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  • About
  • The Global ETD Search service is a free service for researchers to find electronic theses and dissertations. This service is provided by the Networked Digital Library of Theses and Dissertations.
    Our metadata is collected from universities around the world. If you manage a university/consortium/country archive and want to be added, details can be found on the NDLTD website.
1

Hepatocyte suspension for liver cell transplantation : consequences of cryopreservation/thawing and evaluation of the infusion related pro-coagulant activity

Stéphenne, Xavier 08 November 2007 (has links)
La transplantation d’hépatocytes est une nouvelle approche thérapeutique pour le traitement des maladies métaboliques. Elle peut être proposée en alternative à la transplantation de foie entier ou, à tout le moins, en attente de celle-ci chez les patients instables, à risque de décompensation métabolique. Les essais cliniques effectués chez 9 patients aux cliniques St Luc ainsi que ceux publiés dans la littérature démontrent l’intérêt de la transplantation de cellules hépatiques à court et moyen terme. La qualité de la suspension cellulaire transplantée reste le premier facteur limitant pour le développement clinique de la technique. La cryopréservation reste le moyen le plus approprié pour la conservation à long terme des cellules. Elle permet de constituer une banque de cellules pouvant être utilisées à tout moment. Nous avons d’abord analysé les protocoles de cryopréservation décrits dans la litérature, ainsi que leurs limites tant au niveau de la préservation de la qualité cellulaire après décongélation in vitro qu’après transplantation in vivo. Dans ce travail, nous avons démontré l’intérêt d’utiliser des cellules cryopréservées/décongelées, afin de stabiliser des patients atteints de maladies du cycle de l’urée, avant la greffe de foie entier. Les tests de contrôle de qualité effectués sur ces cellules ont cependant montré une altération aux niveaux biochimique et cellulaire, après décongélation. Nous avons ainsi démontré une chute des concentrations intracellulaires d’ATP, signe d’une atteinte mitochondriale. Nos travaux ont également permis de mettre en évidence une diminution de la consommation d’oxygène des hépatocytes en suspension, due plus particulièrement à une atteinte du complexe 1 de la chaîne respiratoire. Cette atteinte mitochondriale peut déjà être observée après l’incubation de la suspension cellulaire à –20°C. Aux alentours de cette température critique se fait le passage de l’état aqueux à l’état cristallin suggérant que les dégâts mitochondriaux observés sont dès lors vraisembablement dus à la formation de glace intracellulaire durant le processus de cryopréservation ou de décongélation. Diverses tentatives visant à améliorer les paramètres mitochondriaux affectés par le processus de congélation/décongélation par l’addition d’agents protecteurs du complexe 1 (Bilobalide), d’ inhibiteurs du pore de transition de perméabilité (Ciclosporine A), d’ anti-oxydants ou encore de solutions hyperosmotiques à la solution de cryopréservation, n’ont pas permis d’améliorer la qualité cellulaire. Le tri de sous-types de populations hépatocytaires ou l’isolement de foies hépatectomisés n’ont pas permis de révéler de différences de capacité de résistance à la cryopréservation. Toujours dans le but d’améliorer le rendement de la transplantation d’hépatocytes et d’augmenter l’efficacité d’implantation dans le parenchyme receveur, nous avons démontré dans la deuxième partie de la thèse la capacité des hépatocytes isolés (fraîchement isolés ou cryopréservés/décongelés) à induire un phénomène de coagulation dépendant du facteur tissulaire. Cette activité pro-coagulante, inhibée