A high degreee of structural and sequence homology exists between the EGF -1 domains of the vitamin K-dependent coagulation factors, as well as between the EGF-1 domains of individual vitamin K-dependent coagulation factors from various species. Through studies of protein evolution it has been determined the conserved amino acid residues observed are essential for protein structure while the variable residues have been implicated in specific protein-protein interactions. In the case of FVII, this interaction is the high affinity binding of its cofactor, TF, which initiates the extrinsic pathway of coagulation. 43% of the contact area of the FVII molecule in the FVIIa•TF complex is located within the FVII EGF-1 domain. A series of human FVII variants have been constructed in which the EGF-1 domain has been exchanged, either in its entirety or as single amino acid substitutions, with that of the mouse or rabbit. These species were chosen as it had previously been shown that plasma from mouse or rabbit, when combined with human TF, was able to clot at a significantly greater rate than homologous human plasma. We hypothesized that through these exchanges it might be possible to generate a human FVII variant with increased TF binding. Of the FVII variants generated, 2 human FVII point mutants have shown an increased affinity for human TF after transient expression. A75D and T83K exhibited TF binding at 200% and 150%, respectively, of that seen with wild-type human FVII. Both A75D and T83K exhibited clotting and amidolytic activity that was proportionally increased, with respect to TF binding. Computer-generated structures of these variants predict an additional hydrogen bond between the FVII and TF molecules likely to be responsible for the increased TF affinity in the T83K mutant. Intramolecular forces within the FVII molecule are predicted to have caused a conformational change with the A75D mutation which has lead to its increased TF binding. The greater affinity for TF exhibited by the human FVII point mutants A75D and T83K, as compared to wild-type human FVII, is an important step toward the creation of new class of competitive inhibitors of coagulation which are specifically directed towards its initial stage, rather than later in the cascade. However, more study is needed to determine the ability of either of these mutants to compete against each other for TF both 𝘪𝘯 𝘷𝘪𝘵𝘳𝘰 and 𝘪𝘯 𝘷𝘪𝘷𝘰. If either mutant is successful, it can then be active site inhibited by site-directed mutagenesis or through the use of chloromethyl ketones, in order to decrease its clotting and amidolytic activity. / Thesis / Master of Science (MSc)

Coagulation has and continues to be one of the most important elements in medicine. Issues from a lack of hemostasis range from poorer clinical outcomes to sudden death. The evolution of treatments for hemostasis have evolved from the use of Tamponade with direct pressure and bandages, the use of materials such as cobwebs and dust, the use of heat with hot oil or heated irons, to the use of suture, glues, plasmas, staplers, and electricity. This evolution has continued to bring about the prophylactic use of technology in an effort to prevent blood loss. This change from reactive treatments to proactive continue to be on a localized or superficial basis. One of the largest opportunities to proactively reduce blood loss in surgical patients is during the resection of solid organs such as the liver, kidney, and spleen. Few options have existed to help improve hemostasis short of the complete occlusion of blood supplying the tissue such as in the Pringle Maneuver. Recent studies have begun to show that practices such as this may have a significant detrimental effect on morbidity. It has been found that by applying radio frequency electrical energy in a particular way that large amounts of tissue can be pre-coagulated prior to resection. A series of animal and human clinical work has been completed to help evolve and confirm the method and the device that was created and refined during this effort. During the course of this work fifty-three patients were treated at four institutions on three continents. Average blood loss for liver resections performed with this pre-coagulation technique using the developed device in a multicenter control trail was 3.35 ml/cm2 as compared to 6.09 ml/cm2 (p < 0.05) for resections performed using standard surgical techniques alone. Additionally, the transection time necessary was also reduced from mean value of 27 minutes (2 -- 219 minutes) to 35 minutes (5 -- 65 minutes). Patients treated included those suffering from liver cirrhosis, fatty liver disease, and post chemotherapy fibrosis. From this work the use of pre-coagulation with methods and device developed was shown to be safe and effective for reducing the amount of blood loss and transection time during liver resections.

