This research investigates the protein interactions that occur when soy protein is added to milk and subjected to renneting or heating. Milk was fortified with 20% soy protein and enzymic coagulation studied at 35°C at various pH's and CaCl2 levels. The first part deals with the interaction between milk and soy proteins during rennet-induced milk coagulation. The first goal was to determine how soy proteins affected milk coagulation. The effects of native versus heat-denatured soy proteins on rennet coagulation time and curd firmness were compared. lmmunogold labeling along with transmission electron microscopy was used to identify and localfze soy proteins in coagulated milk. Partitioning of ß-conglycinin and glycinin, the two main soy protein fractions, between cheese and whey was determined by electrophoresis. Soy proteins affected milk coagulation to the greatest extent at pH 6.6. Both heat-denatured and native soy proteins increased rennet coagulation time. Only heat-denatured soy proteins affected final curd firmness. Most of ß-conglycinin was lost in whey, whereas glycinin was retained in curd. Soy proteins existed in the curd as aggregates that were less electron dense than casein micelles. At pH 6.6, heat-denatured soy proteins were fibrous and adhered to the surfaces of casein micelle, preventing direct micelle-micelle contact. This would delay aggregation rate and decrease curd firmness by decreasing the number and strength of links between casein micelles. Native soy proteins did not bind to the casein micelles but rather were physically trapped within curd. Their effect of delaying aggregation is thought to be a function of their binding of calcium. Adding CaCl2 or lowering the pH to 6.3 or 6.0 helped restore coagulation properties. The second goal was to determine what heat-induced interaction occurs between milk and soy proteins, specifically between κ-casein and glycinin. Both κ-casein and glycinin are heat labile and form insoluble aggregates when heated. When glycinin and κ-casein were heated together, some acidic polypeptides of glycinin crosslinked with κ-casein via disulfide linkages. However, when disulfide linkage was prevented by adding ß-mercaptoethanol , non-covalent interactions between κ-casein and both acidic and basic polypeptides of glycinin occurred that prevented the heat precipitation of glycinin. This non-covalent interaction between glycinin polypeptides and κ-casein may explain why the heat-treated soy proteins became attached to the surfaces of casein micelles during rennet coagulation of milk.

Water recycling by membrane treatment is widely accepted as a leading alternative water source. This separation process creates a concentrated stream (called concentrates), containing most of the pollutants in 10%-20% of the flow; and a treated water stream. As nitrogen is a major concern, environmental regulations have become more stringent, requiring additional treatment to meet effluent standards. Other concerns include organic contaminants and potential production of halogenated organics if disinfection of the reject was applied. One option to address the problem of dissolved organic nitrogen and carbon is advanced oxidation. This oxidation could lead to degradation of refractory organic materials, which are poorly removed in conventional treatment. This project aims to evaluate treatment extent and cost of alternatives for organic (particularly nitrogen) removal in reject water addressing the following research gaps: (i) identifying the key organic pollutants present in the concentrated stream, (ii) the effectiveness and optimisation of coagulation, ion exchange and advanced oxidation; (iii) apparent cost of the different treatment methods. The untreated reverse osmosis concentrates were collected from two treatment plants:- Luggage Point, and Bundamba, both near Brisbane, Queensland, Australia. The first contains more colourful of organics than the second plant. Stirred cell fractionation with ultrafiltration membranes was used to characterise the removed key pollutants, as it offers better accuracy and reproducibility compared to centrifugation fractionation. Fluorescence spectral was used to monitor and identify specific organic compounds. The largest fraction was smaller sized <1kDa. This is probably small humic substances and fulvic acids, as indicated by Excitation Emission Matrix (EEM) analysis. A smaller portion of soluble microbial products (SMPs) also contributes to the concentrates. Bundamba contains large non coloured organics including organic nitrogen with elevated ammonia-N. In contrast, Luggage Point has higher colour, inorganic carbon and conductivity with less ammonia-N. Advanced Oxidation Process (AOP) was the most effective treatment method (high removal of organics, e.g. 55% COD of initial), followed by magnetised ion exchange (MIEX) and coagulations. For UV/H2O2 AOP, the optimal operating condition 400mg.L-1 H2O2 and 3.1kWh.m-3 energy input resulted in organics removals up to 55% with complete decolourisation. The effective reduction was found in all size ranges, preferably in >1kDa. Low inorganic carbon and salinity in Bundamba may allow better overall oxidation rates. MIEX also performed better in Bundamba with organic removals up to 43% and 80% decolourisation at the optimum resin dose of 15mL.L-1. Removal was preferential in size range of >3kDa, with more proportional percentage for decolourisation. Similarly, ferric coagulation removed a wider size range of organics. Further, ferric achieved better organic removal in Luggage Point with up to 49%. At the same molar dose (1.5mM), ferric is superior to alum, especially in Bundamba where there were less hydrophobic compounds according to EEM. Alum is poor for treatment of high organics with less coloured water. MIEX with an operational cost (chemicals and power only) of $0.14-$0.20.m-3 treated water seemed to be the most effective treatment overall. The resin achieved better results with a slightly higher cost than coagulation, and had a lower environmental impact due to reduced sludge production. AOP offers better treatment, but at a higher cost ($0.47.m-3 treated). Combined alternatives may benefit the removal effectiveness. Furthermore, more specific identification of contaminants should be investigated separately to choose appropriate treatment for priority chemicals. Another issue is further investigation of costing, including capital, and full environmental impact of treatment.

