Immune response provides vertebrates an important mechanism to fight pathogens and to reduce the incidence of diseases. Defining the molecular basis of antibody response may facilitate genetic improvement in the immune response of animals to pathogens. For almost 4 decades, antibody titers in response to challenge by sheep red blood cells (anti-SRBC) have provided an investigative tool in the efforts to define molecular mechanisms that underlie vertebrate immune response. The overall objective of this dissertation research was to identify DNA markers associated with anti-SRBC response in chickens. Specific objectives were: to develop a resource population for QTL analysis for anti-SRBC, to identify DNA markers and genes associated with primary anti-SRBC, and to evaluate the allelic frequencies in non-selected chicken populations of candidate markers associated with either high or low anti-SRBC response. These objectives tested the hypothesis that genetic control of a chicken's response to SRBC is polygenic. The resource population developed consisted of F1, backcross, and F2 derived from reciprocal crosses of birds from parental lines in the 28th generation of divergent selection for low (L) and high (H) anti-SRBC. The mean anti-SRBC titers of the parental lines were significantly different, with 11.5 for H and 2.6 for L (P<0.05). That for the 4 groups of F2 progeny ranged from 6.3 to 7.5, while those of the 8 groups of backcross progeny ranged from 3.9 to 13.3. Four of 555 random primers used to screen the parental H and L anti-SRBC lines were informative by amplifying seven line-specific fragments (P<0.0025). Each of the 7 line-specific fragments was converted to a sequence characterized amplified region (SCAR) within which single nucleotide polymorphisms (SNPs) were identified and tested for association with anti-SRBC. Only two of the seven SCARs in the parental lines were associated (P<0.05) with anti-SRBC level in the backcross resource population. Additionally, from analysis of the parental L and H anti-SRBC lines using microarrays, a total of 57 line-specific SNPs were also identified. Twenty of the line-specific SNPs were in and/or near genes previously reported to have immunity-related function. Microarray-based gene expression profiling of pooled RNA samples from L and H anti-SRBC birds identified three differentially expressed genes. In summary, this dissertation describes resources that include candidate SCARs and SNPs as well as differentially expressed genes that may be useful for the identification of genes that underlie antibody response. / Ph. D.

The experiment discussed in the next chapter was to confirm the aforementioned bystander effect. In the first experiment we separated hosting and non-hosting mRBCs by the percol purification method and then measured the corner frequencies of them. The mean fc of the distribution is almost the same, and this confirms the effect of the parasite on the non-hosting mRBC. In the next experiment, we have incubated nRBCs in the spent media and measured the corner frequency at six-hours intervals to see how the fc changed with the incubation time. The results showed that within 24 hours, the fc of the incubated nRBCs increases to the level of the iRBCs. The fact that nRBCs are getting affected by the spent media indicates that some substances must be released in the spent media which alter the physical properties of the nRBCs. This kind of effect on non-host mRBCs was previously observed by some earlier works [Dondorp97, Sabolovic91a, Bambardekar08]. It has also been recently shown that the rosetting of the host mRBCs to the non-host mRBCs is also activated by the substances released in the medium [Handunnetti89, Wahlgren89], which are also somewhat similar to the bystander effect observed by us. In addition to this, there are reports which suggest that sickle cell disease also shows binding properties [Roseff08, Zhang12] which may be due to the substances released in the medium. So it was already observed that the released substances induced changes in the properties of RBCs, but our study gives a direct confirmation of the same. The next study was to find out the released substances which were responsible for the observed changes above. We incubated infected and uninfected RBCs in different drugs. Then, we measured them to see what kind of changes occur in the corner frequency of the incubated RBCs. The corner frequency of normal RBCs incubated in db-cAMP shows the maximum change. So the released substance that is responsible for the bystander effect may be due to the db-cAMP. All