in vitro par lea N-acetyl-L-cystéine, pourrait être le point de départ d’une réaction inflammatoire aspécifique influençant ainsi la réussite de la transplantation cellulaire. En conclusion, nous proposons dans ce travail différentes stratégies en vue de l’amélioration du rendement de la thérapie cellulaire. La vitrification, autre technique de cryopréservation, permettrait d’éviter la formation d’eau intracellulaire. Enfin la modulation de l’activité pro-coagulante par la N-acetyl-L-cystéine, due à la transplantation cellulaire, constitue une piste intéressante pour essayer d’améliorer l’implantation des cellules transplantées et ainsi le rendement de la greffe. / Liver cell transplantation provides clinical benefit to patients with congenital metabolic abnormalities and currently represents an alternative to orthotopic liver transplantation or at least an interim measure for unstable patients awaiting transplantation. Our team and others have already demonstrated that transplanted hepatocytes can achieve metabolic control in the short or medium term. The quality of transplanted cells remains the first limiting factor for the success of liver cell transplantation. Because the use of freshly isolated cells is restricted by contemporary organ donation, cryopreservation remains necessary for long-term storage and permanent availability of the cells. In this thesis, we have first reviewed and discussed established hepatocyte cryopreservation protocols, especially the cooling procedure, and have focussed on the in vitro and in vivo assays used for the evaluation of post-thawing hepatocyte quality. Amongst 9 cell transplanted patients in our center, several received exclusively or predominantly cryopreserved/thawed hepatocytes. We demonstrated post-transplantation benefits of using these cells in control patients with congentital abnormalities in the urea cycle, particularly with respect to clear evidence of cell engraftment and de novo appearance of enzyme activity. However, despite these clinical benefits, we found an in vitro relationship between the low post-thawing quality of cryopreserved /thawed hepatocytes and an alteration in their mitochondrial function. This post-thawing mitochondrial damage was already evident after the first −20°C cryopreservation step of our protocol, suggesting it occurrs early in the process, around the nucleation point, by intracellular ice formation. Cellular impairment could therefore be possibly explained by mechanical alteration of mitochondria due to water crystallisation during the cryopreservation process or thawing procedure. We also observed a poor efficacy of cryopreserved/thawed hepatocytes (as compared to freshly isolated cells) when used liver engraftment in two mice transplantation models. The marked reductions in intracellular ATP concentrations and the decreases in oxygen consumption by hepatocytes were therefore used as markers for the evaluation of the effects of several compounds such as bilobalide, hyperosmotic or anti-oxidant molecules, pore transition permeability inhibitors, and for the evaluation of the resistance of selected hepatocyte subtypes to cryopreservation protocols. We also demonstrated that isolated hepatocytes exert tissue factor-dependent pro-coagulant activity, which may contribute to the early loss of infused cells. We observed that the addition of N-acetyl-L-cysteine to hepatocyte suspensions inhibits coagulation activation. In conclusion, this work has identified several ways to improve the clinical benefit of liver cell transplantation, including new cryopreservation strategies, such as vitrification. In addition, modulation of the pro-coagulant activity induced by cell infusion with N-acetyl-L-cysteine might beneficially enhance cell engraftment.
2

