Thesis (M.S.)--Oregon State University, 2010. / Printout. Includes bibliographical references (leaves 42-44). Also available on the World Wide Web.

The U.S. Environmental Protection Agency is in the process of developing a nationwide standard for turbidity in construction site runoff. It is widely expected that this standard cannot be met with conventional erosion and sediment control measures; consequently, innovative practices for managing sediment on construction sites must be developed. The objective of this research was to develop an understanding of how soil characteristics and polymer properties affect the amount of turbidity reduction that can be achieved through flocculation. The polymers used were PAMs, a proprietary product, and chitosan. The charge density of the PAMs ranged from 0% to 50% and the molecular weights ranged from 0.2 to 14 Mg/mol. A protocol for creating modified synthetic stormwater runoff for soil samples was developed and used on soils from seven construction sites. Particle size distributions were used to compare the modified synthetic stormwater runoff with grab samples of stormwater from one site and showed the synthetic runoff was representative of the actual runoff. Flocculation tests were performed on the synthetic runoffs with PAM and chitosan doses from 0.03 to 10 mg/L. The non-ionic PAM, proprietary product, and chitosan were found to be the most effective at reducing the turbidity of all the synthetic runoff below 200 NTU. The high molecular weight anionic PAMs were effective on only two of the seven synthetic runoff samples. Hardness tests were performed indicating interparticle bridging to be the bonding mechanism of the PAM. Electrophoretic mobility tests were performed on two of the soil suspensions and indicated the bonding mechanism of PAM to be interparticle bridging, and the bonding mechanism of chitosan to be a combination of charge neutralization and interparticle bridging. Tests showed as the charge density of the PAM increased, their effectiveness decreased. / text

published_or_final_version / Medicine / Master / Master of Research in Medicine

Haemophilia B is a bleeding disorder caused by various mutations of the coagulation Factor IX gene (F9) resulting in qualitative or quantitative Factor IX protein (FIX) deficiency. Factor replacement therapy is the current standard of care. Cure may be possible in the near future by gene therapy — the transfer of normal copies of F9 to patients with haemophilia, causing establishment of FIX production and correction of the bleeding phenotype. Mesenchymal stem cells (MSC) are potential vehicles for gene delivery through ex vivo gene transfer and subsequent transplantation to the patient. Lentiviral vectors can transduce MSC effectively and mediate long term gene expression. However, gene expression may decline with time due to transgene silencing. Ubiquitous Chromatin Opening Element (UCOE) is a set of genetic sequences cloned from housekeeping genes that can maintain a transcriptionally competent, open chromatin structure and was shown to prevent gene silencing by resisting DNA methylation. We tested human F9 expression and FIX protein secretion by transducing MSC with lentiviral vectors that carry the FIX gene under the control of A2UCOE (A2UCOE-hF9). A2UCOE is a 2.2 kb sequence cloned from the HNRPA2B1–CBX3 gene loci that harbour UCOE function. A2UCOE-eGFP, an enhanced Green Fluorescent Protein (eGFP) gene expression construct, was used to assist in vector titration of A2UCOE-hF9 by flow cytometry and quantitative reverse transcription-polymerase chain reaction (qRT-PCR). MSC were transduced at various Multiplicities of Infection (MOIs) by A2UCOE-hF9 lentiviral vector. Upon transduction, F9 mRNA expression and FIX secretion were measured by qRT-PCR and ELISA respectively. Osteogenic and adipogenic differentiation assay were performed to compare differentiation potential before and after transduction at an MOI of 1. F9 mRNA expression and FIX secretion were both undetectable in untransduced MSC. Upon transduction, vector dose-dependent increase in F9 mRNA expression and FIX secretion were detected at MOIs of 1, 2, 4 and 8. The level of secreted FIX ranges from 20 to 150 μIU in 72 hours. Osteogenic and adipogenic differentiation were not affected post-transduction at an MOI of 1. In conclusion, FIX secretion by MSC was detected upon A2UCOE-hF9 lentiviral transduction. However, the level of FIX appeared to be low compared to published studies. Further studies are required to determine the cause of low FIX expression, develop methods to maximize FIX expression and confirm whether A2UCOE can prevent gene silencing and maintain sustainable gene expression. / published_or_final_version / Paediatrics and Adolescent Medicine / Master / Master of Research in Medicine

