Diss. (sammanfattning)--Stockholm : Karol. inst., 2002. / Härtill 5 uppsatser.

Il est désormais admis que le caractère invasif d'une tumeur est lié, non seulement, au génotype des cellules cancéreuses, mais aussi à leurs interactions avec le microenvironnement tumoral (MT). Au sein du MT, une déstabilisation de la matrice stromale favorise la progression tumorale et la dissémination métastatique. Le remaniement de la matrice extracellulaire est souvent piloté par des enzymes protéolytiques. En revanche, les effets de l'inhibition de la formation de cette matrice sont peu étudiés. C’est dans cette optique que nous nous sommes intéressés à la protéine C (PC) et son récepteur endothélial (EPCR) et à leur rôle dans la tumorigenèse des leucémies et des cancers solides.L’EPCR est exprimé par un grand nombre de lignées cellulaires cancéreuses. Il est aussi détecté dans le compartiment tumoral chez des patients atteints de pathologie tumorale. Son gène est hautement conservé. Il possède cependant plusieurs polymorphismes. Un de ces SNPs (single nucleotide polymorphism) - 6936A/G - se traduit par la libération d'une forme soluble circulante de l'EPCR (EPCRs) résultant de la protéolyse de la forme membranaire. Chez des patients leucémiques, une fréquence élevée du SNP 6936A/G est observée et associée à la survenue de thrombose. D'autre part, l’EPCR est détecté in situ dans la majorité des biopsies tumorales testées et sécrété en grande quantité dans les ascites. La fixation de la PC sur l’EPCR et son activation augmentent la survie et le potentiel migratoire des cellules cancéreuses. Aussi, la PCA est capable de moduler, par communication paracrine, la sécrétion de plusieurs interleukines et cytokines. Ainsi, la stimulation de cellules du cancer de l'ovaire par la PCA induit la synthèse d'une thrombopoéïtine ovarienne fonctionnelle. Cette cytokine étant régulatrice de la production de plaquettes, la PCA semble être de nouveau à l'interface entre troubles de l'hémostase et pathologie cancéreuse. L’élucidation du rôle complexe de la PCA et de son récepteur endothélial dans la carcinogenèse permettrait non seulement de dégager de nouvelles approches thérapeutiques, mais aussi de prévenir le risque de thrombose associée au cancer et d’en réduire la morbidité. / It is now recognized that the invasiveness of tumor cells is not only related to the genotype of these cells but also to their interaction with tumor microenvironment (TM). Within the TM, stromal matrix destabilization promotes tumor progression and metastatic dissemination. The extracellular matrix remodeling is often driven by proteolytic enzymes. However, few studies have investigated the effects of an impairment of the matrix formation. Given these facts and circumstances, we were interested in protein C (PC) and its endothelial receptor (EPCR), as well as in their role in tumorigenesis in leukemia and solid cancers. EPCR is expressed by a wide range of cancer cell lines. It is also detected within the tumor compartment in patients with malignant diseases. EPCR gene is highly conserved but nevertheless contains polymorphisms. One of these SNPs (single nucleotide polymorphism) - 6936A/G – reflects – in the release of a soluble circulating form (EPCRs) resulting from the proteolysis of membrane-associated form. In leukemic patients a high incidence of 6936A/G SNP is observed and associated with thrombosis events. Moreover, EPCR is detected in the majority of tumor biopsies and is abundantly secreted in ascitic fluid. The PC attachment to EPCR and its activation promotes cell survival and migratory potential of tumor cells. Also, APC is able to modulate, by a paracrine manner, interleukins and cytokines secretion. Thus, ovarian cancer cells stimulation by APC induces the synthesis of a functional ovarian thrombopoietin. As this cytokine has a regulatory effect on platelet production, APC may be once again at the interface between hemostasis disorders and coagulation. The elucidation of the intricate role of APC and its endothelial receptor could permit not only to identify new therapeutic approaches but also to prevent cancer-associated thrombosis risk and to decrease morbidity in cancer patients.

Human protein C (HPC) is a vitamin-K dependent plasma glycoprotein which is one of the major components regulating anticoagulation. HPC injection is a promising therapy for several diseases but a heterologous production system would be preferred over purifying HPC from human plasma because of its low concentration (4-5 $ mu$g/ml). A cDNA clone coding for HPC was inserted downstream of the CaMV 35S promoter and of a dimer of the CaMV 35S promoter. Tobacco plants were transformed using Agrobacterium and a binary vector strategy. Kanamycin resistant plants were regenerated and enzyme linked immunosorbent assay determined that HPC, in crude plant extracts, accounted for up to 0.03% of plant soluble proteins. HPC was found to be expressed by R$ sb1$ seedlings suggesting successful integration of the T-DNA into plant genome. A partial protein purification system was developed in order to enrich the protein mixture for HPC. HPC was found to bind tightly at pH 6.0 to Fast Flow Q Sepharose resin.

