Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A maneira mais difundida na observaÃÃo de cÃlulas sanguineas (hemÃcias) à aquela que utiliza microscopia Ãtica convencional. Devido ao limite de resoluÃÃo dos instrumentos Ãticos, novas tÃcnicas de microscopia colocam-se como alternativas para o estudo de cÃlulas, tais como: a microscopia eletrÃnica (de varredura e transmissÃo) e as tÃcnicas de varredura por sonda. Inclui-se neste Ãltimo grupo a microscopia de forÃa atÃmica (AFM). Este trabalho discute as possibilidades de uso da Microscopia de ForÃa AtÃmica ($emph{Atomic Force Microscopy}$ - AFM) em ciÃncias da vida, para ser mais especÃfico, na caracterizaÃÃo de eritrÃcitos. Cinco experimentos envolvendo hemÃcias e AFM estÃo aqui descritos: diferenciaÃÃo entre os grupos sanguÃneos AB+ e O+; anÃlise do perfil da membrana eritrocitÃria de indivÃduos sadios e portadores de SMD; preparaÃÃo de cÃlulas vermelhas para anÃlise em microscopia de forÃa atÃmica; anÃlise volumÃtrica de cÃlulas vermelhas; e monitoramento do envelhecimento de um eritrÃcito ao ar usando o AFM. No primeiro experimento, a rugosidade das membranas celulares dos grupos AB+ e O+ mostraram-se diferentes. Jà no segundo experimento, depressÃes foram encontradas sobre a membrana de pacientes com SMD e indivÃduos sadios, contudo, aparentemente mais profundas no primeiro grupo. O terceiro estudo trouxe à tona a importÃncia da preparaÃÃo adequada dos eritrÃcitos para medidas especÃficas em AFM. A quarta experiÃncia comprovou a capacidade da tÃcnica AFM de fornecer informaÃÃo de volume, o que tambÃm foi utilizado no Ãltimo experimento para monitorar o envelhecimento de uma hemÃcia ao ar. / The optical microscopy is the most employed technique used for visualize red blood cells (RBCs). But, due to its resolution limit, it is necessary to use other complementary techniques to study the cells, such as: the scanning and transmission electron microscopy, and the scanning probing microscopy. The Atomic Force Microscopy (AFM) is included in the last group. This work refers to the possibilities of using AFM in life science, focusing on the erythrocytes characterization. Five experiments involving red blood cells and AFM were carried out: AB+ and O+ blood types differentiation; RBCs membrane study of donors and patients with MDS (Myelodysplastic Syndrome); suitable preparation of red blood cells for AFM analysis; volume study of erythrocytes; and finally aging process observation of RBC in air. The first experiment determined the cell membrane roughness for AB+ and O+ groups, which were different. For the second one, depressions were found on the cell surface of both MSD patients and healthy people. These "holes" were deeper in the first group. The third experiment showed the importance of sample (RBCs) preparation for each AFM specific analysis. The fourth experimental procedure showed the AFM technique capability for providing volume information, which was also used in the last experiment to monitor the aging process of RBCs in air.