Platelets play an important role in the haemostatic process in order to seal damaged blood vessels. The platelets form a platelet plug at the damaged area and prevent blood loss. Once the damage to the vessel wall has been covered, the platelets retract the coagulum, to allow the blood to flow freely in the vessel. Free oscillation rheometry (FOR) can be used for analysis of coagulation as measured by clotting time and changes in clot elasticity (G'). Clot G' provides information about the fibrin network in the coagulum and the platelets’ ability to retract the coagulum. FOR analysis is performed using the ReoRox® 4 instrument. The blood sample is added to a cylindrical sample cup, which is set into free oscillation. The frequency and damping of the oscillation is recorded over time as the blood coagulates. The change in G' is calculated from the frequency and damping measured. Patients with malignant haematological diseases are often thrombocytopaenic and require platelet transfusions to prevent or stop bleeding. To ensure good haemostatic function in the recipient it is important that the quality of the platelets used for transfusion is well preserved. The aim of this thesis was to determine the quality of platelet concentrates (PCs), during storage, using various in vitro methods, including FOR, and to investigate how various preparation processes affect the quality. We also investigated whether FOR can be used to evaluate the haemostatic status in subjects at risk for thrombosis or bleeding as well as how the haemostatic status was affected by a platelet transfusion. We show that FOR can provide information about the coagulation properties in subjects at risk of thrombosis (pregnant women) or bleeding (thrombocytopaenic patients). We also show that the coagulation as measured by FOR is influenced by red blood cells and the fibrinogen concentration. However, the presence of functional platelets accounted for 90% of the G'. Furthermore we present data that FOR can provide information on the haemostatic effect of platelet transfusions and on the function of the transfused platelets. PCs produced by two different cell separators showed similar quality during storage for 7 days as assessed by FOR analysis. Leukocytes in the PCs can cause transfusion-associated graft-versus-host disease which can be prevented by gamma irradiation of the PCs. Gamma irradiation did not affect the quality of PCs during 7 days of storage as analysed by FOR. The clotting time was unchanged during the storage period. The capacity of platelets to retract the coagulum was reduced from days 1 to 5 of storage as seen by a prolonged time to reach maximum G' and the reduced mean change in G' per minute. However, if sufficient time is allowed for the platelets to regain their function, the clot will be fully retracted (as seen by a well maintained maximum G'). The FOR parameters were similar for 5- and 7-day old PCs, which, combined with other in vitro tests (e.g. hypotonic shock response, changes in pH, swirling, lactate and glucose), support the prolongation of the platelet storage period to 7 days. Intercept™ treatment of PCs can be performed to inhibit replication of contaminating bacteria in PCs. Intercept™ treatment of PCs did not diminish the clot-promoting capacity of the platelets as assessed by FOR clotting time. In conclusion, FOR is a promising method for assessing hyper- and hypocoagulability. It can provide information on the haemostatic effect of platelet transfusions and the function of the transfused platelets. FOR was also shown to be useful for analysing PC quality during different preparation and storage conditions.