Thesis (MAppSc (Medical Laboratory Science)) --University of South Australia, 1992

No description available.

Both of the acute phase reactants, C-reactive protein and fibrinogen, as well as neutrophils have been shown to accumulate at sites of tissue injury or inflammation. The association of C-reactive protein with neutrophils and the concomitant degradation of this ligand by a phorbol 12-myristate 13 acetateactivatable membrane-associated neutral protease has been shown in previous studies. Degradation of C-reactive protein by the neutrophil protease was shown to result in peptides with an ability to modulate various immune functions of the neutrophil. The aim of this study has been to investigate specific characteristics of the protease, with respect to cellular distribution and molecular size. The ability of this neutrophil membrane-associated protease to degrade the acute phase protein, fibrinogen was investigated. The mechanism of degradation of both C-reactive protein and fibrinogen during their association with the neutrophil was also examined. The neutrophil protease, capable of degrading C-reactive protein, was also associated with the cytoskeleton and was proposed to be a submembrane protease localised at sites of attachment of the membrane with the cytoskeleton. The protease was found to have a molecular mass of approximately 600 kDa which, on sodium dodecyl sulphate polyacrylamide gel electrophoresis, separated into four bands which migrated to molecular mass values of 209 kDa, 316 kDa, 398 kDa and 501 kDa. This protease also possessed fibrinogenolytic activity. The fibrinogen degradation products generated by this neutrophil membrane-associated protease were distinct from the products generated by the fibrinogenolytic systems of plasmin, human neutrophil elastase and neutrophil lysosomal enzymes and were unclottable through cleavage of the Aα chain from the N-terminus and the Bβ and γ chains from the C-terminus. N-terminal cleavage of the Aα chain by the neutrophil membrane-associated protease generated the Aα1-21 peptide, previously regarded as a unique consequence of elastase activity. Degradation of C-reactive protein and fibrinogen occurred as a result of their interaction with the neutrophil near to the CD11c integrin receptor. This interaction resulted in the egress of proteolytic activity into the extracellular medium. The fibrinogen products generated outside the cell associated with the neutrophil via the β₂ integrin receptors and the IgG Fc receptor. The interaction of the Creactive protein degradation products with the neutrophil could not be determined. Both C-reactive protein and fibrinogen are degraded by non-stimulated neutrophils but activation with phorbol 12- myristate 13 acetate resulted in maximum degradation This upregulation of activity was achieved through activation of H7 and trifluoperazine inhibitable cellular kinases and changes in microfilament assembly. The generation of non-clottable fibrinogen together with possible modulation of neutrophil receptormediated functions by the fibringen degradation products as well as the knowledge that the neutrophil protease generates C-reactive protein peptides with immunomodulatory activity implicates this neutrophil membrane-associated protease in the modulation of various inflammatory processes.

No description available.

Thesis / Doctor of Philosophy (PhD)

