<p>The effect of direction thrombin inhibition on acute deep vein thrombosis (DVT) stability has not been defined and could contribute to pulmonary embolism (PE) risk. Direct thrombin inhibitors (DTIs) effectively inhibit free and clot-bound thrombin, which could potentiate thrombus instability through disruption of platelet, fibrin, and FXIIIa stabilizing mechanisms. This could manifest as increased thrombus embolization. A clinically relevant mouse model of DVT stability could further our understanding of venous thrombosis pathophysiology and define the effect of direct thrombin inhibition on PE. We hypothesized that acute DTI administration would decrease acute DVT stability and potentially increase PE risk. Platelets were labeled <em>in vivo</em>, femoral vein thrombosis was induced using FeCl₃, and lepirudin (8U/g) was administered <em>after</em> clot formation. Using intravital videomicroscopy (IVM), real time embolization was quantified as a measurement of thrombus stability. Thrombus stability increased in the control group and decreased in the lepirudin-treated group over two hours. The decrease in α₂-antiplasmin (α₂-AP) content within lepirudin-treated thrombi, compared to control thrombi, could possibly contribute to the observed decrease in thrombus stability. Continued growth and embolization established the dynamic nature of formed thrombi. In both groups, emboli were detected in the pulmonary artery circulation. Therefore, we successfully developed a mouse model of venous thrombus stability, which imitated the clinical progression of DVT to PE. DTI administration in the acute DVT setting could decrease thrombus stability, demonstrated through increased embolization and PE. This model could be useful in examining the effect of other antithrombotics and risk factors settings on DVT stability.</p> / Master of Science (MSc)

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Alloresponses to blood group antigens result from antigen mismatch between donor and recipient during blood transfusion or transplantation and between mother and fetus during pregnancy. During pregnancy, antigen mismatch can result in haemolytic disease of the newborn (HDN), a disease characterised by the development of potentially harmful alloantibodies, which cross the placenta and mediate the destruction of fetal erythrocytes. This project investigates examples of clinically important alloresponses to blood group antigens and, more specifically, characterises the ymphocytes that either drive or regulate these responses. The main aims or this project were to first map alioreactive T-helper cell epitopes and secondly to clone using a novel method, IL-10 secreting blood group specific regulatory cells. The work focussed on two major antigens, the kell (K) 1 and Rhesus (Rh) D antigens.

Laminitis is one of the most common multifactorial diseases of horse limbs. Recent studies show that 90% of this disease is due to endocrinological diseases such as Cushing's syndrome or equine metabolic syndrome. In available literature, there is not much relevant information on the relationship of laminitis to hematological and biochemical blood parameters. This diploma thesis "Changes in selected blood parameters of horses" evaluates biochemical and haematological blood parameters in connection with laminitis in 27 sick horses. It statistically compares the differences in blood parameters between healthy and sick horses.

Orientador: Luiz Claudio Nogueira Mendes / Coorientador: Lina Maria Wehrle Gomide / Banca: Flavia de Almeida Lucas / Banca: Rafael Resende Faleiros / Resumo: O objetivo do trabalho foi avaliar a influência de diferentes frequências de treinamento sobre o condicionamento físico de equinos através da avaliação das concentrações de lactato plasmático, das concentrações séricas de CK, AST e LDH e dos parâmetros físicos vitais e hematológicos, após exercício físico de alta intensidade e curta duração. Amostras de sangue venoso foram obtidas de 16 equinos da raça Quarto de Milha, que foram divididos em dois grupos: grupo de treinamento regular (GTR) e grupo de treinamento esporádico (GTE), em sete diferentes momentos: 30 minutos antes do exercício (MA), imediatamente após (MD), 30 minutos (M0,5), uma (M1), duas (M2), seis (M6) e 24 (M24) horas após a prova de três tambores. Não foram observadas diferenças entre os grupos. Frente a esse resultado os equinos foram