<p>Thrombosis is associated with the occlusion of a blood vessel and can be triggered by a number of types of injury, such as the rupture of an atherosclerotic plaque on the artery wall, changes in blood composition, or blood stasis. The resulting thrombosis can cause major diseases such as myocardial infarction, stroke, and venous thromboembolic disorders that, collectively, account for the most common cause of death in the developed world. Anticoagulants are used to treat and prevent these thrombotic diseases in a number of clinical and surgical settings. Although commonly prescribed, currently approved anticoagulants have a major limitation of severe drug-induced bleeding that contributes to the high levels of morbidity and mortality associated with use. The "holy grail" for antithrombotic therapy is to identify a drug that inhibits thrombus formation without promoting bleeding. Understanding the differences between thrombosis and hemostasis in the vascular system is critical to developing these safe and effective anticoagulants, as this depends on striking the correct balance between inhibiting thrombus formation (efficacy) and reducing the risk of severe bleeding (safety). While it is commonly thought that the same factors play a similar role in hemostasis and thrombosis, recent evidence points to differing functions for FXI and FXII in each of these settings. Importantly, these factors seem to contribute to pathological thrombus formation without being involved in normal hemostasis.</p><p> The overall goal of this project was to evaluate the inhibition of the intrinsic pathway of coagulation as a potential anticoagulant strategy utilizing the aptamer platform. Aptamers are short, highly structured nucleic acids that act as antagonists by binding to large surface areas on their target protein and thus tend to inhibit protein-protein interactions. High affinity binding aptamers have been isolated that specifically target a diverse range of proteins, including transcription factors, proteases, viral proteins, and growth factors, as well as other coagulation factors. As synthetic molecules, aptamers have a small molecular weight, are highly amenable to modifications that can control their bioavailability, and have not been found to elicit an immune response, thus making them ideal drug candidates. Importantly, aptamers can be rapidly and effectively reversed with either a sequence specific antidote that recognizes the primary sequence of the aptamer or a universal antidote that binds to their backbone and reverses all aptamer activity independent of sequence. This ability lends itself well to their therapeutic application in coagulation, as rapid reversal of a drug upon the onset of bleeding is a key property for increasing the safety of this class of drugs.</p><p> Aptamers targeting FXI/FXIa and FXII/FXIIa were isolated in two separate SELEX (systematic evolution of ligands by exponential enrichment) procedures: the FXII aptamer was isolated in a convergent SELEX approach and the FXIa aptamer was isolated from a purified protein selection. In both processes, 2'fluoropyrimindine modified RNA with a 40-nucleotide random region was incubated with either the plasma proteome (in initial rounds of the convergent SELEX) or the purified protein target (FXII or FXIa). The nucleic acids that did not bind to the target were separated from those that bound, and these molecules were then amplified to generate an enriched pool with increased binding affinity for the target. This process was repeated under increasingly stringent conditions to isolate the aptamer that bound with the highest affinity to the purified target protein. Utilizing biochemical and in vitro coagulation assays, specific, high-affinity binding and functional anticoagulant aptamers were identified for both protein targets, and the mechanism of anticoagulation was ascertained for each aptamer. </p><p> Overall, both aptamers bound to an exosite on their target protein that was able to inhibit downstream activation of the next protein in the coagulation cascade. In order to specifically examine aptamer effects on several parameters of thrombin generation, a new assay was developed and fully characterized using aptamer anticoagulants targeting other coagulation factors. Aptamer inhibition of both FXI and FXII was able to decrease thrombin generation in human plasma. However, limited cross-reactivity in other animal species by both aptamers hindered our ability to assess aptamer inhibition in an in vivo setting. Moving forward, screening aptamers against a larger selection of animal plasmas will hopefully allow us to identify an animal species in which we can analyze aptamer inhibition of the intrinsic pathway for effectiveness and safety in inhibiting thrombosis. The further characterization and use of these aptamers in plasma and blood based settings will allow us to study the diverging functions of the intrinsic pathway in thrombosis and hemostasis.</p><p> A critical need exists for safe and effective anticoagulants to treat and prevent numerous thrombotic procedures and diseases. An ideal anticoagulant is one that strikes the correct balance between inhibiting thrombus formation and reducing drug-induced bleeding. Inhibition or depletion of factors XI and XII of the intrinsic pathway of coagulation have shown reduced thrombus formation without interruption of normal hemostasis in several models of thrombosis. By developing novel RNA aptamer anticoagulants to these factors, we have set the stage for evaluating the net therapeutic benefit of intrinsic pathway inhibition to effectively control coagulation, manage thrombosis, and improve patient outcome. As well as developing a safe anticoagulation, these agents can lead to important biological discoveries concerning the fundamental difference between hemostasis and thrombosis.</p> / Dissertation