Sepsis is a “life-threatening organ dysfunction caused by a dysregulated host response to infection” that has a widespread impact on human life around the world. It affects more than 1.5 million people, killing at least 250,000 each year in the US alone and affects 90,000 people annually, with estimated mortality rates of up to 30% in Canada. Our understanding of the different biochemical pathways that in the progression of sepsis has improved patient care for sepsis patients. One part of patient care is the use of biomarkers for patient prognosis that draws on the full range of relevant and available information to model the possible outcomes for an individual. Numerous biomarkers have been studied for patient prognosis that includes Procalcitonin (PCT), C-reactive protein (CRP), TNF-α, cfDNA, protein C and PAI 1. Using a panel of multiple biomarkers provided more accuracy in patient prognosis than using individual biomarkers and one such panel that was proposed used cfDNA, protein C, platelet count, creatinine, Glasgow Coma Scale [GCS] score, and lactate. Commercial, low cost POC techniques were available for the measurement of all biomarkers besides cfDNA and protein C. The objective of this doctoral thesis was chosen to develop low cost, microfluidic devices for the measurement of protein C and cfDNA using nonspecific fluorescence dyes that would enable the eventual integration of the systems and improve patient prognosis. The measurement of protein C in plasma required the separation of protein C from interfering proteins in plasma. This was done through the development of a two-stage separation process that included the development of tunable agarose isoelectric gates for separating proteins using their isoelectric point and the miniaturization of immobilized metal affinity chromatography and its extension to Barium for the selective binding of proteins using their chemical affinity. This was performed in a xurographically fabricated chip to reduce costs and enable the use of geometric focusing of the electric field to enable the operation of the device at a lower applied voltage. The challenges faced with cfDNA were different due to the different characteristics of the material and less interference from plasma. The requirement was to measure the total cfDNA content with minimal cost in comparison to currently available techniques. This was achieved through the development of thread microfluidic devices that showed the use of thread for automated aliquoting of samples by controlling length and twists of the thread. Preconcentration and use of external apparatus was avoided by showing that thread could be used to amplify fluorescence response to a range that was sufficient for the measurement of cfDNA in sepsis patients. A portable fluorescence imaging setup was developed for this purpose and was used in demonstration for the measurement of cfDNA in plasma with sufficient resolution. In conclusion, we developed technologies for rapid and low-cost measurement of protein C and cfDNA using xurographic and thread-based microfluidics that may serve as valuable in improving patient prognosis. / Thesis / Doctor of Philosophy (PhD) / Sepsis is a major reason for hospitalization and cause of death in hospitals worldwide. Its treatment is highly time sensitive with each hour of delay in diagnosis causing a significant increase in chances of death. Due to the wide range of symptoms that can be caused by sepsis, its diagnosis uses a scoring method that relies on the expertise of the onsite doctors and nurses increasing their workload. A more objective system for detection requires the measurement of the quantities of different biomarkers in blood. Biomarkers are proteins present in plasma that change in quantity due to the body’s reaction to sepsis. Several of these biomarkers have been identified and studied for their use in both diagnosing the presence of sepsis and in predicting the outcome with the current treatment plan. In this PhD study, we chose two of these biomarkers – circulating free DNA (cfDNA) and protein C and developed low-cost techniques for rapidly measuring their concentration in blood plasma. To do this, we made microfluidic devices with techniques that use low-cost materials such as plastic sheets and threads.The device for the measurement of protein C required separating it from many other proteins in plasma. We showed that a device fabricated from stacked plastic sheets and integrated with agarose gels could be used for the measurement of protein C in plasma with sufficient resolution to help with treating septic patients at a cost of less $5 per device. Similarly, we showed that a device that integrated threads with plastic sheets could be used for measuring the quantity of cfDNA in plasma in a portable format within 15 minutes. Overall, we developed tools for rapid measurement of two biomarkers of sepsis using low cost device that cost under $5 to run and could led to improving the quality of care for sepsis patients.

The vitamin K-dependent family of proteins (VKDs) include prothrombin, factors VII, IX, and X, and protein C (hPC) is synthesized in the liver and act to maintains normal hemostasis. such as properly regulated clotting. An imbalance of any of these pro- or anti-clotting proteins result in hemophilia or disseminated intravascular clotting diseases. Therefore, these proteins have a significant therapeutic value. Many of these proteins are not available in sufficient quantity due to the trace amounts found in plasma and limitations encountered with downstream recovery. Protein C, a major regulatory protein of thrombosis and hemostasis, has a potent anticoagulant activity and can be used as an anti-thrombotic agent. The technology for isolating hPC from human plasma is challenged by; (1) its low concentration in plasma, (2) the limited availability of plasma, (3) similar physicochemical characteristics among VKD plasma proteases, and (4) the risk of transmitting viruses such as the human immunodeficiency virus (HIV). This work focuses on the isolation of protein C from alternative sources for the large-scale production and downstream recovery of highly purified and biologically active hPC. The partial characterization of the protein with respect to post-translational modifications which are essential for functionally active, was also evaluated. Several studies were undertaken: 1. Cohn Fraction IV-I, an off-line discard stream during traditional plasma fractionation process is introduced as an affordable starting material for the large-scale production of hPC. More than 90 percent of the total protein C antigen detected in the various Cohn fractions was found to reside in fraction IV-I. The protein C isolated from Cohn IV-I paste using a metal-dependent monoclonal antibody to hPC was found to be biologically active. 2. Recombinant production of hPC in the milk of transgenic pigs, achieved by targeting the synthesis of the protein to the mammary gland, is presented as a model bioreactor system for the synthesis and downstream recovery of complex human proteins. Two major populations of biologically active recombinant hPC (rhPC) were detected and immunopurified by employing conformation specific metal-dependent monoclonal antibodies in the immunopurification process. A high performance thin layer chromatography method was also developed for the detection of total carbohydrate compositions in protein C. / Ph. D.