Made available in DSpace on 2016-05-02T13:54:45Z (GMT). No. of bitstreams: 1 LuisaBarbosaMessora-dissertacao-completa.pdf: 574964 bytes, checksum: f3b5918f00bf09a802e755b11db471d0 (MD5) Previous issue date: 2010-11-21 / LDLr-/ - mice are spontaneously hyperlipidemic and resistant to the development of neointimal lesions. This study determined the influence of plasma levels of high density lipoprotein on cardiovascular inflammation, insulin resistance, and blood cell count in LDL (LDLr-/ -) receptor gene knockout mice. Three groups of 3-month-old male mice were used: Group WT, wild-type mice, Group S, LDLr-/ - mice fed a standard diet, Group HL, LDLr-/ - mice fed a hyperlipidic diet. After 15 days, blood was collected for analysis of plasma lipids, glucose, insulin, and hematological assays. The HOMA index was calculated to determine insulin resistance. The heart and aorta were removed and histologically processed. Heart sections were immunohistochemically processed with the anti-CD40L antibody to evaluate the inflammatory process. Artery sections were stained with hematoxylin-eosin and picrosirius red to assess morphological and morphometric changes. The S mice were resistant to inflammatory, had a low immunoreactivity to CD40L, high HDL plasma levels, and showed no insulin resistance, even with moderate hyperlipidemia in relation to WT. HL mice exhibited severe hyperlipidemia, increased immunoreactivity to CD40L, marked morphological alterations in the aorta wall, and insulin resistance, all associated with a decrease in HDL plasma levels in relation to S. The results showed a negative association between the plasma levels of high density lipoprotein and the total and differential leukocyte and platelet counts in the LDL receptor gene knockout mice. This ratio showed the important influence of the high density lipoprotein on the modulation of the immune and inflammatory response in dyslipidemias. Therefore, the evaluation of the blood cell count results, routinely correlated with the lipid plasma levels, can be promising in the prevention and prognosis of the severity of pathological conditions involving immune responses in dyslipidemias. The high HDL plasma level is a protective factor against the development of cardiovascular inflammation and insulin resistance in LDLr-/- mice, thus preventing the incidence of neointimal lesions. / Os camundongos LDLr-/- são hiperlipidêmicos espontâneos e resistentes ao desenvolvimento de lesões neointimais. O presente estudo teve como objetivo determinar a influência dos níveis plasmáticos de lipoproteína de alta densidade na inflamação cardiovascular, na resistência insulínica e no hemograma de camundongos knockout para gene do receptor de LDL (LDLr-/-). Foram utilizados 3 grupos experimentais de camundongos machos com 3 meses de idade: Grupo WT, camundongos selvagens; Grupo S, camundongos LDLr-/- que receberam ração padrão; Grupo HL, camundongos LDLr-/- que receberam ração hiperlipídica. Após 15 dias, o sangue foi coletado para análises plasmáticas dos lipídeos, glicose, insulina e para análises hematológicas. O índice de Homa foi calculado para determinar a resistência insulínica. O coração e aorta foram removidos e processados histologicamente. Cortes histológicos do coração foram processados imunoistoquimicamente com anticorpo anti-CD40L para avaliar processo inflamatório. Cortes histológicos das artérias foram corados com hematoxilina/eosina e picrosírius red para avaliar alterações morfológicas e morfométricas. Os camundongos S foram resistentes ao processo inflamatório, caracterizado por baixa imunorreatividade para o CD40L, com níveis plasmáticos de HDL elevados, e não desenvolveram resistência insulínica, mesmo com hiperlipidemia moderada em relação aos WT. Os camundongos HL apresentaram uma hiperlipidemia severa, aumento na imunorreatividade cardíaca para o CD40L, pronunciadas alterações morfológicas na parede da aorta e resistência insulínica, associadas a um decréscimo nos níveis plasmáticos do HDL em relação aos S. Os resultados mostraram uma associação negativa entre os níveis plasmáticos de lipoproteína de alta densidade e as contagens total e diferencial de leucócitos e plaquetas nos camundongos knockout para o gene do receptor de lipoproteína de baixa densidade. Essa relação demonstrou importante influência da lipoproteína de alta densidade na modulação da resposta imunológica e inflamatória na dislipidemia. Portanto, a avaliação dos resultados do hemograma correlacionada com os níveis plasmáticos de lipídeos, rotineiramente, pode ser promissora na prevenção e no prognóstico da severidade de quadros patológicos que envolvam respostas imunológicas nas dislipidemias. O nível plasmático elevado de HDL é o fator protetor contra o desenvolvimento de processos inflamatórios cardiovasculares e resistência insulínica nos camundongos LDLr-/-, impedindo o desenvolvimento das lesões neointimais.