Coagulation has and continues to be one of the most important elements in medicine. Issues from a lack of hemostasis range from poorer clinical outcomes to sudden death. The evolution of treatments for hemostasis have evolved from the use of Tamponade with direct pressure and bandages, the use of materials such as cobwebs and dust, the use of heat with hot oil or heated irons, to the use of suture, glues, plasmas, staplers, and electricity. This evolution has continued to bring about the prophylactic use of technology in an effort to prevent blood loss. This change from reactive treatments to proactive continue to be on a localized or superficial basis. One of the largest opportunities to proactively reduce blood loss in surgical patients is during the resection of solid organs such as the liver, kidney, and spleen. Few options have existed to help improve hemostasis short of the complete occlusion of blood supplying the tissue such as in the Pringle Maneuver. Recent studies have begun to show that practices such as this may have a significant detrimental effect on morbidity. It has been found that by applying radio frequency electrical energy in a particular way that large amounts of tissue can be pre-coagulated prior to resection. A series of animal and human clinical work has been completed to help evolve and confirm the method and the device that was created and refined during this effort. During the course of this work fifty-three patients were treated at four institutions on three continents. Average blood loss for liver resections performed with this pre-coagulation technique using the developed device in a multicenter control trail was 3.35 ml/cm2 as compared to 6.09 ml/cm2 (p < 0.05) for resections performed using standard surgical techniques alone. Additionally, the transection time necessary was also reduced from mean value of 27 minutes (2 -- 219 minutes) to 35 minutes (5 -- 65 minutes). Patients treated included those suffering from liver cirrhosis, fatty liver disease, and post chemotherapy fibrosis. From this work the use of pre-coagulation with methods and device developed was shown to be safe and effective for reducing the amount of blood loss and transection time during liver resections.

Diss. (sammanfattning) Stockholm : Karol. inst., 2004. / Härtill 5 uppsatser.

