas não só possuem funções distintas, mas também parecem compor complexos AP-1 com diferentes funções celulares, já que apenas a variante AP-1 contendo ?2, mas não ?1, participa da regulação negativa de CD4 por Nef. Estes estudos contribuem para o melhor entendimento dos mecanismos moleculares envolvidos na atividade de Nef, que poderão também ajudar na melhor compreensão da patogênese do HIV e da síndrome relacionada. Em adição, este trabalho contribui para o entendimento de processos fundamentais da regulação do tráfego de proteínas transmembrana no sistema endo-lisossomal.
The Human Immunodeficiency Virus (HIV) is the etiologic agent of Acquired Immunodeficiency Syndrome (AIDS). AIDS is a disease which has a global distribution, and it is estimated that there are currently at least 36.9 million people infected with the virus. During the replication cycle, HIV promotes several changes in the physiology of the host cell to promote their survival and enhance replication. The fast progression of HIV-1 in humans and animal models is closely linked to the function of an accessory protein Nef. Among several actions of Nef, one is the most important is the down-regulation of proteins from the immune response, such as the CD4 receptor. It is known that this action causes CD4 degradation in lysosome, but the molecular mechanisms are still incompletely understood. Nef forms a tripartite complex with the cytosolic tail of the CD4 and adapter protein 2 (AP-2) in clathrin-coated vesicles, inducing CD4 internalization and lysosome degradation. Previous research has demonstrated that CD4 target to lysosomes by Nef involves targeting of this receptor to multivesicular bodies (MVBs) pathway by an atypical mechanism because, although not need charging ubiquitination, depends on the proteins from ESCRTs (Endosomal Sorting Complexes Required for Transport) machinery and the action of Alix, an accessory protein ESCRT machinery. It has been reported that Nef interacts with subunits of AP- 1, AP-2, AP-3 complexes and Nef does not appear to interact with AP-4 and AP-5 subunits. However, the role of Nef interaction with AP-1 or AP-3 in CD4 down-regulation is poorly understood. Furthermore, AP-1, AP-2 and AP-3 are potentially heterogeneous due to the existence of multiple subunits isoforms encoded by different genes. However, there are few studies to demonstrate if the different combinations of APs isoforms are form and if they have distinct functional properties. This study aim to identify and characterize cellular factors involved on CD4 down-modulation induced by Nef from HIV-1. More specifically, this study aimed to characterize the involvement of AP-1 complex in the down-regulation of CD4 by Nef HIV-1 through the functional study of the two isoforms of ?-adaptins, AP-1 subunits. By pull-down technique, we showed that Nef is able to interact with ?2. In addition, our data from immunoblots indicated that ?2- adaptin, not ?1-adaptin, is required in Nef-mediated targeting of CD4 to lysosomes and the ?2 participation in this process is conserved by Nef from different viral strains. Furthermore, by flow cytometry assay, ?2 depletion, but not ?1 depletion, compromises the reduction of surface CD4 levels induced by Nef. Immunofluorescence microscopy analysis also revealed that ?2 depletion impairs the redistribution of CD4 by Nef to juxtanuclear region, resulting in CD4 accumulation in primary endosomes. Knockdown of ?1A, another subunit of AP-1, resulted in decreased cellular levels of ?1 and ?2 and, compromising the efficient CD4 degradation by Nef. Moreover, upon artificially stabilizing ESCRT-I in early endosomes, via overexpression of HRS, internalized CD4 accumulates in enlarged HRS-GFP positive endosomes, where co-localize with ?2. Together, the results indicate that ?2-adaptin is a molecule that is essential for CD4 targeting by Nef to ESCRT/MVB pathway, being an important protein in the endo-lysosomal system. Furthermore, the results indicate that ?-adaptins isoforms not only have different functions, but also seem to compose AP-1 complex with distinct cell functions, and only the AP-1 variant comprising ?2, but not ?1, acts in the CD4 down-regulation induced by Nef. These studies contribute to a better understanding on the molecular mechanisms involved in Nef activities, which may also help to improve the understanding of the HIV pathogenesis and the related syndrome. In addition, this work contributes with the understanding of primordial process regulation on intracellular trafficking of transmembrane proteins.

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BARABINO, GILDA ANN. « RHEOLOGICAL STUDIES IN SICKLE CELL DISEASE (RED BLOOD, ENDOTHELIAL, ADHERENCE) ». Thesis, 1986. http://hdl.handle.net/1911/15952.