rearranjados em um grupo único, para a avaliação do condicionamento físico desses animais de uma forma geral. Além disso, foram realizados testes de correlação dessas variáveis para melhor observação do comportamento entre elas. Como resultado, houve um aumento significativo no MD das variáveis: lactato, FC, FR, He, VG, Hb e LINF, no entanto todas as variáveis voltaram aos seus valores basais antes de 24 horas após o exercício, indicando que os equinos avaliados possuem condicionamento físico satisfatório. Quanto às correlações, foram encontrados coeficientes de correlação forte e positivo entre as variáveis: He, VG eHb, e entre LEU e GRAN, em todos os momentos, entre LDH e AST ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to evaluate the influence of different frequencies of training on the fitness of horses through the evaluation of plasma lactate, serum concentrations of CK, AST and LDH and clinical and hematological parameters after high intensity and short durationexercise. Venous blood samples were obtained from 16 horses of Quarter Horses, which were divided into two groups: regular training group (GTR) and sporadic training group (GTE) in seven different times: 30 minutes before exercise (MA), immediately after (MD), 30 minutes (M0,5), one (M1), two (M2), six (M6) and 24 (M24) hours after barrel racing. No differences were observed between the groups. In view of this result the horses were rearranged into a single group, to evaluate the fitness of these animals in general. In addition, correlation tests were conducted to observe the interdependence of these variables after exercise. As a result, there was a significant increase in MD of the variables lactate, HR, FR, He, VG, Hb and LINF, however all variables returned to their baseline values within 24 hours after exercise, indicating that the assessed horses have satisfactory physical condition. As for the correlations, strong and positive correlations were found between the variables: He, VG and Hb and between LEUC and GRAN, at all times, between LDH and AST in the M6, AST and CK in M0 and HR and RR in M24. It was concluded that the training frequency did not influence the fitness of the animals, however they... (Complete abstract click electronic access below) / Mestre

Células T de memória são a marca registrada da imunidade adaptativa, e podem ser caracterizadas em central memory (TCM) e effector memory (TEM) T cells. A participação destas células no curso de doenças hematológicas é descrita como mecanismo relacionado à evolução das mesmas. Neste trabalho, analisamos o sangue periférico de doadores de sangue e pacientes com Mielodisplasia (SMD), Mieloma Múltiplo (MM) e Leucemia linfocítica crônica B (LLC), e avaliamos a distribuição das células T CD4+ e CD8+ naive e de memória. SMD e MM não apresentaram resultados significativos, mas na LLC, as células T CD4+ estavam alteradas e dependentes do prognóstico, com aumento das células TCM somente nos pacientes com prognóstico ruim. Essas evidências sugerem que interações imunológicas entre células B da LLC e células T CD4+ possa ser um mecanismo próprio da doença que venha interferir na fisiopatologia e favorecer a geração de células TCM, que podem fornecer sinais de sobrevivência, como citocinas e CD40L, contribuindo assim para o estabelecimento e agressividade da LLC. / Memory T cells are the hallmark of adaptive immunity and are characterized as central (TCM) and effector memory (TEM) T cells. The influence of T cells in the course of hematological malignancies has been described as a mechanism related to the evolution. In this study, we analyzed the peripheral blood of healthy donors and patients with myelodysplastic syndrome (MDS), multiple myeloma (MM) and chronic lymphocytic leukemia B (CLL), and analyzed the distribution of CD4+ and CD8+ naive and memory T cells. MDS and MM revealed no significant difference, but CLL patients showed changes in CD4+ T cell and it were dependent on the prognosis. Patients with poor prognosis presented increased in frequency and absolute number of TCM cells. These evidences show that immunological interactions between CLL and CD4+ T cells could be a disease mechanism that could interfere in pathophysiology and result in the generation of TCM cells, that provide survival signals to the tumor clone, such as cytokines and CD40L, thus contributing to establishment and more aggressive CLL progression.