the experiments above were done using samples cultured only in the lab. Since the environment of the blood taken directly from the patient may differ from the one that is cultured in the lab, it is natural to find out if similar kinds of changes can be observed in the clinical sample or not. The study in chapter 6 was targeted to find out the same. We took clinical samples from BMRI for patients having a confirmed malaria infection by both P. falciparum and P. vivax. This also provided us the opportunity to work with the P. vivax infected sample as it is very difficult to culture them in the lab. The results shown in this chapter clearly indicate that similar kinds of changes occur in the clinical sample also. It is worth noting that even though P. vivax infects only immature RBCs (reticulocytes), changes were also observed in P. vivax samples. This gives us another strong confirmation about the previously observed bystander effect. This also indicates that this technique can be used as a tool to diagnose malaria. Although we cannot differentiate between P. falciparum and P. vivax, this technique combined with other well established techniques can give us more confirmation. So, in all the experiment above we have shown an easy and novel technique which can be used to differentiate between normal and malaria-infected RBCs. We have also observed the bystander effect and tried to find out the released substances which are responsible for this effect. We have shown that this technique can use the bystander effect of malaria to identify malaria. It has also been shown that the RBCs taken from the patient sample also show the same changes as the cultured samples, which gives us the possibility that this technique can be used as a diagnostic tool combined with other technique. This technique can also be used in experiments like the effects of drugs and to find out drugs for diseases like malaria. Future outlook 1. We have observed the changes only for malaria. There may be other diseases like sickle cell anemia which can also alter the corner frequency of the distribution of RBCs. We have to find out the specificity of the observed changes. 1 We can directly measure the elasticity of RBCs using dual traps in optical tweezers to find out the effect of different infections and drugs on the rigidity of RBCs and compare the with the data above. 2 We can also study other cells using the same method to see if we can find out any difference between healthy and unhealthy cells.

Nanotechnology is a rapidly growing field focused on the manipulation and control of materials with dimensions under 100 nm. The novel electronic, optical and mechanical properties observed at the nanoscale have resulted in a number of applications in catalysis, light emitting devices, solar power, self-cleaning surfaces and medicine. Medical applications of nanotechnology (“nanomedicine”) are particularly promising for rapid clinical diagnosis and targeted treatments. Understanding the interactions of nanoparticles with living matter is of fundamental importance for all application areas: manufacture, use and disposal of the growing number of nanoproducts will result in increased environmental exposure in addition to direct exposure through nanomedical applications. However, there is a lack of standard methodologies for assessing these interactions. In this work the stability of silver-based nanoparticles was established by UV- Visible (UV-Vis) spectroscopy, atomic force microscopy (AFM) and transmission electron microscopy (TEM). The presence of a higher valence metal or polymer on the nanoparticle surface was demonstrated to improve stability. A standard methodology was developed to study nanoparticle-cell interactions: an “atlas” of the effects of known drugs on a cell is created, and compared with the effects of a nanoparticle. Escherichia coli was selected as a model organism and the effects of a range of antibiotics were characterised through a combination of microbiological assays and AFM. Susceptibility, population cell growth and individual heights, widths, lengths and volumes of bacteria were obtained on a 2% agarose substrate in air. The methodology was applied and adjusted for silver nanoparticles due to the interactions of silver with the bacterial growth medium. 10 and 30 nm silver nanoparticles and ions were found to kill E. coli through an internal mechanism of action, with a size-specific effect on the height of bacteria. Finally, a novel AFM characterisation method is described to examine the mechanical properties of live bacterial and human cells in liquid.