Στο παρών κείμενο μεταπτυχιακής εργασίας περιγράφεται η κλινική εφαρμογή μαγνητοσυστολικού αισθητήρα για τη μέτρηση του χρόνου πήξης ολικού αίματος. Περιγράφεται η αρχή λειτουργίας της συσκευής και η κατασκευή του πρωτότυπου μοντέλου. Τα επιμέρους κομμάτια της διάταξης σχεδιάστηκαν, μελετήθηκαν και υλοποιήθηκαν ειδικά για τη συγκεκριμένη εφαρμογή. Με τη χρήση της συσκευής, το βιολογικό φαινόμενο της πήξης του αίματος μετατρέπεται σε εύκολα μετρούμενο σήμα ηλεκτρικής τάσης. Η μετατροπή αυτή γίνεται με τη βοήθεια διάταξης επαγωγικών πηνίων (διέγερσης-λήψης), στο εσωτερικό των οποίων τοποθετείται μαγνητοελαστικό υλικό. Στην επιφάνεια του υλικού τοποθετείται σταγόνα (2 μl) τριχοειδικού αίματος. Η μεταβολή των ιξωδοελαστικών χαρακτηριστικών του δείγματος καθώς αυτό περνάει από την υγρή στη στέρεα φάση (θρόμβος), ανιχνεύονται μέσω της αντίστοιχης μεταβολής της διαπερατότητας του υλικού. Το αποτέλεσμα της μέτρησης είναι γράφημα τάσης-χρόνου. Από το γράφημα του κάθε δείγματος που μετρήθηκε με τη συσκευή κατά τη διάρκεια της κλινικής εφαρμογής του στο Ιπποκράτειο Νοσοκομείο Αθηνών, προέκυψε ένας πειραματικός χρόνος, ο χρόνος πήξης tπηξ. Ο χρόνος αυτός αποδείχθηκε ότι έχει στατιστικά σημαντική σχέση με τον εργαστηριακό χρόνο ροής ΒΤ (Bleeding Time) (p<0.01). Η πήξη του αίματος αποτελεί το σημαντικότερο κομμάτι του αιμοστατικού μηχανισμού του ανθρώπινου οργανισμού. Διαταραχές που σχετίζονται με δυσλειτουργίες του μηχανισμού αυτού θεωρούνται ιδιαίτερα κρίσιμες και απαιτούν άμεση διάγνωση και βέλτιστη θεραπευτική προσέγγιση. Υπό το πρίσμα αυτών των απαιτήσεων, η υλοποίηση της συγκεκριμένης αισθητήριας εφαρμογής προσβλέπει στη διερεύνηση μιας νέας απλής και οικονομικής μεθόδου για την εξέταση δειγμάτων αίματος, όσον αφορά την πήξη τους. / The present master thesis describes the principle of operation, the construction, and the clinical evaluation of a whole blood coagulation magnetostrictive sensor. The major parts of the setup where specially designed and constructed for the needs of the present implementation. The function of the sensor relates to the transformation of the biological process of blood, into an easy-to-measure voltage signal. This transformation is feasible with the placement of a magnetoelastic material inside a double coil setup (primary-secondary). A drop of capillary blood (2 μl) is placed on the surface of the material. Viscosity variations of the sample, while it passes from the liquid to the solid phase, are detected through the detection of the corresponding permeability variations of the material. The result of the measurements which took place at the Hippokratio Hospital of Athens, is a V(t) graph. For each sample which was measured, the corresponding graph was used to export the experimental value tcoag. This value was proved to have a statistically significant relationship with Bleeding Time (BT) (p<0.01). Blood coagulation is the most important part of the human hemostatic mechanism. The disorders relating to the dysfunction of this mechanism are considered critical and demand immediate diagnosis and optimum therapeutic approach. Under this view, the realization of the specific sensor apparatus targets to the investigation of a new simple and cost-effective method for blood coagulation testing.

Membrane fouling is a chronic problem for many nanofiltration (NF) membrane plants. Foulant material can range from colloidal, particulate, inorganic minerals and natural organic matter (NOM) (Schäfer et al., 2006). This research project worked with a small community integrated membrane facility (low-pressure membrane followed by high-pressure) in Nova Scotia with membrane fouling concerns associated with dissolved NOM as the primary foulant. Membrane autopsies conducted in our laboratory have demonstrated that NOM deposits on the NF membrane decreased pore space on the membrane (Lamsal et al., 2012). The membrane fouling resulted in a requirement for increased pressure to produce a constant permeate flow. By adding in-line coagulation prior to low-pressure filtration in an integrated membrane system, the goal was to remove more organic material by MF thereby improving the quality of the feed-water entering the NF membranes. Previous work has shown that for some IMS installations there is a need to reduce the amount of dissolved organic matter prior to NF (Cho et al., 2000; Lamsal et al., 2012; Nilson and DiGiano, 1996; Schäfer et al., 2001). An improved membrane feed-water quality reduces fouling on the membrane and membrane operating cost, and increases productivity and lifespan of the membrane (Choi, 2008). A negative aspect to adding in-line coagulation is it adds another step to the treatment process and sludge removal is required. This study examined the use of in-line coagulation using coagulants aluminum sulphate, ferric chloride and polyaluminum chloride to improve membrane feed-water quality. The addition of in-line coagulation prior to microfiltration will remove NOM with the MF producing improved feed water quality for NF. After determining the optimal dose of each coagulant, 20 L of post-coagulation MF permeate was batched and run through the bench-scale NF membrane for 200 hours. The water quality of the feed tank, concentrate and permeate were monitored constantly as well as the operational properties of pressure and flow. To simulate a full-scale plant the operating conditions of Collins Park water treatment plant on Fletchers Lake were used in the bench-scale set-up. After the 200h NF run time the membranes were analyzed to assess the fouling on the membrane and the performance of each coagulant. Coagulation was found to reduce NF pressure fouling by reduction of NOM in the NF feed-water. Ferric chloride was found to perform best of the three coagulants at a low dose of 0.5mg/L of Fe at a pH of 5.0. / n/a