Os girinos de rã-touro, Lithobates catesbeianus, podem ser bons bioindicadores de condições ambientais. O objetivo desse trabalho foi determinar o potencial de toxicidade para L. catesbeianus de alguns dos principais agrotóxicos utilizados no cultivo de arroz irrigado. Foram realizados testes de toxicidade aguda para a determinação da CL50-96h do bentazon, penoxsulam, óleo vegetal, permetrina e carbofuran, separadamente, e da mistura desses agrotóxicos. Com esses resultados foram estimados os índices de segurança dos produtos. Girinos em fase pré-metamorfose foram expostos aos agrotóxicos na própria lavoura de arroz e em laboratório por 21 dias, para avaliar os possíveis efeitos crônicos destas substâncias, separadamente e da mistura, sobre o quadro hematológico, metamorfose (regulada pelo eixo tiroideano), e também o possível potencial mutagênico através do teste do micronúcleo. A CL50-96h para girinos foi de 4530 mg/L para o bentazon; 7,52 mg/L para o penoxsulam + 145,66 mg/L do óleo vegetal; 81,57 mg/L para o óleo vegetal, 0,10 mg/L para a permetrina, 29,90 mg/L para o carbofuran (ingredientes ativos) e, 38,79 vezes a dose utilizada no campo para a mistura desses produtos. Foi determinado risco ambiental apenas para o inseticida permetrina. Nos testes in situ, as águas de irrigação não apresentaram toxicidade aguda para os girinos. A taxa de metamorfose não diferiu entre os tratamentos, demonstrando que os agrotóxicos utilizados nas doses indicadas não tem ação desreguladora do eixo tiroideano. As análises do micronúcleo mostraram aumento significativo de eritrócitos micronúcleoados para os testes in situ e, no laboratório, para o herbicida bentazon e para a mistura dos agrotóxicos. As análises hematológias mostraram diminuição da hemoglobina e número de eritrócitos no teste de campo, retornado aos padrões normais na semana seguinte. No laboratório houve queda na contagem de eritrócitos para o bentazon, aumento do VCM e HCM para o bentazon e penoxsulam; aumento do CHCM para o penoxsulam e para a mistura dos agrotóxicos. Para a série branca não houve diferenças no teste in situ, mas obtivemos aumento dos números de neutrófilos dos girinos tratados com o bentazon. / American bullfrogs, Lithobates catesbeianus could be good environmental indicators. The aim of this study was evaluate the potential toxicity of some principal pesticides used in irrigated rice crops to L. catesbeianus tadpoles. The pesticides Bentazon, Penoxsulam, Vegetable oil, Permetrina, Carbofuran and the mixture of them were assessed. Pre-metamorphose tadpoles were exposed to all of these agrochemicals in the laboratory to determinate de LC50-96h and so estimate the index of security by each product. Animals in the same phase were exposed to these pesticides on the rice crops, in situ and in laboratory per 21 days to evaluate the possible chronic effects of the substances, separated and in the mixture of them. The hematological results, red and white series, the mutagenic potential (micronucleous test), and the metamorphose rate (regulated by thyroid axis) were evaluated. The LC50-96h to tadpoles was 4530 mg/L to Bentazon; 7.52 + 145.66 mg/L to Penoxsulam + vegetable oil; 81.57 mg/L to vegetable oil; 0.10 mg/L to Permetrina; 29.90 mg/L to Carbofuran (active ingredients) and 38.79 times to the dose used in the field to the mixture of the products. Only to Permetrina insecticide was observed environmental risk. The metamorphose rate showed no difference between the treatments suggesting that these pesticides, used on indicated doses did not promote deregulated action on the thyroid axis. In situ tests the irrigated waters showed low mortality to the animals. The red series showed in situ, a decrease in the haemoglobin tax and in the counting of erytrocyte\'s number however return to the normal values in the follow week. In laboratory tests showed a decrease in the counting of erytrocyte\'s number to the animals exposed to Bentazon, an increase in the MCV and MCH to the animals exposed to Bentazon and Penoxsulam, an increase in the MCHC to those exposed to Penoxsulam and to the \"mixture\". The white series showed no difference in situ test however an increase in the neutrophils number was observed to the animals exposed to Bentazon in laboratory. The micronucleous analyze showed significant increase in the erytrocyte\'s micronucleated number in situ and in laboratorial tests to animals exposed to Bentazon and to the \"mixture\".

