A doença de Alzheimer (DA) é uma doença neurodegenerativa e a principal causa de demência em idosos. Os mecanismos fisiopatológicos mais envolvidos na DA são: o acúmulo do peptídeo beta amiloide (A?) em agregados extracelulares, e a formação dos emaranhados neurofibrilares (ENF). A Proteína Precursora do Amiloide (APP) é clivada pelas secretases alfa (ADAM10), beta (BACE1) e y (Presenilina 1 [PSEN1]). As plaquetas contêm 95% da APP circulante e possuem toda a maquinaria necessária para estudar perifericamente a APP e suas secretases. A pesquisa de biomarcadores na DA tem como objetivo identificar, em vida, os indicadores do processo patogênico em fluídos corporais e/ou por métodos de imagem cerebral. O objetivo do presente estudo foi investigar proteínas envolvidas no metabolismo da APP em plaquetas de pacientes com DA e o potencial de modificação destas vias pela ação do tratamento com cloridrato de donepezila. Para tanto foram analisadas amostras de 23 pacientes com DA leve ou moderada, avaliados antes e depois de 6 meses de tratamento e 38 indivíduos idosos cognitivamente saudáveis (controles). As variáveis de desfecho estudadas foram: (1) expressão protéica de ADAM10, BACE1 e PSEN1; (2) expressão protéica dos metabólitos secretados da APP de 110 e 130kDa, possibilitando o cálculo da razão de APP (rAPP); e (3) atividade enzimática das APP-secretases ADAM10 e BACE1. Foram utilizados os métodos de western blotting e o fluorimétrico. Encontramos, nos pacientes com DA pré-tratamento, uma diminuição da rAPP em relação aos controles; porém, não identificamos diferenças após seis meses de tratamento. Os níveis de ADAM10 mostraram-se menores em pacientes com DA na avaliação basal quando comparados aos controles, mas também sem modificação com o tratamento, o tratamento mostrou-se associado a uma redução da expressão de BACE1 em pacientes com DA, embora não tenhamos encontrado diferenças entre pacientes e controles na avaliação basal. A expressão de PSEN1 mostrou-se menor nos pacientes com DA pré-tratamento quando comparada aos controles, sem contudo haver alteração em resposta ao tratamento. Quanto à atividade enzimática de ADAM10 e BACE1, não observamos diferenças nos valores pré e pós-tratamento. Nossos achados reforçam a utilidade da utilização de plaquetas como matriz biológica para o estudo do metabolismo da APP em tecidos periféricos e para a investigação de efeitos modificadores da patogenia da DA a partir do tratamento com drogas antidemência / Alzheimer\'s disease (AD) is a neurodegenerative disease and the major cause of dementia in the elderly. The main mechanisms in AD are: extracellular aggregates of beta amyloid peptide (Abeta) and neurofibrillary tangles formation (NFT). Amyloid Precursor Protein (APP) is cleaved by the secretases alfa (ADAM10), beta (BACE1) and ? (Presenilin 1 [PSEN1]). Platelets containing 95% of the circulating APP and possess all the machinery necessary to study peripherically APP and its secretases. The search for biomarkers in AD aims to identify, in life, the pathogenic process indicators in body fluids and/or brain image methods. The aim of this study was to investigate proteins involved in APP metabolism in platelets of AD patients, and the potential modification of these pathways by treatment with Donepezil hydrochloride. Therefore, 23 patients with mild to moderate AD evaluated before and after 6 months treatment and 38 healthy elderly subjects (controls) were analyzed. Outcome variables were: (1) ADAM10, BACE1 and PSEN1 expression; (2) APP secreted metabolites expression (110 and 130kDa), allowing the APP ratio (rAPP) estimate; (3) APP-secretase ADAM10 and BACE1 enzymatic activity. Western blotting and fluorimetric methods were used. We found in AD patients pre-treatment, a decrease of rAPP compared to controls; however, we did not identify differences of this parameter after six months of treatment. The ADAM10 levels were lower in AD patients at baseline when compared to controls, however