Cerebral malaria (CM) is a severe neurological complication of malaria infection that results from interrelated pathologies. Despite extensive research efforts, the mechanism of the disease is not completely understood. Clinical studies, postmortem analysis, and animal models have been the main research arenas in CM. In this thesis, shotgun proteomics approach was used to further understand the pathology of human and experimental CM. The mechanism by which CM turns fatal is yet to be identified. A clinical proteomics study was conducted on pooled plasma samples from children with reversible or fatal CM from the Gambia. The results show that depletion of coagulation factors and increased levels of circulating proteasomes are associated with fatal pediatric CM. This data suggests that the ongoing coagulation during CM might be a disseminated intravascular coagulation state that eventually causes depletion of the coagulation factors leading to petechial hemorrhages. In addition, the mechanism(s) by which blood transfusion benefits CM in children was investigated. To that end, the concentration and multimerization pattern of von-willebrand factor, and the concentration of haptoglobin in the plasma of children with CM who received blood transfusions were measured. In addition to clinical studies, experimental cerebral malaria (ECM) in mice has been long used as a model for the disease. A shotgun proteomics workflow was optimized to identify the proteomic signature of the brain tissue of mice with ECM.Because of the utmost importance of membrane proteins in the pathology of the disease, sample fractionation and filter aided sample preparation were used to recover them. The proteomic signature of the brains of mice infected with P. berghei ANKA that developed neurological syndrome, mice infected with P. berghei NK56 that developed severe malaria but without neurological signs, and non-infected mice, were compared to identify CM specific proteins. Among the differentially expressed proteins in mice that developed neurological signs, coagulation and acute-phase proteins were enriched. The data are in accordance with data from the clinical study. Taken together, the results support the role of coagulation and platelets adhesion in the pathogenesis of the disease.

Orientador: Priscylla Tatiana Chalfun Guimarães-Okamoto / Resumo: Cães com doença renal crônica (DRC) apresentam risco de eventos tromboembólicos, porém o mecanismo que leva à hipercoagulabilidade e ao risco na população de cães é desconhecido. O objetivo deste trabalho foi avaliar as possíveis tendências trombóticas e sua correlação com o estadiamento da DRC em cães. Trata-se de um estudo observacional transversal de casos-controle de cães atendidos no Hospital Veterinário da FMVZ – UNESP, com sinais clínicos compatíveis com DRC. Os animais incluídos neste estudo foram cães de tutores voluntários, com diagnóstico de DRC proteinúrica (n=19) e animais saudáveis (n=20). Foram avaliados hemograma, perfil bioquímico renal, hepático, lipídico e proteínas, urinálise e razão proteína/creatinina urinária, fibrinogênio, TP, TTPa, tromboelastometria (TEM) e o teste de geração de trombina (TGT). Não houve diferença na geração de trombina entre os grupos, refutando a hipótese de que a geração de trombina contribui para o estado de hipercoagulabilidade em cães com DRC e proteinúria. O fibrinogênio aumentado é o principal achado encontrado em cães com DRC, corroborando com os achados de hiercoagulabilidade na TEM. Estudos adicionais são necessários para explorar uma possível contribuição da trombogenicidade para as manifestações clínicas da DRC. / Abstract: Dogs with chronic kidney disease (CKD) are at risk thromboembolic events, but the mechanism leading to hypercoagulability and the population of dogs at risk are unknown. The aim of this study is to evaluate the possible thrombotic tendencies and their correlation with the staging of CKD in dogs. This is a observational cross-section study of client-owned dogs presented to Small Animal Clinical Service at the School of Veterinary Medicine and Animal Science of São Paulo State University, with clinical signs compatible with proteinuric CKD. The animals included this study were client-owned dogs with a diagnosis of proteinuric CKD (n=19) and healthy case-matched controls (n=20). Complete blood counts, renal, hepatic, lipidic and proteins serum biochemistry profile, urinalysis and urinary ratio protein/creatinine, fibrinogen, PT, aPTT, and thrombin generation test were evaluated. There was no difference in generation of thrombin between groups, refuting the hypothesis that thrombin generation is required for the state of hypercoagulability in dogs with CKD and proteinuria. Increased fibrinogen is the main finding found in dogs with CKD, corroborating with the findings of hypercoagulability in thromboelastometry. Studies are mandatory for the evaluation of thrombogenicity for clinical manifestations of DRC. / Doutor