O curto tempo de armazenamento dos hemocomponentes é um dos fatores que dificulta e limita a quantidade de sangue que pode ser efetivamente armazenada, o que é uma desvantagem na medicina veterinária, pois o acesso a doadores é restrito e a demanda é contínua e cada vez maior na prática de clínicas e hospitais veterinários. Durante o armazenamento do sangue em baixas temperaturas, seja sob a forma de sangue total ou concentrado de eritrócitos, há uma queda intensa de metabólitos importantes para a viabilidade e funcionalidade dos eritrócitos. O desenvolvimento de meios e soluções de preservação sanguínea possibilitou o armazenamento dos eritrócitos e, consequentemente, facilitou o trabalho dos bancos de sangue. Portanto, a busca por melhores formas e soluções para preservação capazes de evitar ou diminuir estes efeitos prejudiciais durante o seu armazenamento é contínua, para que ao final se obtenha uma melhor qualidade do sangue transfundido. O presente trabalho avaliou o concentrado de eritrócitos felino armazenado na solução de CPDA-1 e CPD/SAGM durante 35 dias. Os dados laboratoriais foram comparados entre grupos e ao longo do tempo. Neste experimento foram utilizadas 10 bolsas de concentrado de eritrócitos felino divididos em dois grupos de cinco para avaliação de cada um dos aditivos. Os parâmetros laboratoriais K+, Na+, Cl-, lactato, HCO3-, amônia, glicose e pH foram avaliados nos dias 1, 7, 14, 21, 28 e 35 após a coleta. Vários parâmetros (K+, lactato, HCO3, glicose e cloreto) demonstraram que a solução CPD/SAGM manteve o metabolismo energético do eritrócito mais estável. Com este trabalho, foi possível entender melhor as alterações metabólicas sofridas pelos eritrócitos felinos durante o armazenamento. Concluímos que, apesar da solução CPD/SAGM se mostrar mais eficaz in vitro, são necessários mais estudos com relação aos hemocomponentes em gatos e à sua viabilidade pós-transfusional. / The short shelf life of blood products is one factor that complicates and limits the amount of blood that can be effectively stored, and it is a disadvantage in veterinary practice, because the access to donors is restricted and the demand is continuous and increasing at veterinary clinics and hospitals. During blood storage at low temperatures, either as whole blood or as packed red cells, there is a significant decrease of metabolites that are important for the viability and functionality of erythrocytes. The development of blood preservation solutions has enabled the storage of red blood cells and improved the service at the blood banks. Therefore, the search for better ways and blood preservation solutions to avoid or reduce these harmful effects during the storage conditions is continuous, in order to obtain the best blood product to be transfused. This study evaluated 10 bags of feline erythrocyte concentrate divided into two groups, stored in CPDA-1 and CPD/SAGM solutions during 35 days. The laboratory data were compared between groups and over time. K+, Na+, Cl-, lactate, HCO3-, ammonia, glucose and pH were assessed on days 1, 7, 14, 21, 28, and 35 after collection. On various parameters (K+, Cl-, HCO3-, glucose and lactate) solution of CPD/SAGM kept the energy metabolism of red blood cells more stable. With these results we can better understand the biochemical changes of feline erythrocytes during storage. We conclude that, although the CPD/SAGM solution shown to be more effective, more studies are needed to improve knowledge of feline blood components and post-transfusion viability.

Cryopreservation is a promising approach to long-term biopreservation of living cells, tissues and organs. The use of cryoprotective agents (CPAs) in combination with extremely low temperatures is mandatory for optimum biopreservation. CPAs (e.g., glycerol, trehalose, dimethyl sulphoxide (DMSO)), however, are relatively cytotoxic and compromise biopreserved cell quality. This is usually resultant in oxidative damage, diminishing cell functionality and survival rate. The growing market of cell therapy medicinal products (CTMPs) demands effective cryopreservation with greater safety, of which the currently available CPAs are unable to provide. The present study was aimed at developing cryomedia formulation to enhance the cryopreservation of nucleated and anucleated mammalian cells. Here, eleven compounds of a polyol nature were selected and examined for their cryoprotective properties. These compounds are derived from plants and honey, thereby ensuring their safety for human consumption. The selection was based on their molecular structure and chemical properties. Here, the presented study is divided into three main phases: 1) Screening the compounds panel for cryo-additive effects on cells during and post-cryopreservation and optimising the dose response and time course for trehalose and glycerol with and without the novel compounds; 2) Assessing the influence of biophysical criteria on biospecimen cryopreservation (e.g., biosampling procedure, cell age, donor age); 3) Establishing the mechanisms of action underpinning the modulatory effect of novel CPAs on biological pathways during cryopreservation. For the stated purposes, red blood cells (RBCs) obtained from sheep and humans were used to screen the compounds for novel cryo-additive agents. Cryosurvival rate was employed as an indication of the compounds' cryoprotective performance. Cellular biochemical profiles, including lipid and protein oxidative damage as well as key redox enzymatic activities (e.g., lactate dehydrogenase (LDH), glutathione reductase (GR)) were measured. The study revealed that nigerose (Nig) and salidroside (Sal) were significantly effective in protecting cells during the freeze-thaw cycle and recovery phases. Both compounds promoted the activity of GR and reduced oxidative stress mirrored by diminished LDH activity. This was also reflected in the protein and lipid oxidation levels, which was limited to a comparable level with the cells' prior freezing. Further studies on human leukaemia (HL-60) were carried out to elucidate the molecular and biological pathways associated with cryodamage and the modulatory effects of adding novel CPAs. The proteome profile and the corresponding biological functions were evaluated and iii showed that Nig and Sal protected cells against cryodamage. The additive compounds (Nig and Sal) demonstrated a unique and overlapping modulation effect pattern. Nig was found to highly influence proteins engaged with metabolic and energetic pathways, whereas Sal greatly affected nuclear and DNA-binding proteins. The current study concluded that novel CPAs have high potency in protecting cells and each compound has a unique effect on the cellular proteome. These features can be applied to designing cryomedia formulae with higher protective efficiency for targeted applications in cell based therapy and biopharmaceutical industries.