The disposition of cells whose mechanism of death is related to the age of the cell cannot be appropriately represented by pharmacodynamic (PD) models where the elimination rate is related to the number of cells. In cells with age-related death their disposition is determined by their lifespan. Thus in these cells PD models of cellular response must incorporate a lifespan component. Previous cellular lifespan PD models assumed that the lifespan of cells is predetermined and does not vary over time. However, in many instances these assumptions are inappropriate and thus extensions to the existing models are needed. An important application of these time variant PD models is determining the erythropoiesis rate, since the lifespan of reticulocytes and mature erythrocytes are known to change over time under specific conditions. The objectives us this work were to develop a general time variant lifespan-based PD model of cellular response and to use the model to determine the dynamic changes over time in both the erythrocyte lifespan and erythropoiesis rate under a variety of complex conditions. An initial time variant cellular lifespan model was formulated assuming no variability in lifespans and used to determine the dynamic changes in both the reticulocyte lifespan and erythropoiesis rate in sheep. Subsequently, the time variant model was extended to account for a distribution of cellular lifespans, which resulted in better capturing the physiology of sheep erythrocyte maturation. The model was then further extended to account for the effect of changes in the environment on cell lifespans and used to determine the effect of chemotherapy administration on sheep erythrocytes. In order to conduct studies on erythropoiesis in premature very low birth weight (VLBW) infants the ability to accurately measure erythrocytes and hemoglobin from clinically collected excess blood was validated. Then an in depth analysis of the relationship between erythropoietin, erythrocytes, and hemoglobin was conducted in a clinical study of premature VLBW infants that accounted for the dynamic hematological conditions experienced by these subjects. This analysis indicated that a nearly 4-fold increase in erythropoiesis could be achieved with only a modest increase in plasma erythropoietin concentrations.

Blood flow through microcirculation in both simple and complex geometry has been difficult to predict due to the composition and complex behavior of blood at the microscale. Blood is a dense suspension of deformable red blood cells that is comparable in dimensions to the microchannels that it flows through. As a result, rheological properties at the microscale can vastly differ from bulk rheological properties due to non-continuum effects. To further develop our understanding of blood microflow; experimental techniques should be explored. In this work, we explore micro-particle image velocimetry (μPIV) and two-beam fluorescence cross-correlation spectroscopy (2bFCCS) in the application of characterizing blood in microflow conditions. For the development of the μPIV analysis, a polydimethylsiloxane co-flow channel is used to observe blood flow in controlled conditions. Flow conditions (velocity profile and blood layer thickness) are selected based on an analytical model and compared to experimental measurement. The experimental results presented indicate that current flow conditions are inadequate in providing a controlled rate of shear on the blood layer in the co-flow channel and further optimization are required to improve the measurement of the velocity profile. For the development of the 2bFCCS application for blood flow analysis, a wide glass capillary microfluidic device is used to complete the verification of fluorescence fluid admissibility, the effect of laser intensity on inducing photobleaching and the velocity measurement performance. The experimental measurement of the velocity profile is validated against the theoretical profile for a rectangular channel. Results of the velocity profile of high concentration red blood cells show promise in the technique’s ability to measure blood microflows closer to physiological conditions.