no differences were observed after treatment. Treatment was associated with a reduction of BACE1 expression in AD patients, although we have not found differences between patients and controls at baseline. PSEN1 expression was lower in pre-treatment AD patients compared to controls. No differences were observed after treatment. Concerning to BACE1 and ADAM10 enzymatic activity, we did not observe differences in pre and post-treatment. Our findings strengthen the use of platelets as a biological matrix for the APP metabolism as well as the modifying effects on AD pathogenicity of antidementia drugs

A depressão é o mais comum dos distúrbios afetivos. Afeta ao menos 10% da população idosa do Brasil. Nos idosos, alguns fatores ligados ao metabolismo parecem estar bastante relacionados a esse transtorno, como uma menor concentração de noradrenalina e serotonina (5-HT) e uma maior atividade da monoaminooxidase em relação a adultos jovens. Os inibidores seletivos da recaptação da serotonina (ISRS), principalmente a sertralina, são a primeira opção no tratamento da fase aguda e manutenção dos episódios depressivos em idosos. As plaquetas vêm sendo amplamente utilizadas como modelo para estudar na periferia alterações que ocorrem no sistema nervoso central. A 5-HT apesar de ser primordialmente expressa no cérebro, também pode ser encontrada em plaquetas. Este neurotransmissor está envolvido em inúmeros aspectos do funcionamento normal do cérebro desde a regulação do humor até a regulação hormonal. A deficiência nos níveis de 5-HT pode estar intimamente ligada a alguma anormalidade na atividade da glicogênio sintase quinase 3B(GSK3B). Esta enzima exerce funções no metabolismo celular que vão desde sobrevivência celular, metabolismo e processamento de proteínas, até processos cognitivos. A atividade da GSK3B é estreitamente regulada pela fosforilação. Fosforilação no sítio ser9 inativa a enzima, enquanto que a desfosforilação neste mesmo sítio ativa a enzima. Diversos estudos têm mostrado que a forma inativa da enzima exerce um efeito neuroprotetor. O objetivo do presente estudo foi verificar a influência do tratamento com sertralina, em pacientes idosos com diagnóstico de depressão maior, sobre a 5- HT e GSK3B após 3 e 12 meses de tratamento. A quantificação da 5-HT foi realizada por HPLC e da GSK3B plaquetária, pelos métodos de ELISA e blotting, que se revelaram equivalentes. Após um ano de tratamento encontramos uma diminuição da 5-HT plaquetária nos pacientes com depressão maior com relação aos níveis basais, bem como um aumento da forma total da enzima GSK3B (GSKT), uma diminuição da forma fosforilada (pGSK) e da razão entre pGSK e GSKT (rGSK). Quando comparados os níveis de GSK3B de pacientes tratados por um ano e controles, observamos uma maior expressão de GSKT em pacientes; enquanto a pGSK e rGSK se mostraram equivalentes. Pudemos observar, portanto, uma modulação da 5-HT e da GSK3B pelo uso de sertralina. Essa modulação pode indicar que a ação antidepressiva deste fármaco pode estar associada a essas vias de sinalização / Depression is the most common affective disorders. It affects at least 10% of the elderly population of Brazil. In the elderly, some factors related to metabolism appear to be closely related to this disorder, such as lower concentration of noradrenaline and serotonin (5-HT) and increased monoamine oxidase activity in relation to young adults. The selective serotonin reuptake inhibitors (SSRI), especially sertraline are the first choice in treating acute and maintenance of depressive episodes in the elderly. Platelets have been widely used as a model to study in peripheral changes that occur in Central Nervous System. Although 5-HT is primarily expressed in the brain, it can also be found in platelets. This neurotransmitter is involved in numerous aspects of normal brain function since the regulation of mood to the hormonal regulation. A deficiency in 5-HT levels may be closely related to an abnormality