La Réalité Virtuelle propose un nouvel outil d'investigation des systèmes biologiques complexes : l'expérimentation in virtuo. La modélisation de tels systèmes implique nécessairement celle de la cinétique de réactions biochimiques. Dans ce contexte, nous soutenons la thèse qu'il est possible d'expérimenter in virtuo la cinétique biochimique d'un système biologique complexe par des systèmes multi-agents (SMA) ordonnancés sur la base d'itérations asynchrones et chaotiques. Cette cinétique peut être abordée selon trois échelles de modélisation : macroscopique, mésoscopique et microscopique. Ainsi, nous proposons d'abord un SMA capable de simuler cette dernière à l'échelle macroscopique. Nous proposons ensuite un SMA pour la cinétique à l'échelle microscopique. Afin d'illustrer ces modèles, nous les appliquons à l'exemple de la coagulation du sang. Le modèle macroscopique est illustré sur le test du temps de Quick, tandis que le modèle microscopique est appliqué à la réaction d'activation de la prothrombine en thrombine.

<p>In patients with unstable coronary artery disease, this study evaluated the degree of inflammation and coagulation activity, relations to myocardial cell damage, prognosis, and influences of randomisation to 72 h infusion with three different doses of inogatran, a direct thrombin inhibitor (n=904), or unfractionated heparin (n=305). </p><p>Anticoagulant treatment effects were evaluated with aPT time. In inogatran treated patients with aPT times ≥ 44 s (median), the 7-days event rate - death, myocardial infarction or refractory angina – was 11.6 %, compared to 6.6 % with aPT times < 44 s (p=0.01). Higher aPT times was related to improved outcome during heparin treatment.</p><p>Markers of inflammation, i.e. fibrinogen and C-reactive protein (CRP), and coagulation, i.e. prothrombin fragment 1+2 (F1+2), thrombin-antithrombin complex (TAT), soluble fibrin (SF) and D-dimer were analysed in serial samples (n=320). High fibrinogen, F1+2 and D-dimer levels persisted at 30 days. Patients with myocardial damage, detected by elevated troponin, had higher levels of all markers except TAT.</p><p>Ischemic events occurred at 30 days in 17 % of patients with high (pre-treatment top tertile) and 8.5 % of patients with lower fibrinogen levels (p=0.03), while high CRP levels only were related to increased mortality. At 30 days, patients with high compared to low pre-treatment levels of TAT or SF had 40 % lower event rate. Patients with early decreased compared to raised F1+2 or TAT levels during treatment had 50 % lower 30-days event rate (p<0.05). </p><p>Conclusions: The aPT time is an inappropriate indicator of antithrombotic efficacy. The raise in fibrinogen in the acute phase is sustained, and indicates risk of thrombosis and new ischemic events. The pronounced CRP elevation is transient, but associated with increased mortality. Higher coagulation activity may identify patients with a thrombotic condition as the major cause of instability, who are best responders to anticoagulant therapy. However, reactivation of coagulation activity with raised risk of ischemic events is a concern at cessation of treatment.</p>