O gene humano lysine methyltransferase 2E (KMT2E) pertence ao grupo Trithorax (TrxG) e age como proteína modificadora de histonas envolvida no controle transcricional de genes relacionados a hematopoiese. Foi previamente identificado como supressor tumoral, atuando sobre a diferenciação, proliferação e ciclo celular. DAMM et al. (2011) e LUCENA-ARAÚJO et al. (2014) descreveram a associação entre baixos níveis de expressão do gene KMT2E e desfechos desfavoráveis do tratamento de pacientes com leucemia mielóide aguda (LMA) e leucemia promielocítica aguda (LPA), respectivamente. O objetivo desse trabalho foi estudar os efeitos do aumento da expressão do gene KMT2E na leucemia mielóide aguda (LMA). Foi utilizada a linhagem celular U937, reconhecida como modelo de LMA, e o aumento da expressão do gene de interesse foi obtido por meio da transfecção das células com um vetor lentiviral contendo o cDNA codificante para a isoforma longa do gene (pCDH-MSCV-MCS-EF1-GFP+Puro, aqui chamado pMEG). As partículas lentivirais foram geradas por co-transfecção em células da linhagem HEK 293T, e posteriormente, titulados com a linhagem celular HT 1080. A expressão do gene e a presença da proteína foram confirmadas por qPCR e western blotting, respectivamente. Foram realizados ensaios funcionais de ciclo celular, proliferação, viabilidade, apoptose espontânea e induzida por trióxido de arsênico e luz ultravioleta e diferenciação celular induzida por 12-miristato 13-acetato de forbol (TPA), com as amostras U937 wild type (WT), U937 pMEG (U937 transduzidas com o vetor vazio) e U937 pMEG-KMT2E. Também foram realizadas mensurações da massa tumoral das células inoculadas em camundongos NSG. A expressão relativa do gene KMT2E na célula U937 pMEG-KMT2E foi 1000 vezes mais alta que na célula U937 sem a modificação genética. Os ensaios de diferenciação celular demonstraram que as células U937 pMEG-KMT2E apresentaram maior diferenciação em monócitos/macrófagos que as células controles, quando levada em consideração a marcação para o antígeno CD11c. A expressão induzida de KMT2E em células U937 não alterou a proliferação, viabilidade, ciclo celular, apoptose, ix espontânea ou induzida e o aspecto clonogênico in vitro, porém, foi associado a um maior crescimento tumoral em modelo animal. Nossa hipótese para justificar as diferenças entre os achados in vitro e in vivo é que o aumento da expressão de KMT2E, talvez por meio do aumento de CD11c, facilitou a interação entre as células e o microambiente, estimulando assim o crescimento tumoral in vivo. / The human lysine methyltransferase 2E (KMT2E) gene belongs to the Trithorax (TrxG) group and acts as a histone modifying protein participating in the transcriptional regulation of hematopoiesis-related genes. KMT2E has been previously described as a tumor suppressor, involved in cellular differentiation, proliferation and cell cycle progression. DAMM et al. (2011) and LUCENA-ARAÚJO et al. (2014) described the association between low levels of KMT2E gene expression and poor treatment outcomes in patients with acute myeloid leukemia (AML) and acute promyelocytic leukemia (APL), respectively. The aim of this project was to study the effects of high levels of KMT2E expression in acute myeloid leukemia (AML). For this purpose, the U937 AML cell line was used and an high expression of the gene was obtained by transfecting the cells with a lentiviral vector containing the cDNA encoding the long isoform of the gene (pCDH-MSCV-MCS-EF1- GFP + Pure, here called pMEG). The lentiviral particles were transfected into HEK 293T cells and the viral concentration was determined by titration using HT 1080 cells. The gene expression and the protein presence were confirmed by qPCR and western blotting, respectively. All experiments to determine the biological function of overexpressed KMT2E were conducted with U937 wild type, U937 pMEG (U937 transduced with the empty vector) and U937 pMEG-KMT2E cells. In-vitro the impact of overexpressed KMT2E was studied on cell cycle progression, proliferation and cell viability, spontaneous and induced apoptosis by arsenic trioxide and ultraviolet light and cell differentiation induced by 12-myristate 13-phorbol acetate (TPA). In vivo, the effect of overexpressed KMT2E was detected by comparing the tumor mass growth in NSG mice when inoculating U937 pMEG and pMEG-KMT2E cells in each flank of the same mouse. The relative expression level of the KMT2E gene in pMEG-KMT2E U937 cells was 1000 higher than in the wild type U937 strain. The cell differentiation assay revealed that U937 pMEG-KMT2E cells presented an increased monocyte/macrophage differentiation, when analyzing the CD11c antigen. Induced xi overexpression of KMT2E in U937 cells did not alter cell proliferation, cell viability, cell cycle progression, spontaneous or induced apoptosis or clonogenic appearance in vitro. However, the overexpression of KMT2E resulted in an increased tumor mass formation in vivo. Taking our discrepant in vitro and in vivo results into account, we could hypothesize that the increased expression of KMT2E, possibly caused by the enhanced expression of CD11c, favored the interaction between U937 pMEGKMT2E cells and their microenvironment, thereby stimulating tumor growth in vivo.