A transfusão de sangue continua a ser uma importante ferramenta terapêutica, porém como qualquer outra, apresenta riscos. O sangue após coletado é processado e origina os hemocomponentes, dentre eles o Concentrado de Hemácias (CH). O CH é o hemocomponente mais utilizado na prática clínica. É indicado para o tratamento de anemias e em casos de hemorragia aguda ocorridas em cirurgia ou em decorrência de doenças ou traumas. Dentre os principais riscos da transfusão de CH estão a aloimunização e as reações transfusionais, imediatas ou tardias. Deve-se avaliar a real necessidade de se transfundir um paciente, principalmente, no período intra operatório. A transfusão de CH no intra operatório está associada a um pior prognóstico, maior tempo de internação, sobrevida menor e aumento do risco de infecção. Este estudo tem por objetivo analisar as solicitações de reserva de CH, e a partir desta análise, elaborar um protocolo de reservas cirúrgicas de CH com a finalidade de determinar o quantitativo de bolsas a serem reservadas ou não para a realização procedimentos cirúrgicos realizados no Hospital Regional de Ceilândia, no Distrito Federal. Para a elaboração do protocolo, foi realizado o levantamento de dados referentes às solicitações de reservas realizadas neste hospital entre março e agosto de 2017. Baseado nestes dados, calculou-se o Índice de Pacientes Transfundidos (IPT) que definiu o percentual das reservas que foram utilizadas. Verificou-se que a maioria das reservas solicitadas não foi utilizada. Do total de 216 pacientes que tiveram reservas preparadas, para as quais foram compatibilizadas 438 bolsas, apenas 48 destas bolsas foram transfundidas, ou seja, somente 10,95%. A partir do resultado do IPT, foi definida uma conduta hemoterápica cirúrgica para cada tipo de cirurgia. E, a partir destas, foi elaborado, um protocolo de reservas de CH para cirurgias, com o intuito de racionalizar a solicitação de reservas para ser implementado no hospital. Podendo assim ser um instrumento, para a equipe médica e para a agência transfusional, contribuindo para a economia de recursos financeiros e de sangue. / Blood transfusion continues to be an important therapeutic tool but it presents risks. The blood after collection is processed originating the blood components, among them the Red blood cells (RBC). RBC is the most widely used blood component in clinical practice. It is indicated for the treatment of anemias and in cases of acute hemorrhage occurring in surgery or due to pathologies or traumas. Among the main risks of RBC transfusion are alloimmunization and transfusion reactions. The actual need to transfuse a patient, especially during the intraoperative period, should be evaluated. Intraoperative RBC transfusion is associated with a worse prognosis, longer length of stay, shorter survival and increased risk of infection. The aim of this study was to analyze the RBC reserve requests and then to elaborate a protocol of RBC surgical reserves in order to determine the quantity of blood bags to be reserved or not for surgical procedures performed at the Regional Hospital de Ceilândia (HRC), in the Federal District. For the elaboration of this protocol, the data of the reservations requests performed in HRC March 2017 to August 2017 was carried out. The transfusion Index was calculated from the data, which defined the percentage of reservations that were used. It was found that most of the requested reserves were not used. Of the total of 216 patients who had prepared reservations, for which 438 bags were compatibilized, only 48 of these bags were transfused, that is, only 10.95%. From the result of the transfusion Index, so for each elective surgical procedures a guide surgical hemotherapy was defined. In addition, a reserve protocol of RBC for surgeries was elaborated, with the purpose of rationalizing the reservation request to be implemented in the hospital. This can be an instrument for the medical team and for the transfusion agency, contributing to the rational use of financial and blood resources.