New medical treatments, compounds that affect human health, nutritional supplements, and other substances, are introduced to society every day. The accurate determination of the potential toxicity from these substances is of critical importance to our society. Goals of the modern toxicologist not only involve the determination of the toxic potential of new substances but also: the elucidation of mechanisms; improving existing assays; and developing new assays to study toxicity. This thesis addresses these goals in two topics fundamental to toxicology. Re-evaluating the expression of dose and susceptibility of cells in culture The exposure of cells in culture to drugs, xenobiotics, and other compounds is one of the first tools used to determine the potential for toxicity. Problems can arise when results of these experiments are translated to next-level toxicity experiments (e.g. animals and humans). I hypothesized that "dose" in cell culture can be improved by designing and reporting experiments based on dose in moles per cell. When experiments were compared on an extracellular concentration basis, a large apparent variability in toxicity was observed. However, if these same exposures were expressed as moles per cell, all experiments yielded the same toxicity. In addition to the evaluation of mole per cell, I investigated the susceptibility of various cells to 1,4-benzoquinone. I hypothesized that upon exposure to toxins that bind covalently, larger cells would require more molecules per cell of toxin versus a smaller cell to achieve identical toxicities. I found a linear correlation between cell volume(pL) and ED50 (mole per cell where 50 % cell viability is lost), supporting my hypothesis. This work could improve current cell culture protocols and allow for better and less expensive determination of toxicities. Heritability of the red blood cell storage lesion Blood transfusions are an integral part of modern medicine with 5 million people receiving blood each year in the United States. There is growing evidence that red blood cells (RBCs) stored for longer periods are less therapeutically beneficial and could even be harmful to patients. This phenomenon of diminished RBC function with increased time in storage is called the storage lesion. However, there is great variation between different donors in the severity of the storage lesion in their donated RBCs. I hypothesized that part of this variability in the RBC storage lesion is determined by heritable genetic differences. To test this hypothesis, a study using mono- and di-zygotic twins was performed to determine the heritability of adenosine triphosphate (ATP), glutathione (GSH), glutathione disulfide (GSSG) and hemolysis in stored blood. Major discoveries in this study include: GSH, GSSG, and the half-cell reduction potential (Ehc) are heritable (57 %, 51 %, and 70 %, respectively) in non-stored RBCs. In addition, ATP was found to be heritable in two different storage solutions (62 % in AS-3, 71 % in CP2D); as well as GSH, GSSG, Ehc and hemolysis (59 %, 48 %, 64 %, and 53 %, respectively). These discoveries could eventually be used to develop new genetic tests that would predict the rate of deterioration in stored blood quality on an individual basis.