The successful production of a wide range of polymer latex products relies on the ability to control the rates of particle nucleation, growth and coagulation in order to maintain control over the particle size distribution (PSD). The development of advanced population balance models (PBMs) has simplified this task at the laboratory scale, but commercialization remains challenging as it is difficult to maintain control over the composition (i.e. spatial distributions of reactant concentration) of larger reactors. The objective of this thesis is to develop and test a combined Computational Fluid Dynamics (CFD) -PBM hybrid modeling framework. This hybrid modeling framework can be used to study the impact of changes in process scale on product quality, as measured by the PSD. The modeling framework developed herein differs from previously-published frameworks in that it uses information computed from species tracking simulations to divide the reactor into a series of interconnected zones, thereby ensuring the reactor is zoned based on a mixing metric. Subsequently, an emulsion polymerization model is solved on this relatively course grid in order to determine the time evolution of the PSD. Examination of shear rate profiles generated using CFD simulation (at varying reactor scales) suggests that, dependent on conditions, mechanically-induced coagulation cannot be neglected at either the laboratory or the commercial scale. However, the coagulation models that are formulated to measure the contributions of both types of coagulation simultaneously are either computationally expensive or inaccurate. For this reason the decision was made to utilize a DLVO-coagulation model in the framework. The second part of the thesis focused on modeling the controlled coagulation of high solids content latexes. POLY3D, a CFD code designed to model the flow of non-Newtonian fluids, was modified to communicate directly with a multi-compartment PBM. The hybrid framework was shown to be well-suited for modeling the controlled coagulation of high solid content latexes in the laminar regime. It was found that changing the size of the reactor affected the latex PSD obtained at the end of the process. In the third part of the thesis, the framework was adapted to work with Fluent, a commercial CFD code, in order to investigate the scale-up of a styrene emulsion polymerization reaction under isothermal conditions. The simulation results indicated that the ability to maintain good control of the PSD was inversely related to the reactor blend time. While the framework must be adapted further in order to model a wider range of polymerization processes, the value of the framework, in obtaining information that would otherwise be unavailable, was demonstrated. / Thesis (Ph.D, Chemical Engineering) -- Queen's University, 2012-05-01 07:09:08.362

Background Several case reports and studies have alluded to an increased prevalence of venous thrombosis in human immunodeficiency virus positive (HIV-positive) patients. Although a relationship between HIV infection and thrombotic disease has been suggested, the mechanisms predisposing to thrombosis have not been fully elucidated. Aim A prospective study, to determine possible coagulation factor abnormalities that could explain the predisposition to thrombosis in HIV-infected African (Black) patients, was undertaken. Method African (Black) patients, with acute upper segment deep vein thrombosis (DVT) confirmed by duplex ultrasound, were enrolled. Patients who had recognisable risk factors such as recent surgery, pregnancy or malignancy, were excluded. After informed consent, blood samples were taken for baseline tests as well as a thrombophilia screen. The control group comprised known HIV-positive African (Black) patients without DVT. Patients with DVT who were found to be HIV-negative were also analysed. Analysis was done in 2 parts: HIV-positive patients with and without thrombosis and HIV-positive and negative patients with thrombosis were compared. Results Part A: HIV-positive patients with and without thrombosis Of the 77 patients with DVT, 50 patients tested HIV-positive. These 50 patients (HIV-positive DVT-arm), as well as 56 controls (HIV-positive, no DVT), were enrolled into the study. The groups were well matched with regard to age, sex and cluster designation 4 (CD4) count. On univariate analysis, significant findings in the DVT-arm were a history of active tuberculosis on treatment, low protein C levels and a positive qualitative D-dimer, whereas on multivariate analysis, only tuberculosis and an elevated D-dimer proved to be significant. Part B: HIV-positive and negative patients with thrombosis There were 20 HIV-negative patients with DVT who met our inclusion criteria Limited assessment was done on this group owing to unavailability of some data. The mean age of the HIV positive DVT group was significantly lower than the HIV-negative group with DVT (31.78 vs. 41.45 years; p=0.005). There was no significant difference in the prevalence of tuberculosis between the HIV-positive and HIV-negative patients with thrombosis (p = 0.269). Mean protein C levels were reduced in the HIV-positive group and normal in the HIV-negative group. They were significantly lower in the HIV-positive patients compared to the negative group (p=0.02). Conclusion The findings of the study suggest a relationship between HIV, its complications and DVT. Although this study confirms HIV infection as a risk factor for thrombosis, clear pathogenetic mechanisms remain to be elucidated. In our population, tuberculosis appears to be an important risk factor predisposing patients to the development of DVT, both in the HIVpositive and negative population. Further studies will need to be done to confirm this hypothesis. / Thesis (MMed)-University of KwaZulu-Natal, Durban, 2006.