Orientador: Erich Vinicius de Paula / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Ciências Médicas / Made available in DSpace on 2018-08-19T11:03:42Z (GMT). No. of bitstreams: 1 Quaino_SusanKellyPicoli_M.pdf: 2874691 bytes, checksum: aa96cc1f9d7979751213f3bda6ad0681 (MD5) Previous issue date: 2011 / Resumo: Pacientes com neutropenia febril apresentam risco aumentado de infecções severas e complicações da sepse. A ativação descontrolada da coagulação é uma das características mais marcantes da sepse. O quadro clínico e laboratorial mais característico desta ativação é chamado de coagulação intra-vascular disseminada. Do ponto de vista da fisiopatologia, a coagulação intravascular disseminada é caracterizada por ativação da coagulação pela expressão anômala intravascular de fator tissular, pelo consumo de inibidores naturais da coagulação e pela liberação excessiva de PAI-1, levando a hipofibrinólise. O quadro resultante destes processos é a hipercoagulabilidade. Acredita-se que parte das complicações da sepse, entre elas a chamada falência de múltiplos órgãos, seja decorrente de trombose de microvasos e isquemia tecidual. Assim, o estudo da coagulação intravascular disseminada tem grande relevância clínica. A avaliação laboratorial da hemostasia em pacientes com sepse e coagulação intravascular disseminada é limitada pelo fato de os testes disponíveis não ilustrarem de forma global e completa o resultado de todos estes processos na hemostasia. Além disso, é reconhecido que esta avaliação só é relevante se feita em mais de um momento ao longo da evolução do quadro na medida em que mais importante do que medidas isoladas é a tendência de mudança de variáveis como tempo de protrombina, dímeros D e fibrinogênio. Por este motivo, os chamados testes globais da hemostasia, capazes de avaliar de forma mais completa a interação de todos os processos citados acima, vêm ganhando importância nos últimos anos. Em pacientes com sepse, o teste de geração de trombina já foi avaliado em medidas isoladas, mostrando resultados que indicam lentificação do processo de ativação da coagulação. Estes resultados são distintos daqueles classicamente aceitos de hipercoagulabilidade durante as fases iniciais da sepse. No entanto, o número de estudos realizados nestes pacientes é escasso, sendo que em nenhum deles foi avaliada a variação temporal deste parâmetro. Em nosso estudo avaliamos parâmetros do teste da geração de trombina em pacientes com sepse e neutropenia febril. A avaliação foi feita no tempo basal, no momento da febre e após 48 horas. Além disso, avaliamos os parâmetros clássicos da hemostasia.. Nossos resultados contrariam a hipótese de presença de hipercoagulabilidade nas fases iniciais da sepse. Nenhum parâmetro do teste de geração de trombina mostrou-se capaz de segregar pacientes com maior risco de evolução para choque séptico / Abstract: Patients with febrile neutropenia are at increased risk of severe infections and complications of sepsis. The uncontrolled activation of coagulation is one of the most striking features of sepsis. The clinical and laboratory most characteristic of this activation is called disseminated intravascular coagulation. The pathophysiology of disseminated intravascular coagulation is characterized by activation of coagulation by anomalous intravascular expression of tissue factor, the consumption of natural inhibitors of coagulation and excessive release of PAI-1, leading to hipofibrinolysis. The net resulting picture of these processes is the hypercoagulability. It is believed that some of the complications of sepsis, including the so-called multiple organ failure, is due to thrombosis of microvasculature and tissue ischemia. Thus, the study of disseminated intravascular coagulation has great clinical relevance. Laboratory evaluation of hemostasis in patients with sepsis and disseminated intravascular coagulation is limited because the available tests do not depict in a comprehensive and completely way the results of all these processes in hemostasis. Moreover, it is recognized that this assessment is only relevant if done in more than one time-point along during disease progression to capture the trends of variables such as prothrombin time, D dimers and fibrinogen. For this reason, the so-called global tests of hemostasis, able to assess more fully the interaction of all the above processes are gaining importance in recent years. In patients with sepsis, thrombin generation test has been evaluated on single measures, showing results that show slowing the process of coagulation activation. These results are distinct from those classically accepted which assume that hypercoagulability would be present in early phases of sepsis. However, the number of patients in these studies is scarce, and none of them evaluated the temporal variation of this parameter. In our study we evaluated the test parameters of thrombin generation in patients with sepsis and febrile neutropenia. The evaluation was performed at baseline, at the time of fever and 48 hours thereafter. In addition, we evaluated classical parameters of hemostasis. Our results contradict the hypothesis of the presence of hypercoagulabilit in the early stages of sepsis and deserve further evaluation in larger studies. No parameter of thrombin generation was able to segregate patients with higher risk to progress to septic shock / Mestrado / Ciencias Basicas / Mestre em Clinica Medica

Many different stormwater control measures (SCMs) can be implemented in order to mitigate issues with polluted stormwater flows into receiving water bodies.  The treatment function of  SCMs is commonly based on the removal of particles by sedimentation, thereby also removing pollutants associated with particles. In recent years, more attention has been given to characterizing and understanding of different particle size fractions and their association with pollutants commonly found in stormwater. It has become increasingly clear that the smaller sized particles are very important pollutant transporters and should be considered when designing and implementing SCMs. However, the settling velocities for smaller sized particles are very low and may not be effectively removed in existing SCMs. One treatment process with a proven ability to enhance sedimentation is coagulation/flocculation, widespread in water and wastewater treatment, but with very few accounts of it being used in a stormwater context. This thesis aims to investigate the treatability of stormwater with a coagulation/flocculation process. This includes the determination of operating conditions, the dominating coagulation mechanism and the reduction efficiency of stormwater related pollutants. The objectives of the thesis were achieved in laboratory tests treating stormwater in a jar-testing procedure. An initial screening of primary coagulants and flocculant aids was conducted using an urban snowmelt mixture. Five of the chemicals were then selected for an extended testing regime which was setup up to determine the operating conditions where maximal turbidity reduction was attained by measuring the pH, conductivity, alkalinity and zeta-potential over the tested doses for each coagulant. Criteria used for chemical selection included high turbidity reduction, low dose requirement and low pH/alkalinity impacts. Charge reversal was observed at positive zeta-potential indicating that the dominating coagulation mechanism was charge neutralization. The content of turbidity/total suspended solids, total organic carbon, total metals and hydrocarbons by &gt;90%. Dissolved copper was reduced by 40% on average, and the reduction rates for dissolved zinc were varying with up to a 300% increase, presumably due to changes in pH, leading to a higher mobility. Changes in the particle size distribution after coagulation/flocculation as compared to sedimentation indicated an effect on the size fraction corresponding to smaller particles. The performance of the coagulation/flocculation process was also tested on road runoff collected from a central road in Luleå with a high traffic intensity. Two coagulants were tested, iron chloride and pre-hydrolyzed aluminum chloride. Reduction rates for the total metal fraction were &gt;90% on average for both coagulants, but for the dissolved metal fractions differences could be observed between the coagulants with the iron chloride resulting in higher reductions for dissolved chrome (57% compared to 34%) and copper (47% compared to 30%). Both products increased the dissolved fractions of nickel and zinc due to lower final pH.

