Ulm, Univ. Diss., 2006.

Thesis (Ph. D.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 197-201). Also available on the World Wide Web.

Thesis--Helsinki. / Includes bibliographical references.

Universiẗat, Diss., 2005--Giessen.

Universiẗat, Diss., 2005--Giessen.

Made available in DSpace on 2014-06-11T19:23:07Z (GMT). No. of bitstreams: 0 Previous issue date: 2011-02-28Bitstream added on 2014-06-13T18:50:23Z : No. of bitstreams: 1 donato_pm_me_botfm.pdf: 1472808 bytes, checksum: dd94c6d1ccf0834bf27a62051a637836 (MD5) / Ministério da Saúde / As plaquetas são fragmentos do citoplasma do megacariócito e possuem papel fundamental na manutenção da hemostasia. Ao aderirem umas às outras, ativam-se, mudam de forma e liberam grânulos que contêm fatores de crescimento, proteínas que atuam na divisão celular. Os grânulos α- plaquetários secretam fatores de crescimento que regulam eventos, tais como, síntese de DNA, quimiotaxia e citodiferenciação. A estocagem prolongada das plaquetas a 22ºC é prejudicial à viabilidade e à função, mesmo quando estocadas em condições adequadas nos bancos de sangue. Observa-se diminuição da concentração do glutation intraplaquetário, representando diminuição da proteção contra o stress oxidativo, do qual resulta a perda do grupo sulfidril. As plaquetas desempenham funções com consumo de energia, como exemplo a diapedese, que pode ser recuperada após cinco dias de armazenamento pelo azul de metileno. Durante a divisão celular há necessidade de bloqueio das funções citoplasmáticas com aumento da produção de proteínas de adesão para que ocorra a fixação da célula que vai se dividir. Os resultados mostraram que quando adicionamos azul de metileno, a quantidade de células viáveis diminui com aumento de apoptose e necrose, muito provavelmente devido à liberação de energia / Platelets are megakaryocyte cytoplasmic fragments that play a fundamental role in maintaining hemostasis. When they adhere to each other, they become activated, change shape and release granules containing growth factors, proteins involved with cell division. Platelet α-granules secrete growth factors that regulate events such as DNA synthesis, chemotaxis and cytodifferentiation. Long platelet storage at 22ºC is harmful to their viability and function, even when they are stored under proper conditions at blood banks. A reduced concentration of intraplatelet glutathione is observed, representing decreased protection against oxidative stress, from which the loss of the sulfhydryl group results. Platelets perform actions with energy consumption, such as diapedesis, which can be recovered after five storage days by methylene blue. During cell division, cytoplasmic functions must be blocked by increased adhesion protein so that the fixation of the dividing cell can occur. Results showed that when methylene blue is added, the number of viable cells decreases with apoptosis and necrosis increase, which is very likely due to energy release