Anthracycline Treatment of the Human Monocytic Leukemia Cell Line THP-1 Increases Phosphatidylserine Exposure and Tissue Factor Activity

Boles, Jeremiah C., Williams, Julie C., Hollingsworth, Rachel M., Wang, Jian Guo, Glover, Sam L., Owens, A. Phillip, Barcel, David A., Kasthuri, Raj S., Key, Nigel S., MacKman, Nigel 01 February 2012 (has links)
Introduction: Cancer associated thrombosis is a well-recognized phenomenon that results in considerable patient morbidity and mortality. Malignancy conveys an increased risk for thrombosis and chemotherapy further elevates this risk. The pathophysiological mechanisms underlying this process remain poorly defined. Materials and Methods: A human acute monocytic leukemia cell line (THP-1) was treated with commonly used anthracycline chemotherapeutics at concentrations similar to those found in the plasma of cancer patients. Cells were analyzed for tissue factor (TF) mRNA, protein, and activity. Microparticle (MP) TF activity was also measured. Phosphatidylserine (PS) exposure on cells and MPs was analyzed by flow cytometry. PS levels on MPs was also evaluated in an annexin V capture assay. Results: Anthracycline treatment of THP-1 cells resulted in a concentration-dependent increase in cellular TF activity without a change in TF protein, which was associated with increased PS exposure on the cell surface and apoptosis. The increase in TF activity was abolished by annexin V or lactadherin indicating that PS exposure was required. Anthracycline treatment of THP-1 cells also increased the number of TF-positive MPs. Conclusion: Treatment of THP-1 cells with anthracyclines induces apoptosis and increases cellular TF activity. The increased activity required an increase in exposure of PS. Additionally, anthracyclines increase the release of TF-positive MPs from THP-1 cells. We propose that the increase in cellular TF activity in circulating leukemic cells, combined with increased numbers of TF-positive MPs, may contribute to thrombosis in cancer patients receiving chemotherapy.
3