published_or_final_version / abstract / Paediatrics and Adolescent Medicine / Master / Master of Philosophy

The application of microalgae for biofuel production is a subject of increasing interest as fuel prices continue to fluctuate and the United States aims to secure a reliable, domestic fuel source. Though microalgae have proven to be very efficient at producing oil-rich lipids, the optimum conditions for algae cultivation and methods for harvesting and oil extraction have not been determined. In particular, the harvesting component is especially important to the effectiveness of the overall process because of the large volumes of algae-rich water that must be processed, the strict requirements for downstream lysing, oil extraction and fuel production and the necessity to generate algae biomass with significant post-extraction byproduct value. A number of solid-liquid separation technologies have shown some potential for achieving microalgae/water separation; however, application of these processes to biofuel production requires an evaluation of treatment effectiveness as a function of water quality, algae particle characteristics, and process chemistry. The goal of this research was to identify and evaluate several potentially viable harvesting methods that could be incorporated into end-to-end algae to biofuel production. To achieve this goal, a literature review was conducted to identify the most promising harvesting methods for biofuel applications, and bench scale tests were performed for several harvesting processes. A number of significant findings were identified. Batch algae coagulation experiments with ferric chloride, chitosan, and pH-induced autoflocculation suggest that coagulants can provide effective treatment, but the effectiveness is dependent on water composition and pH. Electrocoagulation experiments indicated that dissolution of the sacrificial electrode led to high metal concentrations in the algae. Pre-oxidation with ozone increased the removal of freshwater Neochloris oleobundans by 20-80% after subsequent flocculation and sedimentation compared with non-ozonated samples. Most notably, this research identified the importance of optimizing water quality and algae particle characteristics for a particular algae harvesting process. Implementing a harvesting process that takes advantage of the natural constituents of a water and the surface characteristics of an algae culture, minimize treatment requirements and enables smoother integration with subsequent processing steps. / text

No description available.

Electrospinning has the capacity to create fibers of natural or synthetic polymers with dimensions that are similar to analogous fibers in native tissue. Mats consisting of fibers of these sub-micron dimensions have shown promise in provoking little immune response and in offering a habitable environment for cell proliferation. Fibrinogen is a natural protein capable of being electrospun and offers the benefit of existing as part of the natural coagulation cascade. Mats of fibrinogen could be utilized as possible hemostatic dressings or as an early scaffold for cell migration for either wound repair or tissue engineering. The addition of antibiotic into such a dressing/scaffold could prevent infection during healing/incorporation. The goal of this study was to determine any effect that the addition of the antibiotic tetracycline hydrochloride (0%, 2.5%, 5%, 10% by weight) would have on the mechanical properties of electrospun fibrinogen (110 mg/mL, 120 mg/mL, and 130 mg/mL concentrations). Also, the time release of tetracycline from electrospun fibrinogen was investigated. The results show no significant effect of tetracycline loading on the mechanical properties of electrospun fibrinogen under the conditions of this study. The results of the release study demonstrate that initial tetracycline release is dependent upon loading percentage. The release data also demonstrate that the amount of tetracycline released is approximately 20-30% of the tetracycline in the original solution and that the release occurs within approximately 4 hours, with no significant release thereafter. This study demonstrates the feasibility of tetracycline in electrospun fibrinogen for the purposes of short term drug release in fibrinogen-based technologies.