in glycogen synthase kinase 3B (GSK3B) activity. This enzyme plays several functions in cell metabolism, ranging from cell survival, metabolism and protein processing, to cognitive processes. The GSK3B activity is tightly regulated by phosphorylation. Phosphorylation on Ser9 site inactives the enzyme, whereas dephosphorylation in the same site actives the enzyme. Several studies have shown that the inactive form of the enzyme plays a neuroprotective effect. The objective of this study was to investigate the influence of sertraline in elderly patients diagnosed with major depression, on platelet 5-HT and GSK3B after 3 and 12 months of treatment. Quantification of 5-HT was performed by HPLC and GSK3B by ELISA and western blotting. The methods for platelet GSK3B determination showed to be equivalent. After one year of treatment we found a decrease of platelet 5-HT in patients with major depression relative to their baseline levels, as well as an increase in the total form of GSK3B enzyme (GSKT), a decrease in phosphorylated form (pGSK) and the ratio between pGSK and GSKT (rGSK). Comparing the levels of GSK3B of patients with one year of treatment and controls, we found a higher GSKT expression in patients; while pGSK and rGSK showed to be equivalent. Therefore we observed a modulation of 5-HT and GSK3B by sertraline. This modulation may indicate that the antidepressant action of this drug may be associated with these signaling pathways

Haemostasis is a complex physiological process involving cellular and plasma protein components that interact to keep the blood fluid under normal conditions and prevent blood loss after vessel injury by promoting clot formation. Primary haemostasis encompasses the activation and aggregation of platelets and is supported by secondary haemostasis, in which the coagulation factors of the plasma interact in a complex series of reactions. Secondary haemostasis is initiated by the exposure of tissue factor (TF) to the blood after vessel injury. TF forms a complex with activated factor VII (FVIIa), which in turn activates factor X (FXa) and ultimately results in fibrin formation. The TF-FVIIa complex and FXa are tightly regulated by tissue factor pathway inhibitor (TFPI), a trivalent Kunitz-type protease inhibitor. The endothelium, consisting of endothelial cells (ECs), constitutes the inner lining of all blood vessels. As such, it is in constant contact with the blood and plays a major role in haemostasis by synthesising and storing both pro- and anti- coagulant substances, including TF and TFPI. Release of TFPI from ECs is increased after exposure to both unfractionated and low molecular weight heparins, though the mechanisms are not clearly defined. TFPI circulates in plasma, predominantly bound to lipoproteins, though the effect of the three major lipoproteins [low density (LDL), very low density (VLDL) and high density (HDL)] on the release of TFPI from ECs is not well established. Furthermore, previous studies have not systematically investigated the effect of these lipoproteins on both TF and TFPI. The initial aim of this project was to establish assays for the measurement of TF activity and TFPI antigen to supplement the TFPI activity assay that is well established in our laboratory. / These assays were then used to determine the effects of heparin and the major lipoproteins on the expression of TF and the release of TFPI on/from ECs. Human umbilical vein endothelial cells (HUVECs) were used as the EC model because their collection and isolation is well established and they have biochemical and physiological properties representative of in vivo conditions. A TF activity assay, based on a previously published method, was successfully modified and validated for the measurement of cell surface TF (standard curve R2 = 0.997). Despite exhaustive attempts, adaptation of this assay for plasma TF was unsuccessful, raising doubts regarding the plasma fractionation procedure of the originally published