During the last 40 years, Drosophila melanogaster has become an invaluable tool in understanding innate immunity. The innate immune system of Drosophila consists of a humoral and a cellular component. While many details are known about the humoral immune system, our knowledge about the cellular immune system is comparatively small. Blood cells or hemocytes constitute the cellular immune system. Three blood types have been described for Drosophila larvae. Plasmatocytes are phagocytes with a plethora of functions. Crystal cells mediate melanization and contribute to wound healing. Plasmatocytes and crystal cells constitute the blood cell repertoire of a healthy larva, whereas lamellocytes are induced in a demand-adapted manner after infection with parasitoid wasp eggs. They are involved in the melanotic encapsulation response against parasites and form melanotic nodules that are also referred to as tumors. In my thesis, I focused on unraveling the mechanisms of how the immune system orchestrates the cellular immune response. In particular, I was interested in the hematopoiesis of lamellocytes. In Article I, we were able to show that ectopic expression of key components of a number of signaling pathways in blood cells induced the development of lamellocytes, led to a proliferative response of plasmatocytes, or to a combination of lamellocyte activation and plasmatocyte proliferation. In Article II, I combined newly developed fluorescent enhancer-reporter constructs specific for plasmatocytes and lamellocytes and developed a “dual reporter system” that was used in live microscopy of fly larvae. In addition, we established flow cytometry as a tool to count total blood cell numbers and to distinguish between different blood cell types. The “dual reporter system” enabled us to differentiate between six blood cell types and established proliferation as a central feature of the cellular immune response. The combination flow cytometry and live imaging increased our understanding of the tempo-spatial events leading to the cellular immune reaction. In Article III, I developed a genetic modifier screen to find genes involved in the hematopoiesis of lamellocytes. I took advantage of the gain-of-function phenotype of the Tl10b mutation characterized by an activated cellular immune system, which induced the formation blood cell tumors. We screened the right arm of chromosome 3 for enhancers and suppressors of this mutation and uncovered ird1. Finally in Article IV, we showed that the activity of the Toll signaling pathway in the fat body, the homolog of the liver, is necessary to activate the cellular immune system and induce lamellocyte hematopoiesis.

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The most widely transfused blood component is red blood cells (RBCs), and voluntary donation is the main resource for RBC transfusion. In the UK, 7,000 units of RBCs are transfused daily but this life-saving cell therapy is completely dependent on donors and there are persistent problems associated with transfusion transmitted infections and in blood group compatibility. Furthermore, the quality, safety and efficiency of donated RBCs gradually decrease with storage time. A number of novel sources of RBCs are being explored including the production of RBCs from adult haematopoietic progenitor cells, erythroid progenitor cell lines and induced pluripotent stem cells (iPSCs). The iPSC source could essentially provide a limitless supply and a route to producing cells that are matched to the recipient. A number of protocols have been described to produce mature RBCs from human pluripotent stem cells but they are relatively inefficient and would be difficult to scale up to the levels required for clinical translation. We tested and evaluated a defined feeder- and serum-free differentiation protocol for deriving erythroid cells from hiPSCs. RBC production was not efficient, the cells that were produced did not enucleate efficiently and they expressed embryonic rather than adult globin. We hypothesised that the production of RBCs from iPSCs could be enhanced by enforced expression of erythroid-specific transcription factors (TFs). Previous studies had demonstrated that Krüppel-like factor 1 (KLF1) plays an important role in RBC development and maturation so we generated iPSC lines expressing a tamoxifen-inducible KLF1-ERT2 fusion protein. Using zinc finger nuclease technology, we targeted the expression cassette to the AAVS1 locus to ensure consistent expression levels and to avoid integration site specific effects and/or silencing. These iKLF1 iPSCs were applied to our defined RBC differentiation protocol and the activity of KLF1 was induced by adding tamoxifen. Activation of KLF1 from day 10 accelerated erythroid differentiation and maturation with an increase in the proportion of erythroblasts, a higher level of expression of erythroid genes associated with maturation and an apparently more robust morphology. However, KLF1 activation had an anti-proliferation effect resulting in significantly less cell generated overall and HPLC analysis demonstrated that KLF1-activated cells expressed higher levels of embryonic globin compared to control iPSCs-derived cells. Many of the effects that were observed when KLF1 was activated from day 10 were not observed when activated from day 18. We therefore concluded that activation of exogenous KLF1 is able to promote erythroid cell production and maturation in progenitors (day 10) but not at the later stage of erythropoiesis (day 18). We hypothesised that KLF1 might require a co-factor to regulate RBC maturation and adult globin expression at the later stage of erythropoiesis. The TF, B-cell lymphoma/leukaemia 11a (BCL11A), plays a key role in the suppression of foetal globin expression, thereby completing globin switching to adult globin. Preliminary data showed that iPSC-derived erythroid cells were able to express adult globin when transduced with a BCL11A-expressing lentiviral-vector. Based on that finding we then generated an iPSC line expressing tamoxifen-inducible BCL11AERT2 and KLF1-ERT2 fusion proteins, applied this iBK iPSC line to our differentiation protocol. Activation of both TFs from day 18 slightly increased the expression of genes associated with RBC maturation and the inclusion of BCL11A appeared to eliminate the anti-proliferation effect of KLF1. Most importantly, activation of both BCL11A and KLF1 from day 18 of the differentiation protocol increased the production of α- globin (foetal / adult globin) indicating that some definitive-like erythroid cells might be generated by activation of both TFs at the later stage of erythroid differentiation. Collectively, these findings demonstrate that enforced expression of erythroid TFs could be a useful strategy to enhance RBC maturation from iPSCs.