By specifically analysing for the various active principles of plasma, platelets, tissues and their fractions, much new information has been obtained concerning the role of lipoids and platelets in blood coagulation and in the hemostatic mechanisms in health and disease. Analysed components are studied in artificial clotting systems, especially a two-stage thrombin- forming system. Some 86 cases of bleeding disorders, 32 new born normal infants and their mothers, and many normal adult bloods have been analysed with respect to components of the clotting and hemostatic functions. The detailed considerations embodied in the thesis are encompassed under the following heads: 1) the importance of certain lipoids, especially cephalin 2) the normal need, in plasma clotting, for platelets, 3) the particular significance of a platelet component, which has many analogies to cephalin, in the thromboplastic system, 4) potentiation of the thromboplastic actions of cephalin, of platelets, and of tissue thromboplastin (to some extent) by a variety of experimental additives. Part of this may be explained as a 'thromboplastin generation' through co-participation of certain plasmatic components (antihemophilic globulin, PTC" etc. ) . Part, however, may be the result of certain proteolytic enzymes, particularly trypsin, 'disaggregating' lipoproteins and thus rendering their phospholipid (and sometimes calcium) available for participation in the clotting reactions, 5) possible Ca-containing and lipid-containing 'intermediates’ in the thrombin-forming reactions, 6) myelin figure formation as an explanation of ‘alterations' of platelets and certain other formed elements such as thrombocytes, megakaryocytes, and stromatolytic erythrocytes 7) the multiplicity of factors which platelets may contribute to the blood clotting and hemostatic mechanisms 8) the occurrence of many clinical disorders due to deficiency of platelet functions. Thrombocytopenias denote deficient numbers ('counts' and total bulk in body). Thrombocytopathies are deficiencies of specific platelet components, e . g. thromboplastic factor, accelerator, vasoconstrictor (5-hydroxy tryptamine), or retractor factor. Such deficiencies can be clinically significant even when the platelet count is normal. Bleeding in leukaemias, uremias, etc. may often be accounted for in these terms, 9) the nature and modes of action of heparin and other 'antithromboplastic’ inhibitors, and of some antiproteases, in relation to the mechanisms discussed 10) the ‘cephalin availability theory' of the author, as a useful working hypothesis to explain the importance of the natural thromboplastic phospholipid. Lipid release from platelet, tissue, or possibly plasma sources may very well be the long-obscure 'trigger mechanism' which initiates blood coagulation.

No description available.

Objective – Sepsis is a syndrome in which infection triggers a systemic inflammatory and procoagulant response. Cell-free DNA (CFDNA) is elevated in sepsis, and correlates with mortality. This DNA may come from nuclear, mitochondrial, or bacterial sources. CpG motifs on bacterial and mitochondrial DNA can stimulate inflammatory responses via TLR9. CFDNA can activate coagulation via the contact pathway. CFDNA may thus play an important pathogenic role in sepsis. This study elucidates the relative effects of nuclear, mitochondrial, and bacterial DNA on inflammatory and pro-coagulant pathways with relevance to sepsis. Results – Mitochondrial DNA as well as nuclear DNA are elevated in plasma from septic patients compared to healthy controls. Bacterial, but not mitochondrial or nuclear, DNA increased neutrophil IL-6 secretion. Both mitochondrial and bacterial DNA increased neutrophil viability. Nuclear, mitochondrial, and bacterial DNA increased thrombin generation in both platelet-poor plasma and platelet-rich plasma to a similar degree. This effect was reduced by addition of corn-trypsin inhibitor and in FXII-depleted plasma, and abolished in FXI-depleted plasma, indicating dependence on the intrinsic pathway of coagulation. Independently of coagulation, DNA from all three sources was capable of causing activation of platelet integrin αIIbβ3. Conclusions – CFDNA from nuclear, mitochondrial, and bacterial sources have varying pro-inflammatory effects, although all three have similar pro-coagulant potential. The pathophysiological effects of CFDNA in sepsis may vary with the source of DNA. / Thesis / Master of Science (MSc)