assay [Fukuda, C., Iijima, K. and Nakamura, K. (1989). "Measuring tissue factor (factor III) activity in plasma." Clinical Chemistry 35(9): 1897‐1900]. A novel insect cell expression system was used to produce well defined recombinant TFPI standards for use in TFPI activity and antigen assays. For the first time, truncated TFPI variants, containing the first Kunitz domain only, the first and second Kunitz domains only, and the first through third Kunitz domains minus the carboxyl terminus, were successfully produced in insect cells, though the full length molecule was not. Possible reasons for this include codon bias, protein instability and/or the signal peptide used. An ELISA to measure TFPI antigen was designed using a monoclonal anti‐TFPI antibody directed against the N‐terminus for protein capture and a polyclonal anti‐ TFPI antibody for detection. The assay was successfully optimised (standard curve R2 = 0.978, intra‐assay CV = 4.8%), however it produced inaccurate results (normal range = 498.7 ± 156.3 ng/mL), probably due to the antibody combination used. / TF and TFPI activity assays were used to determine the effect of both unfractionated and low molecular weight heparins (UFH and LMWH, respectively) on the release of TFPI and the expression of TF from/on ECs. A significant increase in the secretion of functional TFPI from ECs due to heparin (0 U/ml vs 1 and 10 U/mL) was demonstrated only in the presence of serum (UFH: 9.0 mU/mL vs 18.3 and 18.4 mU/mL, p < 0.0001; LMWH: 8.8 mU/mL vs 13.3 and 21.4 mU/mL, p < 0.05), suggesting, for the first time, that a component of serum is required for the heparin‐dependent release of TFPI. The effect of LDL, VLDL and HDL on the release of TFPI and the expression of TF from/on ECs was also investigated. All three lipoprotein fractions increased the secretion of functional TFPI after one hour incubation (LDL: 12.5 μg/mL, p < 0.01; 25 μg/mL, p < 0.05; VLDL: 50 μg/mL, p < 0.01; HDL: 50 μg/mL, p < 0.05). This is the first data to demonstrate a HDL‐dependent increase in released TFPI. After 24 hours, both LDL and VLDL decreased levels of secreted functional TFPI (LDL: 25 μg/mL, p < 0.01; 50 μg/mL, p < 0.01; VLDL: 12.5 μg/mL, p < 0.01), probably due to the oxidation and subsequent association of both lipoprotein species with TFPI. Surprisingly, both LDL and VLDL decreased cell surface TF, though this effect was not dose dependent. These results suggest that the major lipoproteins have a short term anticoagulant effect which is reversed in the longer term due to lipid oxidation. In summary, this thesis describes the successful adaptation of a chromogenic assay for the measurement of cell surface TF activity and the production of truncated TFPI variants. / Both will be used for the measurement of TF and TFPI, their association with thrombus formation and propagation, and investigations into potential therapeutic applications of TFPI. The results presented in this thesis extend the current knowledge on the expression and release of TF and TFPI on/from ECs by heparin, highlighting the importance of serum in the heparin dependent release of TFPI in vitro. Furthermore, it describes for the first time the effects of the major lipoprotein fractions on TFPI release and TF expression. The data support novel mechanisms by which LDL and VLDL are procoagulant, and HDL anticoagulant. This study provides a foundation for future research of the TF pathway in cellular models, which is critical in increasing the understanding of the pathogenesis and treatment of thrombotic disease. vitro. Furthermore, it describes for the first time the effects of the major lipoprotein fractions on TFPI release and TF expression. The data support novel mechanisms by which LDL and VLDL are procoagulant, and HDL anticoagulant. This study provides a foundation for future research of the TF pathway in cellular models, which is critical in increasing the understanding of the pathogenesis and treatment of thrombotic disease.