Made available in DSpace on 2016-08-15T20:31:08Z (GMT). No. of bitstreams: 1 RejaneSS_DISSERT.pdf: 2305528 bytes, checksum: 47bde97e4a35896152b153a7383736d8 (MD5) Previous issue date: 2012-07-04 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / This study evaluated the clinical, hematological, biochemical and blood gas responses and the oxidative stress in sheep submitted to hiperacute anemia and subsequently underwent to homologous transfusion with whole blood, either fresh, or stored for 15 or 35 days. Eighteen adult Santa Inez crossbred sheep, males and females, were used, weighing on average 52kg. The animals were submitted to a single phlebotomy to remove 40% of blood volume and equally divided into three experimental groups: G0 - animals receiving fresh blood; G15 receiving blood stored for 15 days; and G35 - receiving blood stored for 35 days. The animals were submitted to clinical examination and blood collection 24 h after induction of anemia (T0), 30 minutes after transfusion (T30), six, twelve, twenty-four, forty-eight, seventy-two and ninety-six hours post-transfusion (T6, T12, T24, T48, T72 and T96, respectively), and eight and sixteen days post-transfusion (T8d and T16d, respectively). The blood bags stored for 35 days showed an increase in K, pCO2, pO2, lactate, plasma hemoglobin and decreased plasma pH, sodium and leukocytes. The sheep transfused from all groups had increased GV, red cell count and total hemoglobin in the T30. The animals of the G35 had higher plasma hemoglobin in T12, a significant decrease in blood pH indicating a mild metabolic acidosis on T96. With respect to oxidative stress, was observed a decreased on catalase values of the G35 at T30, T6, T12 and T24, suggesting the occurrence of hemolysis witch was supported by the concomitant increase in the total bilirubin values at the same periods. Animals that received blood stored for 35 days showed higher alteration on hematological, blood gas, biochemical and oxidative stress parameters / O presente trabalho objetivou avaliar as respostas clínicas, hematológicas, bioquímicas, hemogasométricas e o estresse oxidativo de ovinos induzidos à anemia superaguda e transfundidos com sangue total fresco ou armazenado por dois diferentes períodos (15 e 35 dias). Foram utilizados 18 ovinos, machos e fêmeas, com idade entre 3 e 4 anos, pesando em media 52kg. Os animais foram submetidos a uma única flebotomia para retirada de 40% do volume sanguíneo e divididos em três grupos experimentais, sendo o G0 composto por animais que receberam sangue fresco, G15 e G35 animais que receberam sangue armazenado em bolsas CPDA-1 por 15 e 35 dias, respectivamente. Foi realizado exame clínico e coleta de amostras de sangue 24 horas pós-indução da anemia (T0), 30 minutos pós-transfusão (T30), seis, 12, 24, 48, 72 e 96 horas após à transfusão (T6, T12, T24, T48, T72, T96, respectivamente) e oito e dezesseis dias após à transfusão (T8d e T16d, respectivamente). O sangue armazenado por 35 dias apresentou aumento do potássio, pCO2, pO2, lactato, hemoglobina plasmática e diminuição do pH, sódio e leucócitos. Ovinos transfundidos no T30 apresentaram aumento significativo do VG, hemácias e hemoglobina total. Os animais do G35 apresentaram maiores valores de hemoglobina plasmática no T12 e diminuição do pH sanguíneo, caracterizando leve acidemia metabólica no T96. Com relação ao estresse oxidativo o G35 apresentou redução da catalase no T30, T6, T12 e T24, indicando a ocorrência de hemólise, o que foi corroborado pelo aumento concomitante da bilirrubina. Os animais que receberam sangue armazenado por 35 dias apresentaram maiores alterações hematológicas, hemogasométricas, bioquímicas e oxidativas