Atividade pró-coagulante da toxina ExoU de Pseudomonas aeruginosa: efeito sobre a expressão do fator tissular em células epiteliais respiratórias / Procoagulant activity of Pseudomonas aeruginosa toxin ExoU: effect on the expression of tissue factor by airway epithelial cells

Luís Filipe Pereira Feliciano 16 July 2008 (has links)
Para avaliar a capacidade da toxina ExoU de P. aeruginosa de induzir a expressão do fator tissular (FT) por células epiteliais respiratórias da linhagem BEAS-2B, células infectadas pela cepa PA103, produtora da toxina, foram comparadas com outras infectadas por cepa mutante obtida por deleção do gene exoU e com células controles não infectadas quanto a i) expressão do mRNA do FT, por RT-PCR; ii) expressão da proteína FT em lisados celulares, por ensaio imunoenzimático (ELISA), e na superfície celular, por citometria de fluxo; iii) atividade pró-coagulante das células, pela determinação da capacidade de indução de coagulação de plasma humano normal e do lisado celular, através de ensaio colorimétrico; iv) presença de FT solúvel no sobrenadante das culturas, por ELISA; v) liberação de micropartículas expressando FT e fosfatidilserina (FS), por citometria de fluxo. Nossos resultados mostraram que ExoU foi responsável pelo aumento da expressão do mRNA e da concentração da glicoproteína FT tanto nos lisados quanto na superfície celular. Esse aumento foi revertido quando as bactérias foram tratadas com uma droga inibidora de PLA2 (MAFP), comprovando-se a dependência da atividade fosfolipásica A2 de ExoU para a modulação da expressão do FT. Células infectadas pela cepa PA103 induziram uma diminuição no tempo de coagulação do plasma humano normal e o aumento da hidrólise do substrato sintético utilizado no teste colorimétrico em comparação com as células infectadas com a cepa mutante, mostrando que o FT expresso era funcionalmente ativo. Foi também detectado um aumento na concentração de FT solúvel presente nos sobrenadantes de culturas infectadas por PA103 e no número de micropartículas expressando, simultaneamente, FT e FS em relação à cultura infectada pela cepa PA103∆exoU. Os resultados obtidos nos testes in vitro foram validados pela demonstração de que a concentração de FT no parênquima pulmonar de camundongos infectados, por via intratraqueal, com a cepa selvagem foi significativamente superior à detectada nos animais infectados com a cepa mutante. / To evaluate the capacity of the P. aeruginosa toxin ExoU to induce the expression of tissue factor (TF) by epithelial respiratory cells from the BEAS-2B cell line, cells infected with the ExoU-producing PA103 bacterial strain were compared with cells infected with a mutant obtained by deletion of the exoU gene and with control non-infected cells in their i) expression of the TF mRNA, by RT-PCR; ii) expression of the protein TF in cell lisates and surfaces, by enzyme immunoassay (ELISA) and flow cytometry, respectively; iii) procoagulant activity by determining the ability of intact cells to induce the coagulation of normal human plasma and the ability of cell lysates to cleave the synthetic substrate of a chromogenic assay; iv) presence of soluble TF in cell culture supernatants, by ELISA and v) release of microparticles simultaneously expressing TF and phosphatidylserine, by flow cytometry. Cells infected with the wild type bacteria exhibited increased expression of TF mRNA 1 hour after infection and a positive modulation of TF expression in both cell lysates and cell surfaces. The enhancement of TF expression was inhibited when cells were infected with bacteria previously treated with a PLA2 inhibitor (MAFP), confirming that the ability of ExoU to modulate TF expression depended on its phospholipase A2 activity. Newly expressed TF was shown to be functionally active, by both the decrease in the clotting time of human plasma and the enhancement of the hydrolysis of the chromogenic assay substrate. Cells infected with the ExoU-producing bacteria exhibited also higher concentrations of soluble TF and of TF and PS bearing microparticles in the cell culture supernatants. These in vitro results were validated by our finding of increase TF concentrations in the lung parenchyma of mice infected intratracheally with the ExoU producing-bacteria at 24 h post-infection.
4