Alzheimer's disease (AD) is the most prevalent form of progressive dementia and is characterized by the accumulation of amyloid beta (Aß) peptide in the brain and in the cerebral vessels forming cerebral amyloid angiopathy (CAA). As previously reported, an active immunization strategy of mice with Aß1-42 peptide results in decreased Th1 and increased Th2 cytokine responses as well as an effectively clearance of CNS Aß. This approach has also yielded favorable results for many patients, unfortunately, a small percentage of these study participants developed severe aseptic meningoencephalitis likely secondary to CNS invasion of activated T-cells. We have previously demonstrated that disruption of CD40-40L pathway reduces Aß plaque load, promotes Th2 response, and rescues from cognitive impairments. However, direct blockage of the CD40 pathway by passive vaccination with anti-CD40L antibody leads to immunosupression. Therefore, in its current form this therapeutic strategy poses an unacceptable risk to the recipient of treatment, aged individual. For those reasons, the identification and characterization of alternative modulators/inhibitors of CD40 signaling may be necessary for the development of safe and effective AD immunotherapy. This proposal introduces novel immunomodulatory therapies that are based on previous vaccination strategies or cell based therapies across medial field. We showed that transcutaneous vaccination can both be efficacious and safe, thus clearly demonstrating that the right combination of the antigens, adjuvants, and the routes of administration are crucial for the right vaccine. Furthermore, we demonstrated that the effects of current Aß vaccine strategies could be enhanced by a simultaneous blockade of CD40-40L signaling. As an alternative approach, we explored the possibility of cell-based therapies and showed that human umbilical cord blood cells, which are currently used as a treatment for systemic lupus erythematosus and leukemia, and currently investigated against stroke, amyotropic lateral sclerosis, age-related macular degeneration, multiple sclerosis, and Parkinson's disease, and showed that not just they improved the AD like pathology in transgenic animals but altered both the brain and peripheral inflammation levels. Lastly, we discussed the involvement of microglia, one of the key players in both AD pathogenesis and Aß clearance and suggesed that microglia in actuality has a continuum of physiological activation states that contribute to proinflammation, antiinflammation, and phagocytosis.

Nine hemoglobins have been isolated from the blood of cutthroat trout. All nine hemoglobins bind oxygen cooperatively and appear to be tetramers with molecular weights of -64,000. The oxygen equilibria and subunit structures of the purified hemoglobins were studied. In addition, the red blood cells of cutthroat trout were examined for the presence of ATP and GTP, which are known to be physiological modulators of hemoglobins in fishes. Five hemoglobins with isoelectric points from 9.1 to 7.0 are classified as cathodal hemoglobins. These five hemoglobins have identical oxygen binding properties by the criteria tested. All have oxygen equilibria which are unaffected by protons and ATP and essentially independent of temperature, with overall enthalpies of oxygenation ~0. Two hemoglobins with isoelectric points near 6.5, classified as a nodal hemoglobins, have oxygen binding properties distinctly different from those of the cathodal hemoglobins. Both are characterized by a Root effect, displaying non-cooperative oxygen binding and low oxygen affinity at pH 6.2. ATP causes a large reduction in the oxygen affinity without affecting the cooperativity of oxygen binding. GTP has a similar but slightly larger effect on both hemoglobins. The oxygen equilibria of the anodal hemoglobins are temperature dependent, with the oxygen affinity being reduced as temperature increases. The overall enthalpy of oxygenation is -14 kcal/mol for both hemoglobins. The two remaining hemoglobins represent only a small percentage of the total hemoglobin. These hemoglobins are tentatively designated as embryonic hemoglobins based primarily on a comparison of their properties to those observed for hemoglobins from newly-hatched rainbow trout (Iuchi, I. (1973) Comp. Biochem. Physiol. 44B, 1087-1101). These two hemoglobins have isoelectric points near 5.9 and oxygen binding properties similar to those of the cathodal hemoglobins. With the possible exception of one of the embryonic hemoglobins (for which globins were not obtained), all the hemoglobins are composed of two different types of globin chains. Six are ∝_2 β_2 tetramers, while two of the cathodal hemoglobins are hybrid tetramers of the type 〖∝∝'β〗_2 and ∝∝'ββ. Red blood cells of cutthroat trout contain both ATP and GTP, suggesting that, in contrast to rainbow trout, both nucleotides may be important physiological modulators of hemoglobin oxygen affinity in this fish.