Made available in DSpace on 2016-08-15T20:31:26Z (GMT). No. of bitstreams: 1 RejaneSS_DISSERT.pdf: 2305548 bytes, checksum: 418124494d2c13cd40a5d285bfaec106 (MD5) Previous issue date: 2012-09-04 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / This study evaluated the clinical, hematological, biochemical and blood gas responses and the oxidative stress in sheep submitted to hiperacute anemia and subsequently underwent to homologous transfusion with whole blood, either fresh, or stored for 15 or 35 days. Eighteen adult Santa Inez crossbred sheep, males and females, were used, weighing on average 52kg. The animals were submitted to a single phlebotomy to remove 40% of blood volume and equally divided into three experimental groups: G0 - animals receiving fresh blood; G15 receiving blood stored for 15 days; and G35 - receiving blood stored for 35 days. The animals were submitted to clinical examination and blood collection 24 h after induction of anemia (T0), 30 minutes after transfusion (T30), six, twelve, twenty-four, forty-eight, seventy-two and ninety-six hours post-transfusion (T6, T12, T24, T48, T72 and T96, respectively), and eight and sixteen days post-transfusion (T8d and T16d, respectively). The blood bags stored for 35 days showed an increase in K, pCO2, pO2, lactate, plasma hemoglobin and decreased plasma pH, sodium and leukocytes. The sheep transfused from all groups had increased GV, red cell count and total hemoglobin in the T30. The animals of the G35 had higher plasma hemoglobin in T12, a significant decrease in blood pH indicating a mild metabolic acidosis on T96. With respect to oxidative stress, was observed a decreased on catalase values of the G35 at T30, T6, T12 and T24, suggesting the occurrence of hemolysis witch was supported by the concomitant increase in the total bilirubin values at the same periods. Animals that received blood stored for 35 days showed higher alteration on hematological, blood gas, biochemical and oxidative stress parameters / O presente trabalho objetivou avaliar as respostas clínicas, hematológicas, bioquímicas, hemogasométricas e o estresse oxidativo de ovinos induzidos à anemia superaguda e transfundidos com sangue total fresco ou armazenado por dois diferentes períodos (15 e 35 dias). Foram utilizados 18 ovinos, machos e fêmeas, com idade entre 3 e 4 anos, pesando em media 52kg. Os animais foram submetidos a uma única flebotomia para retirada de 40% do volume sanguíneo e divididos em três grupos experimentais, sendo o G0 composto por animais que receberam sangue fresco, G15 e G35 animais que receberam sangue armazenado em bolsas CPDA-1 por 15 e 35 dias, respectivamente. Foi realizado exame clínico e coleta de amostras de sangue 24 horas pós-indução da anemia (T0), 30 minutos pós-transfusão (T30), seis, 12, 24, 48, 72 e 96 horas após à transfusão (T6, T12, T24, T48, T72, T96, respectivamente) e oito e dezesseis dias após à transfusão (T8d e T16d, respectivamente). O sangue armazenado por 35 dias apresentou aumento do potássio, pCO2, pO2, lactato, hemoglobina plasmática e diminuição do pH, sódio e leucócitos. Ovinos transfundidos no T30 apresentaram aumento significativo do VG, hemácias e hemoglobina total. Os animais do G35 apresentaram maiores valores de hemoglobina plasmática no T12 e diminuição do pH sanguíneo, caracterizando leve acidemia metabólica no T96. Com relação ao estresse oxidativo o G35 apresentou redução da catalase no T30, T6, T12 e T24, indicando a ocorrência de hemólise, o que foi corroborado pelo aumento concomitante da bilirrubina. Os animais que receberam sangue armazenado por 35 dias apresentaram maiores alterações hematológicas, hemogasométricas, bioquímicas e oxidativas