<p>In 101 patients with ST-elevation myocardial infarction treated with streptokinase the additional effects of lmw-heparin (dalteparin) were investigated. The prognostic value of troponin-T (TnT) was elucidated and the development of myocardial damage was investigated with Positron Emission Tomography (PET).</p><p>Dalteparin tended to provide a higher rate of TIMI grade 3 flow in the infarct-related artery at 24 h compared to placebo. In patients with signs of early reperfusion there was a higher rate of TIMI grade 3 flow in the dalteparin group compared to placebo. There were significantly fewer patients with ischemic episodes at 6-24 h in the dalteparin compared to placebo group.</p><p>The increase in coagulation activity was attenuated in the dalteparin group. There was a tendency to more ischemic episodes and lower frequency of TIMI grade 3 flow in patients with persistent elevation of coagulation activity at 18 h. Among deceased patients the coagulation activity was significantly higher than in survivors. </p><p>The association between elevated TnT on admission and long-term mortality might be explained by longer delay, episodes of chest pain during the last 24 h, less non-invasive signs of reperfusion at 90 minutes, and lower patency in the infarct-related artery at 24 h. </p><p>Eight patients were investigated with PET at 3h, 24 h and after 3 weeks. PET outlines the infarct region with reduced perfusion and metabolism. The oxidative metabolism in the infarct region at 3 h correlated with the water-Perfusable Tissue Fraction (PTF) and its improvement over time.</p><p>Dalteparin seems to improve maintenance of coronary patency, which can be explained by attenuation of the increased coagulation activity. Elevated TnT level on admission is associated with a worse outcome, which can partly be explained by less successful fibrinolytic treatment. PET investigations might to be a useful method in future trials evaluating new agents in the treatment of acute myocardial infarction.</p>

<p>The laparoscopic way of performing general surgical procedures was introduced all over the Western world in a few years around 1990. No previous scientific studies of the safety of this new way of performing general surgery had been undertaken.</p><p>In an animal study, it was shown that carbon dioxide pneumoperitoneum (CO₂PP) causes an increase in inferior caval vein (ICV) pressure, although there were no effects on the ICV blood flow. There were gradual increases in systemic, pulmonary and ICV vascular resistance, which remained after exsufflation. These effects on vascular resistance could not be reproduced in a second animal study, presumably due to a different form of anesthesia. In this study, there was only indirect evidence of CO₂ PP decreasing urine output. No increase in vasopressin, which is commonly seen during CO₂ PP, was found, indicating that vasopressin may play a role in the decreased urine output during CO₂ PP but that there must be other contributing factors as well. Only brief effects on the renal arterial blood flow were seen.Renal venous pressure increased to that of the ICV.</p><p>A literature review indicated that thromboembolic complications do occur after laparoscopic cholecystectomy (LC). The relative frequencies indicated an underreporting of deep vein thrombosis (DVT) in relation to pulmonary embolism (PE).</p><p>In a clinical study, activation of the coagulation after LC was demonstrated. There were differences between the groups receiving dextran and low molecular weight heparin as prophylaxis. A further clinical study showed the incidence of DVT, as demonstrated by phlebography, to be 2.0 % (95 % confidence interval 0-6.0 %) 7-11 days after LC, even though thromboembolism prophylaxis was given in shorter courses than those scientifically proven to be effective against DVT. D-dimer values increased at the first postoperative day and even further at the time of phlebography, suggesting that the effects of LC on coagulation and/or fibrinolysis may be of longer duration than previously known.</p>