O sangue total passa por processo de centrifugação a fim de que o paciente receba apenas o componente sanguíneo do qual necessita. O controle de qualidade, inicia com inspeção visual, onde são avaliados: coloração, lipemia, presença de coágulos e vazamentos. No Hemocentro Coordenador do Paraná, a principal causa de descarte de plasma fresco (PF) é a lipemia. Os PF turvos são descartados e os respectivos concentrados de hemácias (CH) permanecem em estoque. Durante o armazenamento, os CH passam por processo de alterações bioquímicas e morfológicas conhecido como lesão de armazenamento, cuja última etapa é a hemólise. É comprometida a função terapêutica e segurança transfusional. Alguns autores relatam a relação entre lipemia e hemólise. Lipemia é o aspecto turvo do plasma, devido à presença de lipoproteínas e tem relação principalmente com a dieta do doador. O objetivo deste estudo foi comparar os parâmetros de qualidade dos CH que tiveram os plasmas descartados por lipemia com os CH de plasmas límpidos. Os PF lipêmicos foram separados e foi realizado registro por fotografia e dosagem dos triglicerídeos. Conforme o grau de turbidez os plasmas foram classificados como: ligeiramente turvos, moderadamente turvos, intensamente turvos ou leitosos. Conforme a concentração de triglicerídeos foram classificados como normal (<175 mg/dl), limítrofe (175 a 199 mg/dl), elevado (200 a 499 mg/dl) ou muito elevado (>500 mg/dl). Os respectivos CH foram avaliados durante o período de validade. Os experimentos nos CH foram realizados entre 1º e 10º, entre 11º e 22º, entre 23º e 34º e entre 35º e 42º dias de armazenamento. Foram avaliados: esterilidade, índices hematimétricos, ERO, TBARS, metemoglobina. Nos sobrenadantes dos CH foram avaliados: Na+, K+, Cl-, lactato, glicose, pH e grau de hemólise. Os experimentos também foram realizados com controles, CH de plasmas límpidos do mesmo dia de doação do grupo teste. Houve aumento da hemólise mais expressivo nos CH teste, demonstrando que a lipemia impacta negativamente na qualidade do CH durante o armazenamento. A partir dos resultados do estudo foram elaboradas estratégias para processamento, controle de qualidade, modificação e distribuição dos CH de doações com lipemia, garantindo a distribuição de um hemocomponente seguro e eficaz, minimizando descarte por hemólise e efeitos adversos à transfusão. / The blood processing allows the patient receives only the blood component that he needs. The quality control starts with visual inspection and are evaluated: coloration, lipemia, clots and leaks. At the HEMEPAR, the main loss of fresh plasma (FP) is caused by lipemia. The lipemic F are discarded and the related red blood cell (RBC) remain in stock. During storage, RBCs go through the process of biochemical and morphological changes known as storage lesions, and the last one hemolysis. Therapeutic function and transfusion safety are compromised. Some authors report the relationship between lipemia and hemolysis. Lipemia is the turbid aspect of plasma, due to the presence of lipoproteins and is mainly related to the diet of the donor. The objective of this study was to compare the quality parameters of the RBCs from lipemic donations with RBC from clear donations. Lipemic FP were photographed and quantificated to triglycerides. According to the degree of turbidity the plasmas were classified as: slightly cloudy, moderately cloudy, intensely turbid or milky. Triglyceride concentrations were classified as normal (<175 mg / dl), borderline (175 to 199 mg / dl), high (200 to 499 mg / dl) or very high (> 500 mg / dl). The respective RBCs were evaluated during the period of validity. The CH experiments were performed between 1 and 10º, 11º and 22º, 23º and 34º and 35º and 42º days of storage. The following: sterility, hematimetric indexes, ROS, TBARS and methemoglobin were evaluated. In the supernatants Na +, K +, Cl-, lactate, glucose, pH and degree of hemolysis were evaluated. The experiments were also carried out with controls, RBC from clear donations of the same day of donation of the test group. There was an increase in hemolysis in the RBC test. It shows that lipemia negatively impacts the quality of RBC during storage. From the results of the study, were established strategies for processing, quality control, modification and distribution of RBC of donations with lipemic plasma to ensure the use of safe and effective blood product for transfusion.