Orientador: Leonardo Perez Faverani / Coorientador: Osvaldo Magro Filho / Banca: Roberta Okamoto / Banca: Ana Paula Farnezi Bassi / Banca: Marisa Aparecida Cabrini Gabrielli / Banca: Adriano Rocha Germano / Resumo: O objetivo deste estudo foi avaliar a influência da Fibrina Rica em Plaqueta (FRP) no processo inflamatório em defeitos críticos em calotas de ratos e sua consequente reparação tecidual. Foram utilizados 128 (cento e vinte e oito) ratos (Rattus norvegicus, albinus, Wistar), adultos, com peso corporal entre 450 e 500g. Os animais foram divididos aleatoriamente em 4 grupos equitativos que compuseram a amostra do trabalho, onde no grupo coágulo (GC) foi realizado o defeito ósseo de tamanho crítico preenchido com coágulo sanguíneo; grupo anti-inflamatório não esteroidal (AINE) que teve os defeitos de tamanho crítico preenchidos com coágulo sanguíneo e administrado cetoprofeno (10mg/kg dia); o grupo fibrina rica em plaquetas (FRP) com defeitos de tamanho crítico preenchidos com preparado de fibrina rica em plaquetas autóloga; e o grupo fibrina rica em plaquetas mais AINE (FRP + AINE) com defeitos de tamanho crítico preenchidos com preparado de fibrina rica em plaquetas autóloga e administrado cetoprofeno (10mg/kg dia). Cada grupo foi avaliado nos períodos de 2, 7, 14 e 28 dias e os espécimes analisados através da histometria, micro-CT e teste ELISA para presença de TNFα. Os dados quantitativos foram submetidos aos testes estatísticos ANOVA 1/2 fatores ou Kruskal-Wallis e pos Tukey e Dunn (p<0,01). Os resultados histométricos e microtomográficos evidenciaram maior formação óssea para o grupo PRF em comparação aos demais grupos (p<0,05) e menor presença de TNF-alfa no período inicia... (Resumo completo, clicar acesso eletrônico abaixo) / The objective of this study was to evaluate the influence of Platelet Rich Fibrin (PRF) on the inflammatory process in critical defects in rat calvaria and its consequent tissue repair. One hundred twenty-eight adult rats (Rattus norvegicus, albinus, Wistar) with body weight between 450 and 500g were used for the study. The animals were randomly divided into 4 equitable groups that composed the work sample. In the clot group (GC) the critical size defect was filled with blood clot; the non-steroidal anti-inflammatory group (NSAID) had the critical size defects filled with blood clot and ketoprofen was given pos-operatively (10mg / kg day); the platelet-rich fibrin group (FRP) had the critical-size defects filled with autologous platelet-rich fibrin preparation; and platelet-rich fibrin group plus NSAIDs (FRP + NSAID) had the critical-size defects filled with autologous platelet-rich fibrin preparation and administered ketoprofen post-operatively (10mg / kg day). Each group was evaluated at 2, 7, 14 and 28 days after the surgical procedures. The samples were evaluated through histometry, micro- CT and ELISA for the presence of TNFα. The quantitative data were submitted to the statistical test ANOVA 1/2 factors or Kruskal-Wallis and post Tukey and Dunn (p <0.01). The histometric and microtomographic results showed higher bone formation for the PRF group compared to the other groups (p <0.05) and lower TNF-alpha in the initial period in the PRF group compared to the control group (p <0.05). It was concluded that the PRF was favorable since the beginning through the later periods, aiding in the inflammatory response and bone neoformation. (Complete abstract electronic access below) / Doutor

Megakaryocytes (Mks) are unique bone marrow cells, which produce platelets. Dysregulated Mk development can lead to abnormal platelet number and the production of functionally defective platelets, causing bleeding, thrombotic events, and leukaemia. Understanding the molecular mechanisms driving megakaryopoiesis may yield insights into the molecular genetics and cellular pathophysiology of a diversity of disorders. The primary aim of this thesis was to gain insight into the molecular events required for normal Mk development. As transcription factors and cytokines play a central role in driving Mk development, both of these processes were investigated. Fli-1 and GATA-1 are key transcription factors regulating Mk-gene expression, alone and co-operatively. To understand the mechanism of transcriptional synergy exerted by Fli-1 and GATA-1, in vitro assays were carried out investigating the interactions between Fli-1, GATA-1 and DNA that mediate synergy. A novel mechanism of synergy was identified, where Fli-1 DNA binding is not