Foram utilizadas 750 poedeiras comerciais de linhagens brancas com 23 semanas ao início do período experimental, distribuídas num delineamento em blocos completos aleatorizados sendo estes caracterizados pela oncatenação das linhagens com coluna de gaiolas; os tratamentos foram cinco densidades ou taxas de lotação na gaiola (321,43; 375; 450; 562,50 e 750 cm2/ave) com seis repetições, totalizando 150 parcelas experimentais. As dietas experimentais foram à base de milho e farelo de soja, formuladas para suprir as exigências nutricionais das linhagens em todas as fases. O desempenho produtivo e econômico foram avaliados através do peso dos ovos (g), percentagem de postura (%), massa de ovos (g/ave/dia), consumo de ração (g/ave/dia), conversão alimentar por dúzia (kg/dz) e por quilo de ovo (kg/kg). A qualidade de ovos foi avaliada através da gravidade específica (GE), unidades Haugh (UH), percentagens de gema, albúmen e casca, espessura da casca (EC), resistência à quebra (RQ), coloração da gema e índice gema (IG). O bem-estar das aves foi avaliado através de indicadores clínicos, imunológicos e fisiológicos. Para efeito da avaliação dos resultados, foram estabelecidos seis blocos com repetições internas. Os dados foram analisados com auxílio do SAS, sob modelo misto, considerando os efeitos da densidade, período e a interação entre estes, como fixos, além dos efeitos aleatórios de bloco e resíduo. Por se tratar de medidas repetidas longitudinalmente, buscou-se a melhor estrutura de covariância para cada variável. Quando adequado foi aplicado o teste de tukey-kramer para a comparação de médias (p&lt;0,05). O menor consumo de ração, CA/dz de ovos e melhor desempenho econômico, foram obtidos pela densidade 321,4 cm2/ave. Altas densidades de alojamento não influenciaram a qualidade interna de ovos, a % casca e RQ, mas essas variáveis tiveram efeitos dos períodos, com os melhores valores nos primeiros períodos. Altas densidades tiveram efeitos negativos sobre a GE e EC, principalmente no pico de postura das aves. Os ovos avaliados encontravam-se dentro dos valores desejados, mesmo nas maiores densidades, podendo ser classificados como de excelente qualidade. A densidade de alojamento não interferiru (p&gt;0,05) na avaliação clínica das aves, mas o maior espaço proporcionado nas gaiolas do sistema covencional conferiu menor freqüência de lesões nas aves, indicando promoção e melhora ao seu bem-estar geral. Dados de freqüência cardíaca e temperatura da cloaca estavam dentro dos padrões de normalidade para a espécie. Não foram observadas diferenças (p&gt;0,05) das densidades na resposta imunológica das aves, com os títulos dos anticorpos vacinais contra as doenças de Gumboro e Newcastle acima dos níveis do ponto de corte (cutoff). Os tratamentos não modificaram o perfil sanguíneo das aves e não foi possível caracterizar um padrão de corticosterona no plasma e de excreção de metabólitos fecais de glicocorticoides. A técnica de enzimoimunoensaio, empregada utilizando anticorpo primário contra corticosterona e anticorpo secundário anticoelho de cabra, foi capaz de detectar as variações nas concentrações de corticosterona nesses tecidos, contudo, o entendimento do significado desses achados ainda necessita de novas investigações. / 750 commercial laying hens of white strains aged 23 weeks at the beginning of the experimental period were used, distributed in a randomized complete block design, these being characterized by the concatenation of the strains with column of cages; the treatments were five densities or stocking rates in the cage (321,43, 375, 450, 562.50 and 750 cm2/bird) with six replications, amounting to 150 experimental plots. The experimental diets were on the basis of corn and soybean meal, formulated to meet the nutritional requirements of the strains in all the phases. Both the productive and economic performances were evaluated through the weight of the eggs (g), percentage of laying (%), egg mass (g/bird/day), feed intake (g/bird/day), feed conversion per dozen (kg/dozen) and per kg of egg (kg/kg). Egg quality was determined through specific gravity (SG), Haugh units (HU), percentages of yolk, albumen and shell, shell thickness (ST), resistance to breaking (BR), yolk coloration and yolk index (YI). The welfare of the birds was evaluated through