Atividade pró-coagulante da toxina ExoU de Pseudomonas aeruginosa: efeito sobre a expressão do fator tissular em células epiteliais respiratórias / Procoagulant activity of Pseudomonas aeruginosa toxin ExoU: effect on the expression of tissue factor by airway epithelial cells

Luís Filipe Pereira Feliciano 16 July 2008 (has links)
Para avaliar a capacidade da toxina ExoU de P. aeruginosa de induzir a expressão do fator tissular (FT) por células epiteliais respiratórias da linhagem BEAS-2B, células infectadas pela cepa PA103, produtora da toxina, foram comparadas com outras infectadas por cepa mutante obtida por deleção do gene exoU e com células controles não infectadas quanto a i) expressão do mRNA do FT, por RT-PCR; ii) expressão da proteína FT em lisados celulares, por ensaio imunoenzimático (ELISA), e na superfície celular, por citometria de fluxo; iii) atividade pró-coagulante das células, pela determinação da capacidade de indução de coagulação de plasma humano normal e do lisado celular, através de ensaio colorimétrico; iv) presença de FT solúvel no sobrenadante das culturas, por ELISA; v) liberação de micropartículas expressando FT e fosfatidilserina (FS), por citometria de fluxo. Nossos resultados mostraram que ExoU foi responsável pelo aumento da expressão do mRNA e da concentração da glicoproteína FT tanto nos lisados quanto na superfície celular. Esse aumento foi revertido quando as bactérias foram tratadas com uma droga inibidora de PLA2 (MAFP), comprovando-se a dependência da atividade fosfolipásica A2 de ExoU para a modulação da expressão do FT. Células infectadas pela cepa PA103 induziram uma diminuição no tempo de coagulação do plasma humano normal e o aumento da hidrólise do substrato sintético utilizado no teste colorimétrico em comparação com as células infectadas com a cepa mutante, mostrando que o FT expresso era funcionalmente ativo. Foi também detectado um aumento na concentração de FT solúvel presente nos sobrenadantes de culturas infectadas por PA103 e no número de micropartículas expressando, simultaneamente, FT e FS em relação à cultura infectada pela cepa PA103∆exoU. Os resultados obtidos nos testes in vitro foram validados pela demonstração de que a concentração de FT no parênquima pulmonar de camundongos infectados, por via intratraqueal, com a cepa selvagem foi significativamente superior à detectada nos animais infectados com a cepa mutante. / To evaluate the capacity of the P. aeruginosa toxin ExoU to induce the expression of tissue factor (TF) by epithelial respiratory cells from the BEAS-2B cell line, cells infected with the ExoU-producing PA103 bacterial strain were compared with cells infected with a mutant obtained by deletion of the exoU gene and with control non-infected cells in their i) expression of the TF mRNA, by RT-PCR; ii) expression of the protein TF in cell lisates and surfaces, by enzyme immunoassay (ELISA) and flow cytometry, respectively; iii) procoagulant activity by determining the ability of intact cells to induce the coagulation of normal human plasma and the ability of cell lysates to cleave the synthetic substrate of a chromogenic assay; iv) presence of soluble TF in cell culture supernatants, by ELISA and v) release of microparticles simultaneously expressing TF and phosphatidylserine, by flow cytometry. Cells infected with the wild type bacteria exhibited increased expression of TF mRNA 1 hour after infection and a positive modulation of TF expression in both cell lysates and cell surfaces. The enhancement of TF expression was inhibited when cells were infected with bacteria previously treated with a PLA2 inhibitor (MAFP), confirming that the ability of ExoU to modulate TF expression depended on its phospholipase A2 activity. Newly expressed TF was shown to be functionally active, by both the decrease in the clotting time of human plasma and the enhancement of the hydrolysis of the chromogenic assay substrate. Cells infected with the ExoU-producing bacteria exhibited also higher concentrations of soluble TF and of TF and PS bearing microparticles in the cell culture supernatants. These in vitro results were validated by our finding of increase TF concentrations in the lung parenchyma of mice infected intratracheally with the ExoU producing-bacteria at 24 h post-infection.
5