required, although an interaction between Fli-1 and GATA-1, and GATA-1 DNA binding is required. Importantly, the results demonstrate that Fli-1 DNA binding is not essential for promoting Mk-gene expression in primary murine bone marrow cells. Thrombopoietin (TPO) is the primary cytokine responsible for Mk and platelet development. Identifying novel TPO gene-targets may provide invaluable information to aid the understanding of the complex and unique processes required for Mk development. Using microarray technology, IFI16 was identified as a TPO-responsive gene that has not previously been studied in the Mk lineage. This work demonstrated that IFI16 is expressed in CD34+ HSC-derived Mks, and that the Jak/STAT pathway is essential for the activation of IFI16 by both TPO and IFN-??. Of biological significance, IFI16 was found to regulate both the proliferation and differentiation of primary Mks, suggesting that IFI16 may control the balance between these two essential processes. In conclusion, the data in this thesis presents a novel mechanism through which Fli-1 and GATA-1 regulate the synergistic activation of Mk genes. The identification and functional characterisation of a novel TPO-inducible gene, IFI16, involved in regulating the proliferation and differentiation of Mks is also described. These findings have implications for several congenital and malignant conditions affecting Mk and platelet development, and possibly a mechanism for IFN-induced thrombocytopaenia.

Cellular prion protein (PrPc) is a GPI–anchored protein, of unknown function, found in a number of cells throughout the body. It is now widely believed that a mis-folded, protease resistant form of this protein is responsible for a group of fatal neurodegenerative diseases called transmissible spongiform encephalopathies (TSE), including Creutzfeldt-Jakob disease (CJD) and kuru in humans, scrapie in sheep, chronic wasting disease (CWD) in deer and elk and bovine spongiform encephalopathy (BSE) in cattle. Although the exact function of PrPc is unknown it has been implicated in copper binding, signal transduction and cell adhesion. The pathogenesis of prion diseases is poorly understood, however it is known that PrPc must be present in order for the disease to progress. Platelets have been shown to be the largest reservoir of PrPc in peripheral blood cells and previous studies in animal models have suggested platelets may also be involved in TSE infectivity. In this study, we determine the exact location of PrPc within human platelets, examine the mobilization and release of PrPc from activated platelets on both microvesicles and exosomes and suggest a possible role for platelets in prion infectivity. In addition we examine the role of PrPc within normal platelet functions including aggregation, signal transduction and adhesion. / May 2005

Simulating blood flow in microvessels is a major challenge because of the numerous blood cells suspended in the blood. Furthermore, red blood cells (RBCs), which constitute 45% of the total blood volume, are highly deformable. RBCs deformation and RBC-RBC interactions determine the complex rheology of the blood. In this research, we simulate the blood flow in periodic two dimensional channels and conduct numerical experiments on the cell-free layer which appears near the wall. We use the boundary integral method and the smooth particle mesh Ewald method to represent the blood flow, and cells are modeled as deformable capsules. In the numerical experiments, we examine four possible mechanisms that may contribute to the cell-free layer: RBC deformation, RBC aggregation, configuration constraint, and the lubrication mechanism. Our simulations correctly represent hemodynamic phenomena such as the blunt velocity profile and the Fåhræus effect. We observed that more deformable RBCs migrate more away from the wall, and, consequently, the thickness of the cell-free layer increases. However, RBC aggregation increased the cell-free layer thickness by only 5%. In the experiment on the configuration constraint, no cell-free "layer" was detected when we removed cells which intersected an artificial constraint in the microvessel. In the last experiment on the lubrication mechanism, the cell-free layer disappeared at a no-shear stress boundary, and the hematocrit profile was similar to that in the constraint test. Therefore, this research clearly shows that the cell-free layer is generated by the lateral migration of deformable RBCs due to the lubrication mechanism. / text