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.

Master of Science / Department of Clinical Sciences / Thomas Schermerhorn / Tonicity is property of a solution that is defined as the total effective (impermeable) osmole concentration that drives fluid movement across a semipermeable membrane via osmosis. Tonicity is related to but distinct from solution osmolality, which is a summation of all solute concentrations, regardless of the solute membrane permeability. In the mammalian body, tonicity is tightly regulated at both a cellular and systemic level; tonic derangements cause rapid change in cell and tissue volume leading to significant dysfunction. Input from the central nervous, circulatory, endocrine, gastrointestinal, and urinary systems are integral to osmoregulation, so many diseases in veterinary medicine are associated with tonicity disorders. However, because the homeostatic mechanisms that control tonicity overlap with those regulating electrolyte and acid-base balance as well as hydration and vascular volume, tonic consequences of disease can be difficult to isolate. Understanding of disease-associated changes in tonicity is further complicated by the fact that the tonic contributions of many solutes that accumulate in disease are unknown. Additionally, direct assessment of tonicity is difficult because tonicity is not just a physiochemical property, but it implies a physiologic effect. Thus, simple summation of osmole concentrations is an inadequate measurement of tonicity. The following report includes three studies investigating various aspects of tonicity as it applies to veterinary patients. Chapter 2 reports a study that examines the tonic effects of ketoacids and lactate using two different in vitro red blood cell assays. Results demonstrated that the ketoacids, beta-hydroxybutyrate and acetoacetate, behave as ineffective osmoles while the tonic behavior of lactate is variable, implying a more complex cellular handling of this anion. Two additional studies examine whether the mean corpuscular volume difference (dMCV) is a novel clinical marker for hypertonicity in dogs. Results of separate retrospective (Chapter 3) and prospective (Chapter 4) studies provide evidence that dMCV is a useful clinical marker for hypertonicity in dogs.

Dans ce travail, nous développons un cadre de calcul dédié à la simulation d'écoulements à plusieurs fluides. Nous présentons des validations et vérifications de ces méthodes sur des problèmes de capture d'interfaces et de simulations de bulles visqueuses.Nous montrons ensuite que ce cadre de calcul est adapté à la simulation d'objet rigides en écoulement.Puis, nous étendons ces méthodes à la simulation d'objets déformables simulant le comportement des globules rouges : les vésicules. Nous validons aussi ces simulations.Enfin nous appliquons les précédents modèles à des problèmes ouverts de microfluidique tels que la séparation d'une suspension dans une bifurcation microfluidique et la rhéologie en milieu confiné. / In this work, we develop a framework dedicated to the simulation of multi-fluid systems. We present validations and verifications of these methods on interface capture problems and viscous bubbles simulations.We then show that this framework is well fitted for the simulation of the rigid bodies flow.Next, we extend these methods to the simulation of deformable objects reproducing the behavior of red blood cells: the vesicles. We also validate these simulations.Finally, we apply the previous models to open micro-fluidic problems such as the splitting of a suspension at a bifurcation and the rheology in a confined environment.

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