Extra??o, caracteriza??o e atividades biol?gicas de prote?nas da esp?cie cnidoscolus urens (L.) Arthur

Menezes, Yamara Arruda Silva de 04 July 2013 (has links)
Made available in DSpace on 2014-12-17T14:16:35Z (GMT). No. of bitstreams: 1 YamaraASM_DISSERT_Parcial.pdf: 1235608 bytes, checksum: 64be8e29311055ebff593313fa2f1681 (MD5) Previous issue date: 2013-07-04 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior / The extraction, chemical and structural characterization of a wide variety of compounds derived from plants has been a major source of bioactive molecules. Several proteases have been isolated in the plant kingdom, with numerous pharmacological and biotechnological applications. Among the proteases isolated from plants, are the fibrinogenolytic, with relevant application in the treatment of disorders in the coagulation cascade, in addition to potential use as a tool in clinical laboratories. In this study, in addition to evaluating the effects of the protein extract of Cnidoscolus urens (L.) Arthur (Euphorbiaceae) in the coagulation cascade also investigates the presence of antimicrobial activity and characterizes the proteolytic activity detected in this extract, aiming to determine their potential pharmacological and biotechnological application. In this way, crude protein extracts obtained from the leaves of C. urens in Tris-HCl 0.05M, NaCl 0.15M, pH 7.5, were precipitated in different concentrations of acetone, and assessed for the presence of proteolytic activity in azocase?na and fibrinogen. The most active fraction (F1.0) in these tests was chosen for assessment of biological activity and biochemical characterization. The A? chain and B? of fibrinogen were completely cleaved at a concentration of 0.18 ?g/?L of protein fraction in 4 minutes. Fibrinogenolytic activity presented total inhibition in the presence of E-64 and partial in the presence of EDTA. The fraction demonstrated coagulant activity in plasm and reduced the APTT, demonstrating acting on the factors coagulation of the intrinsic pathway and common, not exerting effects on the PT. Fibrinolytic activity on plasma clot was detected only in SDS-PAGE in high concentrations of fraction, and there were no defibrinating. Although several proteases isolated from plants and venomous animals are classically toxic, the fraction F1.0 of C. urens not expressed hemorrhagic nor hemolytic activities. Fraction F1.0 also showed no antimicrobial activity. In proteolytic activity on the azocasein, the optimal pH was 5.0 and optimum temperature of 60?C. The enzyme activity has been shown to be sensitive to the presence of salts tested, with inhibition for all compounds. The surfactant triton did not influence the enzyme activity, but the tween-20 and SDS inhibited the activity. In the presence of reducing agents increase in enzyme activity occurred, a typical feature of enzymes belonging to the class of cysteine proteases. Several bands with proteolytic activity were detected in zymogram, in the region of high-molecular-weight, which were inhibited by E-64. In this study, we found that C. urens presents in its constitution cysteine proteases with fibrinogenolytic and procoagulant activity, which may be isolated, with potential application in treatment of bleeding disorders, thrombolytic and clinical laboratory / A extra??o, caracteriza??o qu?mica e estrutural de uma grande diversidade de compostos derivados de plantas tem sido uma fonte importante de mol?culas bioativas. Diversas proteases t?m sido isoladas no reino vegetal, com in?meras aplica??es farmacol?gicas e biotecnol?gicas. Dentre as proteases isoladas de plantas, est?o as fibrinogenol?ticas, com relevante aplica??o no tratamento de dist?rbios na cascata da coagula??o, al?m do uso em potencial como ferramenta em laborat?rios cl?nicos. Neste trabalho, al?m de avaliar os efeitos do extrato prot?ico de Cnidoscolus urens (L.) Arthur, pertencente ? fam?lia Euphorbiaceae, na cascata de coagula??o, tamb?m se investigou a presen?a de atividade antimicrobiana e caracterizou a atividade proteol?tica detectada neste extrato, tendo como objetivo determinar sua potencial aplica??o farmacol?gica e biotecnol?gica. Desse modo, extratos prot?icos brutos obtidos das folhas de C. urens em tamp?o Tris-HCl 0,05M, NaCl 0,15M, pH 7,5, foram precipitados em diferentes concentra??es de acetona, e avaliados quanto a presen?a de atividade proteol?tica em azocase?na e fibrinog?nio. A fra??o mais ativa (F1.0) nestes testes foi escolhida para realiza??o de avalia??o de atividade biol?gica e caracteriza??o bioqu?mica. As cadeias A? e B? do fibrinog?nio foram completamente clivadas na concentra??o de 0.18 ?g/?L de prote?na da fra??o em 4 minutos. A atividade fibrinogenol?tica apresentou inibi??o total em presen?a de E-64 e parcial em presen?a de EDTA. A fra??o demonstrou atividade coagulante sobre o plasma e reduziu o tempo de tromboplastina parcial ativada, indicando atuar sobre os fatores da via intr?nseca e comum da coagula??o, n?o exercendo efeitos sobre o tempo de protrombina. A atividade fibrinol?tica sobre o co?gulo de plasma foi detectado apenas em SDS-PAGE em concentra??es elevadas da fra??o, e apesar da atividade fibrin(ogen)ol?tica, n?o foi observada atividade defibrinogenante in vivo. Apesar de v?rias proteases de plantas e animais pe?onhentos serem classicamente t?xicas, a frac??o F1.0 n?o expressou atividade hemorr?gica nem hemol?tica. A fra??o F1.0 tamb?m n?o demonstrou atividade antimicrobiana. Na avalia??o da atividade proteol?tica sobre a azocase?na, o pH ?timo de rea??o foi 5.0, e a temperatura ?tima igual a 60?C. A atividade enzim?tica demonstrou ser sens?vel ? presen?a dos sais testados, com inibi??o para todos os compostos. O tensoativo triton n?o influenciou a atividade enzim?tica, por?m o tween-20 e SDS inibiram tal atividade. Em presen?a de agentes redutores ocorreu aumento da atividade enzim?tica, caracter?stica t?pica de enzimas pertencentes ? classe das ciste?no proteases. Diversas bandas prot?icas com atividade proteol?tica foram detectadas em zimograma, na regi?o de elevada massa molecular, que foram inibidas por E-64. Neste trabalho, foi revelado que C. urens apresenta fra??o enriquecida com ciste?no-proteases que apresentam atividade fibrinogenol?tica e procoagulante, que podem ser isoladas, com potencial aplica??o no tratamento de dist?rbios hemorr?gicos, como trombol?tico e em laborat?rio cl?nico / 2020-01-01

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