The regulated interaction of leukocyte with endothelium is of key importance during normal immune surveillance and leukocyte infiltration to sites of infection in the inflammatory response. This thesis is concerned with the structure and function of CD31 (platelet-endothelial cell adhesion molecule-1), one of the adhesion molecules implicated in these processes. Previous work has shown both in vivo and in vitro that CD31 is involved in the final step of leukocyte recruitment, transmigration across the endothelial monolayer. CD31 mediated adhesion is complex, since it is capable of mediating multiple adhesive interactions, both to itself (homophilic adhesion) and to other ligands (heterophilic adhesion). In order to study homophilic adhesion, an heterologous cell-protein assay was used in combination with recombinant chimeric CD31Fc fusion proteins, ICAM-3/CD31 chimeras and chimeras between human and murine CD31. These reagents located the homophilic binding site to the NH₂-terminal domains 1 and 2, but also define a non-binding accessory role for the membrane proximal domains. Using site-directed mutagenesis to target all of the exposed charged residues in domain 1 and a subset of charged residues in domain 2, five residues were identified, mutations in which resulted in inhibition of homophilic adhesion. These residues map to both faces of the domain 1 immunoglobulinlike fold, suggesting that each molecule of CD31 interacts with two others. A novel zipper model of homophilic adhesion involving CD31 lateral association analogous to that seen amongst cadherins is proposed on the basis of these results. Evidence for lateral association of CD31 to form dimers was obtained from biophysical, biochemical and molecular biology techniques. These show that Cd31 exists in an equilibrium between monomeric and dimeric forms both in solution as soluble recombinant protein, and at the cell surface. In solution the affinity of the interaction was calculated to lie in the range 12-14μM. A large panel of anti-CD31 monoclonal antibodies were generated and tested for their ability to effect homophilic adhesion. Inhibitory antibodies were identified, mapping throughout the extracellular domain, away from the ligand binding site. In addition possible stimulating antibodies mapping to the membrane proximal domains were also identified. This indicates that CDS 1 may be induced to undergo conformational changes which effect homophilic adhesion, and it is proposed that these conformational changes may be linked to the ability of CD31 to form laterally associated dimers. Using the reagents described above, a screen of haematopoietic cell lines identified a novel heterophilic interaction, which was shown to be mediated by the integrin αvβ3. Proteinprotein assays were used to confirm a direct physical association between CD31 and αvβ3, and to map the integrin binding site to the third immunoglobulin-like fold of CD31. The functional significance of this interaction was assessed in neutrophil transmigration assays, in which both anti-CD31 and anti-αvβ3 antibodies were found to partially inhibit neutrophil transmigration.

Abnormalities in the structure of fibrin fibers and blood platelets have been broadly studied and well correlated to functional abnormalities in the coagulation system due to disease, genetics or environmental factors. The importance of these and further ultrastructural investigations of fibrin fiber networks and platelets is therefore paramount in understanding iii the aetiology of haemorrhagic or thrombogenic tendencies and disorders. To study structural abnormalities and variations of fibrin fibers and platelets, electron microscopy is essential; unfortunately the use of electron microscopy necessitates several preparation steps to make a biological specimen stable enough to withstand the high vacuum environment of an electron microscope and also electron beam irradiation. The two most common procedures to accomplish this is chemical fixation and freeze fixation. Chemical fixation entails the chemical alteration of a specimen by means of the introduction of chemical bonds and cross-links that keep molecules and structures in place, followed by several rinsing and dehydration steps. Freeze fixation preserves biological specimens by the removal of thermal energy from the specimen at an extremely fast rate (> 105 In this study, methods to fixate fibrin fiber and platelet networks by freeze fixation was developed, optimised and subsequently compared to chemical fixation methods to ascertain the optimum preparation technique for transmission and scanning electron microscopy for ultrastructural studies of platelets and fibrin networks. K/s) allowing the water in a biological specimen to reach a super-cooled stabilized state (vitrification). The general consensus in the scientific community is that ultrastructural preservation by high pressure freeze fixation is superior to that of chemical fixation, although the facts are that different fixation methodologies have dissimilar chemical and physical interactions with different specimens and as a result different artefact introductions. Therefore the best possible specimen preparation method to ensure an accurate likeness of the fixated specimen to its in vivo condition needs to be ascertained and used. In this study, methods to fixate fibrin fiber and platelet networks by freeze fixation was developed, optimised and subsequently compared to chemical fixation methods to ascertain the optimum preparation technique for transmission and scanning electron microscopy for ultrastructural studies of platelets and fibrin networks. K/s) allowing the water in a biological specimen to reach a super-cooled stabilized state (vitrification). The general consensus in the scientific community is that ultrastructural preservation by high pressure freeze fixation is superior to that of chemical fixation, although the facts are that different fixation methodologies have dissimilar chemical and physical interactions with different specimens and as a result different artefact introductions. Therefore the best possible specimen preparation method to ensure an accurate likeness of the fixated specimen to its in vivo condition needs to be ascertained and used. Ultimately it was found that high pressure freezing coupled with freeze substitution is a superior method for fine structure preservation of fibrin fiber networks and platelets when utilizing transmission electron microscopy. Contrastingly for scanning electron microscopy ultrastructural studies it was found that chemical fixation is the more optimal method for the preparation of fibrin networks and platelets. / Dissertation (MSc)--University of Pretoria, 2013. / gm2014 / Anatomy / Unrestricted