Orientador: Áureo Evangelista Santana / Resumo: A anemia é considerada um dos fatores para avaliar progressão da doença renal e diminuição da qualidade de vida do paciente. Conforme a doença renal progride, ocorre aumento gradativo na produção de toxinas urêmicas que reduz a meia vida dos eritrócitos circulantes por interferir na estabilidade da membrana eritrocitária. Para tanto, utiliza-se a contagem de reticulócitos para classificar a anemia como regenerativa ou não regenerativa. Objetivou-se neste estudo avaliar a resistência da membrana das hemácias, utilizando-se do teste de Fragilidade Osmótica Eritrocitária (FOE) em cães com doença renal crônica (DRC) e avaliação de reticulócitos. Foram avaliados 43 cães provenientes da rotina do Serviço de Nefrologia e Urologia do Hospital Veterinário Governador Laudo Natel da Faculdade de Ciências Agrárias e Veterinárias - UNESP - campus Jaboticabal. As referidas unidades experimentais foram distribuídas em três grupos, quais sejam, G0 (n=13), composto por cães hígidos e G1, DRC estádios 1 e 2 (n=14) e G2, DRC estádios 3 e 4 (n=16), classificados de acordo com o recomendado pela International Renal Interest Society. A fim de definir os critérios de inclusão dos cães foram feitos, além do exame físico, a avaliação de pressão arterial, hemograma, contagem de reticulócitos, exames bioquímicos, urinálise e relação proteína/creatinina urinária (UP/C). Para execução do teste de FOE as hemácias foram diluídas em concentrações decrescentes de cloreto de sódio e analisadas por citometria ... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Anemia is considered one of the factors to assess the kidney disease progress and the decrease in patient's quality of life. As kidney disease progresses, there is a gradual increase in urinary toxin production that shortens the circulating erythrocyte half-life by interfering with erythrocyte membrane stability. To do this, use a reticulocyte count to classify anemia as regenerative or non-regenerative. The objectives of this study were to evaluate the resistance of the red blood cells, using the Erythrocyte Osmotic Fragility test in dogs with chronic kidney disease (CKD) and to evaluate reticulocytes. Forty-three dogs were charged, followed by the routine of the Nephrology and Urology Service of the Governor Laudo Natel Veterinary Hospital of the Faculty of Agricultural and Veterinary Sciences - UNESP - Campus Jaboticabal. The experimental units were divided into three groups, namely, G0 (n = 13), consisting of healthy dogs and G1, CKD stages 1 and 2 (n = 14) and G2, CKD stages 3 and 4 (n = 16), classification proposed by the International Renal Interest Society. In order to define the inclusion criteria of dogs made, in addition to physical examination, an assessment of blood pressure, blood count, reticulocyte count, biochemical tests, urinalysis, and urinary protein/creatinine ratio. To perform the erythrocyte osmotic fragility test, red blood cells were diluted in decreasing sodium chloride filters (0.9 to 0.0%) and analyzed by flow cytometry. As creatinine serum concen... (Complete abstract click electronic access below) / Mestre

In this exploratory study, we investigated total erythrocyte carbonic anhydrase (CA) estrase activity as well as CA I isozyme concentration in patients with diabetes mellitus type II (DM) and healthy individuals of Howard University Hospital community. Total estrase activity of CA was measured spectrophotometrically using p-nitrophenol acetate before and after inhibition with acetazolamide. CA I isozyme was measured by radial immunodiffusion using monoclonal antibody (CA I) in agarose plates. The study involved 20 consented participants; 10 normal (N) and 10 (DM), 21 to 84 years of age. The study was approved by the Howard University Institution Review Board. The CA activity was measured following lysis of cells as U/min/mL and CA I concentration as mg/l. We observed CA activity as 46.3±4(N) and 25±2.1 (DM) whereas CA I concentration as 1896±125 (N) and 1104 ±63 (DM). We speculate that the change in the CA activity may of fundamental importance in the regulation of intracellular; pHi for the basic control of metabolism in diabetes mellitus. Further, we propose that CA activity is a good candidate for a biomarker of diabetes mellitus for the early detection of insulin resistance because the CA activity variation was proportional to the severity of the diabetes.