clinical, immunological and physiological indicators. For the purpose of evaluation of the results, six blocks with internal replications were established. The data were analyzed with the aid of SAS, under a mixed model, considering the effects of density, period and the interaction between them, as fixed, in addition to the random effects of block and residue. For se tratar de measures longitudinally repeated, the best covariance structure for each variable was sought. When appropriate, the Tukey-Kramer test was used for comparison of means (p&lt;0.05). The lowest feed intake, FI/dozen of eggs and best economic performance, were obtained at the density of 321.4 cm2/bird. High housing densities did not influence the internal quality of the eggs, % shell and BR, but these variables had effects from the periods, with the best values in the early periods. High densities had negative effects on SG and ST, mainly at the laying peak of the birds. The eggs checked lie within the desired values, even at the highest densities and can be classified as of excellent quality. The housing density did not interfere (p&gt;0.05) in the clinical evaluation of the birds, but the greater space provided in the cages of the conventional system conferred a lower frequency of lesions in the birds, indicating promotion and improvement to their general welfare. Data of heart rate and cloacal temperature were within the normal limits for the species. No differences (p&gt;0.05) of the densities in the immune response of the birds, with the titers of the vaccine antibodies against Gumboro and Newcastle diseases above the cutoff levels were found. The treatments did not modify the blood profile of the birds and it was not possible to characterize a standard of plasma corticosterone and excretion of fecal metabolites of glucocorticoids, but the enzyme immunoassay technique, using primary antibody against corticosterone and secondary goat anti-rabbit antibody, was capable of detecting variations in the corticosterone concentrations in these tissues, however, the understanding of the meaning of these findings still requires further investigation.

Traumatic brain injury (TBI) is a major cause of death among trauma patients and is associated with a high rate of mortality due to complications such as bacterial pneumonia, sepsis, and subsequent coagulopathies. While severe TBI is positively associated with the development of pneumonia, mild traumatic brain injury (mTBI) results in a paradoxical decrease in mortality following bacterial pneumonia via an unidentified mechanism. New evidence suggests that mTBI stimulates vagus nerve signaling resulting in an anti-inflammatory state that is mediated by neurotransmitters (NT) such as acetylcholine (ACh) and substance P (Sub P). This anti-inflammatory state induced by mTBI has been correlated with an increased resistance to pneumonia (PNA) in mice and has been shown to be mediated in part by increased bacterial clearance in the lungs via enhanced neutrophil recruitment. However, it has not been investigated whether this reduced mortality is due to alterations in the coagulation system and if they have any effect on either the severity or occurrence of disseminated intravascular coagulation (DIC), a common sequelae of pneumonia-induced sepsis. Our study investigates whether administration of mTBI prior to pneumonia challenge in mice decreases mortality by ameliorating DIC. We assess and define DIC in our mouse models by changes in plasma coagulation parameters including fibrinogen, D-dimer, and plasminogen activator inhibitor-1 (PAI-1) Our study found that mTBI administration prior to pneumonia significantly decreased mortality in mice gavaged with high concentrations of Pseudomonas aeruginosa (Psd.). We also found that mTBI administration prior to pneumonia rescued fibrinogen levels and increased D-dimer levels in plasma, suggesting a compensated fibrinolytic state and amelioration of DIC. Circulating neutrophil and absolute leukocyte counts were also increased in mTBI/pneumonia models compared to those with pneumonia alone, supporting previous evidence implicating mTBI as the origin of enhanced bacterial clearance in lungs. Taken together, these data suggest that the increase in survival seen in patients with mTBI is in part due to alterations in coagulation.