[Truncated abstract] Vitamin E is comprised of a family of tocopherols (TOH) and tocotrienols. The most studied of these is [alpha]-tocopherol ([alpha]-TOH), as this form is retained within the body and any deficiency of vitamin E is corrected with this supplement. [alpha]-TOH is a lipid-soluble antioxidant required for the preservation of cell membranes and potentially acts as a defense against oxidative stress. Individuals who have a primary vitamin E deficiency such as low birth weight infants, secondary vitamin E deficiency due to fat malabsorption such as in abetalipoproteinaemia, or a genetic defect in TOH transport require supplementation. There is debate as to whether vitamin E supplementation in other patient groups is required. Vitamin E supplementation has been recommended for persons with FHBL, a rare disorder of lipoprotein metabolism that leads to low serum [alpha]-TOH and decreased LDL cholesterol and apolipoprotein B concentrations. We examined the effect of truncated apoB variants on vitamin E metabolism and oxidative stress in persons with heterozygous FHBL. We used HPLC with electrochemical detection to measure [alpha]- and [gamma]-TOH in serum, erythrocytes, and platelets, and GC-MS to measure urinary F2-isoprostanes and TOH metabolites as markers of oxidative stress and TOH intake, respectively. Erythrocyte [alpha]-TOH was decreased, but we observed no differences in lipid-adjusted serum TOHs, erythrocyte [gamma]-TOH, platelet [alpha]- or [gamma]-TOH, urinary F2-isoprostanes, or TOH metabolites. Taken together, our findings do not support the recommendation that persons with heterozygous FHBL should receive vitamin E supplementation. ... Sesame lignans are natural components of sesame seed oil and there is evidence that these lignans can inhibit CYP450 enzymes, in particular, those responsible for vitamin E metabolism. We hypothesised that sesame seed ingestion would increase serum [gamma]-TOH, lower plasma lipids and inhibit platelet function in human subjects with at least one cardiovascular risk factor. We used HPLC with electrochemical detection to measure [alpha]- and -TOH in serum and GC-MS to measure F2-isoprostanes and TOH metabolites as markers of oxidative stress and TOH intake, respectively. We used high-sensitive C-reactive protein as a measure of systemic inflammation. Platelet function was assessed using the PFA-100 platelet aggregation assay. Although serum [gamma]-TOH increased by 17%, we observed no effect on lipid metabolism, markers of inflammation, oxidative stress or platelet function following treatment with ~25 g/day sesame seeds for five weeks. Our findings challenge the hypothesis that sesame seed ingestion provides beneficial cardiovascular effects. In summary, we have studied the metabolism and transport of both [alpha]- and [gamma]-TOH in humans to evaluate the requirements for supplementation and the effects of vitamin E on platelet function and CYP3A4 activity. Specialised techniques using HPLC were developed to measure serum and cellular TOH concentrations both in supplemented and un-supplemented individuals. We also used GCMS to provide a sensitive, accurate assessment of TOH metabolites and midazolam pharmacokinetics in humans after vitamin E supplementation. We have examined the role vitamin E has on important biochemical endpoints, with emphasis on the implications for TOH supplementation in subjects at risk of CVD.

<p>Background. Platelets (thrombocytes) are the smallest cells in the blood. Platelet fulfils functions as formation of blood clots when bleeding. Low levels leads to bleeding while high levels increase the risk of thrombosis (obstruction of the circulatory flow system). Platelet transfusions may be required for patients with systemic bleeding and for patients at higher risk of bleeding because of coagulation defects, sepsis (presence of bacteria in the bloodstream), or platelet dysfunction related to medication or disease. A pathogen-reduction system for platelet components would be a useful method since it reduces the risk of bacterial, protozoa, viral and white blood cell contamination. The Intercept Blood System method (IBS) for platelets, destroys DNA and RNA and was validated against the routine method in order to reduce pathogen transmission risk during transfusion. The validation of IBS, the trombocyte count for100 buffy coat concentrates from 2007 were compared to values for 100 buffy coat concentrates from 2006 that had been treated with gamma-radiation. Akademiska sjukhuset in Uppsala has a requirement that 75% of the platelet concentrates contain at least 300*10 9 platelets per unit. IBS fulfilled to 94% compared to 98% for the routine method.</p><p>Thus, the IBS-method was well above the required value and is now used at</p><p>Akademiska sjukhuset in Uppsala.</p>

Background. Platelets (thrombocytes) are the smallest cells in the blood. Platelet fulfils functions as formation of blood clots when bleeding. Low levels leads to bleeding while high levels increase the risk of thrombosis (obstruction of the circulatory flow system). Platelet transfusions may be required for patients with systemic bleeding and for patients at higher risk of bleeding because of coagulation defects, sepsis (presence of bacteria in the bloodstream), or platelet dysfunction related to medication or disease. A pathogen-reduction system for platelet components would be a useful method since it reduces the risk of bacterial, protozoa, viral and white blood cell contamination. The Intercept Blood System method (IBS) for platelets, destroys DNA and RNA and was validated against the routine method in order to reduce pathogen transmission risk during transfusion. The validation of IBS, the trombocyte count for100 buffy coat concentrates from 2007 were compared to values for 100 buffy coat concentrates from 2006 that had been treated with gamma-radiation. Akademiska sjukhuset in Uppsala has a requirement that 75% of the platelet concentrates contain at least 300*10 9 platelets per unit. IBS fulfilled to 94% compared to 98% for the routine method. Thus, the IBS-method was well above the required value and is now used at Akademiska sjukhuset in Uppsala.