Red blood cells (RBCs) are the predominant cell type in blood and have a two-layered outer shell which is composed of a cytoskeleton network tethered to a cytoplasmic membrane. In this thesis, I study the structure and mechanical properties of the RBC’s cytoplasmic membrane (RBCcm) on the nanoscale and utilize this knowledge to functionalize this biological structure on a molecular level. In a first case study, I measure the membrane’s bending rigidity from thermal fluctuations observed in X-ray diffuse scattering (XDS) and Neutron Spin Echo (NSE) experiments, as well as Molecular Dynamics (MD) simulations. I provide evidence of the RBCcm's highly deformable nature with a bending rigidity that is substantially softer as compared to synthetic membranes. The methods are applied to RBCs that were stored for up to 5 weeks. I demonstrate that storage of RBCs leads to an increased fraction of liquid ordered membrane domains and an increased bending rigidity. RBCs are ideal for pharmaceutical applications as they provide access to numerous targets in the body, however lack specificity. Functionalizing the cytoplasmic membrane is thus a prerequisite to use these cells in biotechnology. I develop protocols throughout two studies to tune the membrane's lipid and protein composition. I investigate the impact of synthetic lipid molecules on the membrane's structure and demonstrate that small molecules can be encapsulated into liposomes that are formed from these hybrid membranes. Further, I provide direct evidence that the SARS-CoV 2 spike protein can be anchored into the RBCcm through a detergent mediated insertion protocol. These virus-like particles are observed to trigger seroconversion in mouse models, which demonstrates the potential of functionalized RBC in biotechnology. / Thesis / Doctor of Philosophy (PhD)

<div>Red blood cells (RBCs) make up 40-45% of blood and play an important role in oxygen transport. That transport depends on the RBC distribution throughout the body, which is highly heterogeneous. That distribution, in turn, depends on how RBCs are distributed or partitioned at diverging vessel bifurcations where one vessel flows into two. Several studies have used mathematical modeling to consider RBC partitioning at such bifurcations in order to produce useful insights. However, these studies assume that the vessel wall is a flat impenetrable homogeneous surface. While this is a good first approximation, especially for larger vessels, the vessel wall is typically coated by a flexible, porous endothelial surface layer (ESL) that is 0.5-1 microns thick. To better understand the possible effects of this layer on RBC partitioning, a diverging capillary bifurcation is analyzed using a flexible, two-dimensional RBC model. The model is also used to investigate RBC deformation and penetration of the ESL region when ESL properties are varied. The RBC is represented using interconnected viscoelastic elements. Stokes flow equations (viscous flow) model the surrounding fluid. The flow in the ESL is modeled using the Brinkman approximation for porous media with a corresponding hydraulic resistivity. The resistance of the ESL to compression is modeled using an osmotic pressure difference. The study includes isolated cells that pass through the bifurcation one at a time with no cell-cell interactions and two cells that pass through the bifurcation at the same time and interact with each other. A range of physiologically relevant hydraulic resistivities and osmotic pressure differences are explored.</div><div><br></div><div>For isolated cell simulations, decreasing hydraulic resistivity and/or decreasing osmotic pressure difference produced four behaviors: 1) RBC distribution nonuniformity increased; 2) RBC deformation decreased; 3) RBCs slowed down slightly; and 4) RBCs penetrated more deeply into the ESL. The presence of an altered flow profile and the ESL's resistance to penetration were primary factors responsible for these behaviors. In certain scenarios, ESL penetration was deep enough to present a possibility of cell adhesion, as can occur in pathological situations.</div><div><br></div><div>For paired cell simulations, more significant and complex changes were observed. Three types of effects that alter partitioning as hydraulic resistivity is changed are identified. Decreasing hydraulic resistivity in the ESL produced lower RBC deformation. Including cell-cell interactions tended to increase deformation sharply compared to isolated cell scenarios. ESL penetration generally decreased for lower hydraulic resistivities except in scenarios with significant cell-cell interactions. This was primarily due to changes in flow profiles induced by the altered hydraulic resistivity levels.</div>

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Microvesicle generation is an integral part of the aging process of red blood cells in vivo and in vitro. Extensive vesiculation impairs function and survival of red blood cells after transfusion, and microvesicles contribute to transfusion reactions. The triggers and mechanisms of microvesicle generation are largely unknown. In this study, we combined morphological, immunochemical, proteomic, lipidomic, and metabolomic analyses to obtain an integrated understanding of the mechanisms underlying microvesicle generation during the storage of red blood cell concentrates. Our data indicate that changes in membrane organization, triggered by altered protein conformation, constitute the main mechanism of vesiculation, and precede changes in lipid organization. The resulting selective accumulation of membrane components in microvesicles is accompanied by the recruitment of plasma proteins involved in inflammation and coagulation. Our data may serve as a basis for further dissection of the fundamental mechanisms of red blood cell aging and vesiculation, for identifying the cause-effect relationship between blood bank storage and transfusion complications, and for assessing the role of microvesicles in pathologies affecting red blood cells.

No description available.