<p>This thesis describes a new <i>in vitro</i> slide chamber model that makes it possible to conduct studies of molecular and cellular interactions between whole blood and biomaterials. The model proved to be a suitable tool for detection of cell and platelet binding to a biomaterial surface. It was possible to monitor activation of the blood cascade systems and cells in the fluid phase and detect surface-bound molecules.</p><p>One finding was that thrombin generation is primarily triggered by FXII on a biomaterial surface since corn trypsin inhibitor, inhibited thrombin generation in blood.</p><p>Another finding was that thrombin generation was dependent on variety types of blood cells, since thrombin generation was almost negligible in platelet-rich plasma. When various preparations of blood cells were used to reconstitute platelet-rich and platelet-poor plasma, erythrocytes were shown to be the most efficient cell type in triggering thrombin generation. Inhibition of platelet aggregation with aspirin and Ro44-9883 was associated with a decrease in thrombin generation, confirming that platelet activation is necessary for normal coagulation activation. These findings suggest that the central events consist of an initial low-grade generation of thrombin that involves erythrocytes and possibly leukocytes which leads to activation of platelets; and a second platelet-dependent amplification loop that produces most of the thrombin.</p><p>Titanium exposed to whole blood produced high amounts of thrombin. Stainless steel and PVC, generated lower amounts. This indicates that titanium might be less suitable as a biomaterial in devices that are in direct contact with blood for prolonged time. Considering the superior osteointegrating properties of titanium and titanium's response to blood, a correlation between high thrombogenicity and good osteointegration seems to exist.</p><p>Compstatin, that binds to complement component C3, effectively inhibited the generation of C3a and sC5b-9 and the binding of C3/C3 fragments to the surface. Our results suggest that a biomaterial is able to activate complement through both the classical and alternative pathways and that the classical pathway alone is able to maintain a substantial bioincompatibility reaction. The results show that complement activation is a prerequisite for activation and binding of PMNs to the surface in the <i>in vitro</i> model.</p>

<p>During the past decade clinical islet transplantation has become a viable strategy for curing type 1 diabetes. The limited supply of organs, together with the requirement for islets from multiple donors to achieve insulin independence, has greatly limited the application of this approach. </p><p>The islets are infused into the liver via the portal vein, and once exposed to the blood, the grafted tissue has been shown to be damaged by the instant blood-mediated inflammatory reaction (IBMIR), which is characterized by coagulation and complement activation as well as leukocyte infiltration into the islets. Islet revascularization is a subsequent critical step for the long-term function of the transplanted graft, which may partially be impeded by the IBMIR. </p><p>In this thesis, we have explored novel strategies for circumventing the effects of the IBMIR and facilitating islet revascularization.</p><p>Systemic inhibitors of the IBMIR are typically associated with an increased risk of bleeding. We therefore evaluated alternative strategies for modulating the islets prior to transplantation. We demonstrated, using an adenoviral vector, that a high level of expression and secretion of the anticoagulant hirudin could be induced in human islets. An alternative approach to limiting the IBMIR was developed in which anticoagulant macromolecular heparin complexes were conjugated to the islet surface. This technique proved effective in limiting the IBMIR in both an in vitro blood loop model and an allogeneic porcine model of islet transplantation. An increased adhesion of endothelial cells to the heparin-coated islet surface was demonstrated, as was the capacity of the heparin conjugate to bind the angiogenic factors VEGF and FGF; these results have important implications for the revascularization process.</p><p>The outcome of the work in this thesis suggests that modulation of the islet surface is an attractive alternative to systemic therapy as a strategy for preventing the IBMIR. Moreover, the same techniques can be employed to induce revascularization and improve the engraftment of the transplanted islets. Ultimately, improved islet viability and engraftment will make islet transplantation a more effective procedure and increase the number of patients whose diabetes can be cured.</p>