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The abnormal adherence of sickle erythrocytes to endothelial cells (EC) has been hypothesized to play a role in the initiation of vaso-occlusion in sickle cell anemia. Erythrocyte/endothelial cell interactions were studied under controlled flow conditions for normal (AA), homozygous sickle cell (SS), sickle trait (AS), mechanically injured normal, and "high reticulocyte control" red blood cells (RBC). Human umbilical vein endothelial cells grown to confluence on glass slides formed the base of a parallel plate flow chamber into which RBC suspensions were perfused at a constant flow rate, producing a wall shear stress of 1 dyne/cm('2). Adhesion was monitored using video microscopy, and the number of adherent RBC was determined at ten-minute intervals during a wash-out period. Results indicate that SS RBC were more adherent than AA RBC. Mechanically injured (sheared) RBC were also more adherent than control normal cells, but less adherent than SS RBC. AS RBC did not differ significantly in their adhesive properties from normal RBC. Less dense (younger) RBC were more adherent to EC than dense (older) cells for normal, SS and "high reticulocyte control" RBC. The average velocity of individual SS RBC in the region near the EC monolayer was approximately half that of AA RBC at the same bulk volumetric flow rate, as determined using image analysis techniques. The influence of several factors on the adherence of normal and sickle cells to endothelial cells was examined: (1) increasing shear rates resulted in decreased adhesion, (2) pretreating EC with a chemotactic agent had little effect on adherence properties, (3) treating RBC with pentoxifylline diminished the adherence of sickle RBC, but had no effect on AA RBC and (4) suspending RBC in a protein free suspending medium did not affect the demonstration of adherence differences between SS and AA RBC. These findings suggest that the increased adhesion of sickle RBC is at least partially related to the increased numbers of young RBC present. Increased adherence of young cells to the EC lining vessel walls could contribute to microvascular occlusion by lengthening vascular transit times of other sickle cells. Pentoxifylline may aid in the treatment of sickle cell disease by decreasing these obstructive RBC-EC interactions that may play a role in vaso-occlusion.

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Ilboudo, Yann. « The genetics of red blood cell density, a biomarker of clinical severity in sickle cell disease ». Thèse, 2016. http://hdl.handle.net/1866/18661.

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Bridgemohan, Roshini. « Red cell membrane abnormalities in hereditary spherocytosis patients of KwaZulu-Natal ». Thesis, 2006. http://hdl.handle.net/10413/2055.

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Hereditary Spherocytosis (HS) is a common inherited haemolytic anaemia with variable clinical expression. Fifty subjects with HS from KwaZulu-Natal were studied with the aim of providing further information on the protein abnormalities of the red blood cell (RBC) membrane and their relationship with clinical presentations. Haematological and biochemical tests were performed by routine procedures. Mean Corpuscular Haemoglobin Concentration ( MCHC) in the HS group was 35.1g /dl. This was significantly higher than in normal control subjects (33.6g /dl) (p value < 0.001); indicating its usefulness for the screening of HS. Mean Red Cell Distribution Width (RDW) was also significantly higher in subjects with HS (p<0.001); thus providing an additional screening tool. Erythrocyte membrane proteins from 21 subjects were analysed by SDS - polyacrylamide gel electrophoresis (SDS-PAGE) using the Laemmli and Fairbanks methods. The most common abnormality was a deficiency of band 3 (10 subjects), followed by a combined spectrin and ankyrin deficiency in five subjects. One subject had increased band 6 and in five cases no abnormality was detected. A decreased ratio of protein 4.1a / 4.1b on the Laemmli SDS PAGE correlated with an increased reticulocyte count. The degree of haemolysis and clinical findings did not correlate with the type of red cell membrane protein defect. In this study red cell membrane analysis did not contribute further to the initial laboratory diagnosis. In addition it did not influence clinical management. The presence of red cell membrane abnormalities, either single or multiple, did not correlate with disease severity. Red cell membrane analysis, however, will play an important role for future management such as gene therapy. Red cell membrane analysis is also useful as a research tool to determine the underlying molecular defect and to assess racial or ethnic differences. It is also of value as a differential diagnostic tool in cases where the clinical and laboratory findings are not conclusive for HS.
Thesis (M.Med.Sci)-University of KwaZulu-Natal, Durban, 2006.

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