Neuroinflammation is an important contributor to the pathogenesis of many neurological diseases. A key component of the innate immune response in the central nervous system is the migration of neutrophils into the brain parenchyma, where they exacerbate neuronal injury and worsen clinical outcome. A greater understanding of the mechanisms underlying neutrophil influx into the brain may aid the development of novel therapeutic interventions for the variety of diseases to which neutrophils contribute, notably including stroke and epilepsy. In vitro evidence implicates the pro- inflammatory cytokine, interleukin-1α (IL-1α), derived from platelets as a key mediator of cerebrovascular inflammation and neutrophil migration across brain endothelial cells.The aim of the work in this thesis was to test if this mechanism is important in vivo.We investigated the contribution of platelets and IL-1 in a murine model of neutrophil migration into the peritoneal cavity in response to injection of lipopolysaccharide (LPS). Depletion of platelets abrogated the migration of neutrophils in response to LPS- induced peritonitis, indicating an important role for platelets in the process. Genetic knockout of IL-1 had no effect on neutrophil influx, demonstrating that migration in the peritoneum occurs independently of IL-1.The discovery that neutrophil migration in LPS-induced peritonitis was independent of IL-1 contrasted with the finding that platelet-derived IL-1 was a mediator of neutrophil influx across mouse brain endothelial cells in vitro. The question arose as to whether IL-1 was required as a mediator of neutrophil migration in extra-cerebral tissues. Hence, we tested the contribution of platelets and IL-1 in two further in vivo models of neutrophil migration: LPS injection into a subcutaneous air pouch, and acute lung injury induced by LPS inhalation. Platelet depletion significantly reduced neutrophil migration into the air pouch in response to LPS, yet had no effect in acute lung injury. This indicated that neutrophil migration into the air pouch was dependent on platelets, and that migration into the lungs was platelet-independent. LPS induced the same degree of neutrophil migration in wild-type and IL-1 knockout mice, demonstrating that IL-1 was not required for neutrophil migration in either model.To determine the contribution of platelets and IL-1 to neutrophil migration in response to cerebrovascular inflammation, we injected LPS into the mouse striatum. In this model, neutrophil influx to the brain parenchyma in response to LPS was reduced by depletion of circulating platelets, and inhibition of the platelet adhesion molecule, GpIb. Genetic knockout of IL-1α significantly reduced the number of invading neutrophils induced by LPS. These data confirmed that both platelets and IL-1α were important contributors to cerebral neutrophil migration in vivo. To determine whether platelets in systemic circulation may be the source of IL-1α, we treated mice with IL-1 receptor antagonist or anti-IL-1 antibodies to block systemic IL-1 action. Neither intervention affected cerebral neutrophil migration in response to LPS, suggesting that the IL-1α that mediates neutrophil migration may originate in the brain.Overall, these data demonstrate that IL-1α and platelets make an important contribution to neutrophil migration to the brain, yet independently of each other. Our data also suggest there may be specific mechanisms driving innate immune responses in vivo even in response to the same inflammatory stimulus.

京都大学 / 0048 / 新制・論文博士 / 博士(医科学) / 乙第13332号 / 論医科博第5号 / 新制||医科||7(附属図書館) / 大阪大学大学院生命機能研究科生命機能専攻 / (主査)教授 河本 宏, 教授 濵﨑 洋子, 教授 髙折 晃史 / 学位規則第4条第2項該当 / Doctor of Medical Science / Kyoto University / DFAM