The functional group composition of acid polysaccharides was determined after isolation using cross-flow ultrafiltration, radiolabeling with 234Th(IV) and other isotopes, and separation using isoelectric focusing (IEF) and polyacrylamide gel electrophoresis (PAGE). Phosphate and sulphate concentrations were determined from cultured bacterial and phytoplankton colloid, particulate and colloidal samples collected from the Gulf of M??xico (GOM). Characterization of the 234Th(IV)-binding biomolecule was performed using ion chromatography (IC), and gas chromatography-mass spectrometry (GC-MS). Radiotracer experiments and culture experiments were conducted in determining the binding environment of the 234Th(IV)-binding ligand (i.e., sorption onto suspended particles), as well as the origin of the ligand in seawater systems. In all samples, 234Th(IV) isoelectric focusing profiles indicated that 49% to 65% of the 234Th(IV) labeled EPS from Roseobacter gallaeciensis, Sagittula stellata, Emiliania huxleyi, Synechococcus elongatus and GOM Station 4-72m was found at a pHIEF of 2 in the IEF spectrum. The carboxylic acid group appeared at the same pHIEF as 234Th(IV) for EPS from Roseobacter gallaeciensis, Emiliania huxleyi, Synechococcus elongatus and GOM colloidal organic matter sample. The phosphate group appeared at the same pHIEF as 234Th(IV) for EPS from Roseobacter gallaeciensis, and Synechococcus elongatus sample. The sulphate group was found at the same pHIEF as 234Th(IV) for EPS from S. elongatus and GOM colloidal organic matter sample. The total polysaccharide content was only 14% and 8%, uronic acids were approximately 5.4% and 87.1%, and total protein content was 2.6% and 6.2% of total carbon content of Sagittula stellata and Synechococcus elongatus, respectively. Monosaccharides identified in both Sagittula stellata and Synechococcus elongatus were galactose, glucose, and xylose in common. In addition, Sagittula stellata contained mannose and Synechococcus elongatus had galactoglucuronic acid. Thus, depending on the species, the size, structural composition, and functional groups of the 234Th(IV)-binding, acidic polysaccharides will vary. From these observations, it is concluded that the steric environment and not necessarily the exact functional group might actually be responsible for thorium-234 complexation to macromolecular organic matter. This research helped to improve our understanding of the observed variability in POC/234Th ratios in the ocean and provided insights into factors that regulate organic carbon export fluxes.

Myocardial infarction (MI) is often caused by a disruption of an atherosclerotic plaque with activation of coagulation, platelets and inflammation. The aims were; to investigate whether the oral direct thrombin inhibitor, ximelagatran affected markers for coagulation, platelet and inflammation in a patient cohort with recent MI and if the coagulation markers could identify patients with increased risk of new ischemic events; to evaluate some of the mechanisms involved in formation of platelet-monocyte aggregates (PMAs). In a biomarker substudy patients with recent MI were randomized to 24-60 mg of ximelagatran or placebo for six months. There was a persistent dose-independent reduction of coagulation markers (F1+2, D-dimer) by ximelagatran treatment. 60 % reduced their D-dimer levels after one week and that group had less ischemic events during treatment. There was an early increase of the platelet activation marker and ximelagatran in higher doses attenuated these increased levels. Both in vivo and in vitro the direct thrombin inhibitor diminished procoagulant activity and tissue factor (TF) presenting microparticles. In contrast, the inflammatory markers increased after six months of ximelagatran treatment. The PMA-levels were elevated for long-term after MI. In vitro thrombin inhibition diminished formation of PMAs. Formation of PMAs in stimulated whole blood was P-selectin dependent and induced TF expression through phosphorylation of the Src-family member Lyn in monocytes. Addition of an oral direct thrombin inhibitor reduces coagulation and platelet activation markers for long-term after a MI together with reduced procoagulant activity which may contribute to the clinical benefit of the drug. Early reduction of D-dimer levels seems to be suitable to identify patients with reduced risk of new ischemic events independent of antithrombotic treatment. Circulating PMAs persist after a MI connecting coagulation to inflammation. Within these aggregates P-selectin induces TF, the main initiator of coagulation, partly through phosphorylation of Lyn.