Introdução. O transporte é um procedimento crítico na manutenção da cadeia do frio do sangue. Falhas neste procedimento podem comprometer a sua qualidade e a segurança transfusional. Objetivo. Avaliar as não conformidades (NC) do Hemocentro de Ribeirão Preto relacionadas ao transporte de concentrado de hemácias (CH). Analisar caixas térmicas e gelos recicláveis submetidos à temperatura de 20°C e 30°C e propor melhorias neste processo. Métodologia. Estudo retrospectivo de NC relacionadas ao transporte de CH no software do sistema da qualidade do Hemocentro de Ribeirão Preto e pesquisa experimental realizada com três caixas diferentes em combinação com três gelos recicláveis distintos, simulando uma situação de transporte, nas temperaturas externas de 20°C e 30°C. Realizou-se uma estatística descritiva com os dados. Utilizou-se a metodologia de análise de variância (ANOVA), com o auxílio do software SAS® 9, utilizando a PROC GLM. Para as comparações foram utilizados contrastes ortogonais baseados na distribuição t. Resultados. Os resultados mostraram diferenças significativas no desempenho entre as caixas e entre os gelos recicláveis. No entanto, as maiores diferenças ocorreram entre as temperaturas externas de 20°C e 30°C, indicando a interferência da temperatura externa na temperatura do conteúdo interno. Outra diferença encontrada foi entre pontos de aferição das temperaturas, dentro da mesma caixa, indicando que a distribuição do ar, no interior da caixa não é homogênea. Conclusão. São vários os interferentes existentes durante o transporte de CH, que podem comprometer a qualidade do sangue. O transporte deve ser validado, padronizando o maior número de variáveis possíveis, para minimizar as adversidades que ocorrem durante o procedimento / Introduction. The transport is a critical procedure in the maintenance of the blood cold chain. Failures in this procedure can compromise the blood quality and transfusion safety. Purpose: Evaluate the non-conformances (NC) of Hemocentro de Ribeirão Preto related to the Red Blood Cell (RBC) transport. Verify coolers, recycle ice at a temperature of 20°C and 30°C and propose improvements in this process. Methodology: Retrospective study of NC related to RBC transport in Hemocentro de Ribeirão Preto quality system software. Experimental research performed with three different insulated boxes combined with three different ice packs, simulating a situation of transport in the external temperatures of 20°C and 30°C. It was made a descriptive statistic of the data. The methodology of analysis of variance was used, through the software SAS® 9, using a PROC GLM. To do the comparisons orthogonal contrasts based on t distribution were used. Results: The results shown significant differences between insulated box and ice packs. However, the larger differences occurred between the temperatures of 20°C and 30°C, indicating that the external temperature interfere in the temperature inside the box. Another difference found was between points of temperature, inside the same insulated box, indicating that the air distribution inside the insulated box is not homogeneous. Conclusion. During RBC transport, many interfering exist that can compromise the blood quality. The transport must be validated, standardizing the largest number of details that is possible, to minimize adversity that occur during the procedure

O sangue total passa por processo de centrifugação a fim de que o paciente receba apenas o componente sanguíneo do qual necessita. O controle de qualidade, inicia com inspeção visual, onde são avaliados: coloração, lipemia, presença de coágulos e vazamentos. No Hemocentro Coordenador do Paraná, a principal causa de descarte de plasma fresco (PF) é a lipemia. Os PF turvos são descartados e os respectivos concentrados de hemácias (CH) permanecem em estoque. Durante o armazenamento, os CH passam por processo de alterações bioquímicas e morfológicas conhecido como lesão de armazenamento, cuja última etapa é a hemólise. É comprometida a função terapêutica e segurança transfusional. Alguns autores relatam a relação entre lipemia e hemólise. Lipemia é o aspecto turvo do plasma, devido à presença de lipoproteínas e tem relação principalmente com a dieta do doador. O objetivo deste estudo foi comparar os parâmetros de qualidade dos CH que tiveram os plasmas descartados por lipemia com os CH de plasmas límpidos. Os PF lipêmicos foram separados e foi realizado registro por fotografia e dosagem dos triglicerídeos. Conforme o grau de turbidez os plasmas foram classificados como: ligeiramente turvos, moderadamente turvos, intensamente turvos ou leitosos. Conforme a concentração de triglicerídeos foram classificados