It has been found that naturally hydrophobic carbonaceous materials such as coal and graphite can be selectively coagulated and separated from hydrophilic impurities without the use of oily agglomerants, flocculants or electrolytes. The coagulation occurs at ζ-potentials significantly higher than those predicted by the classical DLVO theory, suggesting that it is driven by a hydrophobic interaction energy. Thus, the process is referred to as the selective hydrophobic coagulation (SHe) process. The fundamental development of this process is the focus of this study. In this study, the energy barriers for the coagulation of two different coal samples and a graphite sample have been calculated using the extended DLVO theory, which incorporates the hydrophobic interaction energy in addition to the dispersion and the electrostatic energies. Stability diagrams have been developed from the data, which show that the maximum ζ-potential at which a given coal can coagulate decreases as surface hydrophobicity decreases. For the coagulation of minerals present in coal, the classical DLVO theory has been used for the energy barrier calculations. The results of these calculations provide an excellent correlation with the results from a series of SHC tests conducted with run-of-mine coal. The strength of the coal aggregates have also been investigated by measuring the coagula size distributions under different hydrodynamic conditions. The coagula size distributions were measured using an in-situ particle size analyzer. These results have been used along with models for coagulation rate and breakage rate to determine strength characteristics of the aggregates and to verify the primary parameters controlling the aggregate size. The study found that the coal and graphite aggregates incurred a substantial reduction in size when a small amount of mechanical agitation was applied. Based on this outcome, quiescent continuous processes have been successfully designed and developed to separate the coagulated hydrophobic particles from the dispersed hydrophilic particles. / Ph. D.

<p>The coagulation system is initiated when subendothelial tissue factor is exposed to circulating blood, and culminates in the production in the enzyme thrombin, which catalyzes clot formation. However, the failure of built-in controls to limit thrombin generation to sites of vascular damage, leads to pathological clot formation, termed thrombosis. Anticoagulants, such as heparin and warfarin, are used to prevent thrombosis; however, these can cause bleeding. As a result, there is a need for novel anticoagulants that limit clot formation without the risk of bleeding. DNA aptamers are small oligonucleotides designed to bind a specific target with high affinity. HD1 is a DNA aptamer that was developed by Griffin and Leung in 1993 to bind thrombin. The focus of this work was to better characterize the anticoagulant properties of HD1.</p><p>We demonstrate that HD1 binds a subdomain of exosite 1 that is fully developed in prothrombin, unlike other exosite 1-directed ligands, such as Hir⁵⁴⁻⁶⁵(SO₃⁻) and fibrin, which do not bind prothrombin. As a result, HD1 binds prothrombin with high affinity and inhibits prothrombin activation by prothrombinase with an IC₅₀ value of 115 nM. HD1 competes with tV a for binding to prothrombin. Based on its capacity to inhibit both thrombin and prothrombin, HD1 is more potent than Hir⁵⁴⁻⁶⁵(SO₃⁻) at inhibiting standard plasma clotting assays, but not in the thrombin clotting time. Furthermore, HDl inhibits thrombin generation by prolonging the lag time, reducing peak thrombin, and reducing the ETP to a greater extent than Hir⁵⁴⁻⁶⁵(SO₃⁻). Based on thrombin generation assays conducted in cofactor-deficient plasma, we demonstrate that HD 1 inhibits thrombin feedback activation of fV, but not fVIII, and that inhibition of prothrombinase accounts for the reduction in both peak thrombin and prolongation of the lag time. HDI is neutralized in buffer-based and plasma-based coagulation assays by an antisense oligonucleotide, antiHD1. AntiHD1 preferentially targets free HD1 because HD1 bound to either thrombin or prothrombin is protected from neutralization by antiHD1. Based on our data, HD1 may be a useful anticoagulant due to its capacity to inhibit both prothrombin and thrombin. Positioned at the junction of the intrinsic and extrinsic pathways prothrombin could be a useful target for aptamer development into anticoagulant applications.</p> / Thesis / Doctor of Philosophy (PhD)

No description available.

No description available.

No description available.