Atherothrombosis can induce acute myocardial infarction and stroke by progressive stenosis of a blood vessel lumen to full occlusion. The goal of this research is to determine what shear rates are pertinent to an occluding blood vessel, the rate of thrombus growth relative to wall shear rates, and to develop a predictive model for estimating length of time to thrombus occlusion for a given atherosclerotic lesion. Computational studies of severely stenotic idealized vessels were performed to investigate the wall shear rates that may exist. The study shows that maximum shear rates in severe short stenoses were found to exceed 250,000 1/s (9,500 dynes/cm2). We utilize an in vitro experiment consisting of blood flow through a collagen coated stenosis to study the rate of thrombus growth. Growth is monitored through light microscopy and a camera. Computational fluid dynamics are used to determine shear rates along the thrombus surface as it grows. We found a strong positive correlation between thrombus growth rates and shear rates up to 6,000 1/s after a log-log transformation (r=0.85, p<0.0001). Growth rates at pathologic shear rates were typically 2-4 times greater than for physiologic shear rates below 400 s-1. To determine whether transport or kinetic binding limits the rate of thrombus growth, a computational model of platelet transport was developed. The model allows for thrombus growth by occluding computational cells. We show that thrombus is transport rate-limited for shear rates below 6,000 1/s, while it is more likely to be kinetic rate-limited for higher shear rates. Predictions of occlusion times based on the model demonstrate that increases in stenosis severity results in decreased time to occlusion.

In order to simulate cellular blood, a coarse-grained spectrin-link (SL) red blood cell (RBC) membrane model is coupled with a lattice-Boltzmann (LB) based suspension solver. The LB method resolves the hydrodynamics governed by the Navier--Stokes equations while the SL method accurately models the deformation of RBCs under numerous configurations. This method has been parallelized using Message Passing Interface (MPI) protocols for the simulation of dense suspensions of RBCs characteristic of whole blood on world-class computing resources. Simulations were performed to study rheological effects in unbounded shear using the Lees-Edwards boundary condition with good agreement with rotational viscometer results from literature. The particle-phase normal-stress tensor was analyzed and demonstrated a change in sign of the particle-phase pressure from low to high shear rates due to RBCs transitioning from a compressive state to a tensile state in the flow direction. Non-Newtonian effects such as viscosity shear thinning were observed for shear rates ranging from 14-440 inverse seconds as well as the strong dependence on hematocrit at low shear rates. An increase in membrane bending energy was shown to be an important factor for determining the average orientation of RBCs, which ultimately affects the suspension viscosity. The shear stress on platelets was observed to be higher than the average shear stress in blood, which emphasizes the importance of modeling platelets as finite particles. Hagen-Poiseuille flow simulations were performed in rigid vessels for investigating the change in cell-depleted layer thickness with shear rate, the Fåhraeus-Linqvist effect, and the process of platelet margination. The process of platelet margination was shown to be sensitive to platelet shape. Specifically, it is shown that lower aspect ratio particles migrate more rapidly than thin disks. Margination rate is shown to increase with hematocrit, due to the larger number of RBC-platelet interactions, and with the increase in suspending fluid viscosity.

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.

A computational model of thrombus initiation and aggrandizement was proposed. The model separated the thrombotic process into three mechanisms, including shear enhanced diffusivity, platelet margination, and platelet adhesion. The model indicates that transport mechanisms may be the rate limiting condition of thrombus formation at physiological shear rates and that at higher shear rates; platelet binding becomes the rate limiting condition. Additionally a wall shear rate of 20000 s-1 and above should be considered as a new criterion for prophylactic treatment of an atherosclerotic lesion.