como normal (<175 mg/dl), limítrofe (175 a 199 mg/dl), elevado (200 a 499 mg/dl) ou muito elevado (>500 mg/dl). Os respectivos CH foram avaliados durante o período de validade. Os experimentos nos CH foram realizados entre 1º e 10º, entre 11º e 22º, entre 23º e 34º e entre 35º e 42º dias de armazenamento. Foram avaliados: esterilidade, índices hematimétricos, ERO, TBARS, metemoglobina. Nos sobrenadantes dos CH foram avaliados: Na+, K+, Cl-, lactato, glicose, pH e grau de hemólise. Os experimentos também foram realizados com controles, CH de plasmas límpidos do mesmo dia de doação do grupo teste. Houve aumento da hemólise mais expressivo nos CH teste, demonstrando que a lipemia impacta negativamente na qualidade do CH durante o armazenamento. A partir dos resultados do estudo foram elaboradas estratégias para processamento, controle de qualidade, modificação e distribuição dos CH de doações com lipemia, garantindo a distribuição de um hemocomponente seguro e eficaz, minimizando descarte por hemólise e efeitos adversos à transfusão. / The blood processing allows the patient receives only the blood component that he needs. The quality control starts with visual inspection and are evaluated: coloration, lipemia, clots and leaks. At the HEMEPAR, the main loss of fresh plasma (FP) is caused by lipemia. The lipemic F are discarded and the related red blood cell (RBC) remain in stock. During storage, RBCs go through the process of biochemical and morphological changes known as storage lesions, and the last one hemolysis. Therapeutic function and transfusion safety are compromised. Some authors report the relationship between lipemia and hemolysis. Lipemia is the turbid aspect of plasma, due to the presence of lipoproteins and is mainly related to the diet of the donor. The objective of this study was to compare the quality parameters of the RBCs from lipemic donations with RBC from clear donations. Lipemic FP were photographed and quantificated to triglycerides. According to the degree of turbidity the plasmas were classified as: slightly cloudy, moderately cloudy, intensely turbid or milky. Triglyceride concentrations were classified as normal (<175 mg / dl), borderline (175 to 199 mg / dl), high (200 to 499 mg / dl) or very high (> 500 mg / dl). The respective RBCs were evaluated during the period of validity. The CH experiments were performed between 1 and 10º, 11º and 22º, 23º and 34º and 35º and 42º days of storage. The following: sterility, hematimetric indexes, ROS, TBARS and methemoglobin were evaluated. In the supernatants Na +, K +, Cl-, lactate, glucose, pH and degree of hemolysis were evaluated. The experiments were also carried out with controls, RBC from clear donations of the same day of donation of the test group. There was an increase in hemolysis in the RBC test. It shows that lipemia negatively impacts the quality of RBC during storage. From the results of the study, were established strategies for processing, quality control, modification and distribution of RBC of donations with lipemic plasma to ensure the use of safe and effective blood product for transfusion.

Major challenges in the management of blood supply chain are related to the shortage and wastage of the blood products. Given the perishability characteristics of blood which can be stored up to a limited number of days, if hospitals and blood centers keep an excessive number of blood units on inventory, wastages may occur. On the other hand, if sufficient number of blood units are not stored on inventory, shortages of this resource may cause the cancellations of important activities and increase the fatality rates at hospitals. Three mathematical models have been developed with the goal to improve the efficiency of blood related activities at blood centers and hospitals. The first model uses an integer programming (IP) approach to identify the optimal order levels that minimizes the total cost, shortage and wastage levels of blood products at a hospital within a specified planning horizon. The IP model explicitly considers the age of blood inventory, uncertain demand, the demand for two types of patients and crossmatch-to-transfusion ratio. The second model formulates the different shortage and inventory distribution strategies of a blood center supplying blood products to multiple hospitals. The third model develops a vehicle routing problem for blood centers to minimize the daily distance travelled by bloodmobiles during the blood collection process. Optimal routing for each bloodmobiles is identified using CPLEX solver, branch \& bound and column generation algorithms and their solution times are compared.

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