Characterization of a plasminogen activator from human melanoma cells cultured in vitro

In this thesis I describe the work that I have done on the isolation and characterization of a plasminogen activator, Mel-PA, that is released by human melanoma cells cultured in vitro. This enzyme was compared to the urinary plasminogen activator, urokinase. The human melanoma cell line, RPMI-7272, (also referred to as the "Bowes" melanoma cell line) released large amounts of Mel-PA into the surrounding medium when cultured under serum-free conditions. A subline of these cells (Bowes II) was developed that was capable of continuous growth in the absence of serum. These cells released only one type of plasminogen activator with a molecular weight of approximately 70 000 daltons. A technique was developed in which plasminogen activators were separated electrophoretically and detected in polyacrylamide gel slabs containing the co-polymerized substrates, plasminogen and gelatin. The technique was compared with the zymographic procedure developed by Granelli-Piperno and Reich (62) using fibrin-plasminogen-agarose underlays. Mel-PA was concentrated and partially purified by affinity chromatography on benzamidine-sepharose. This preparation was used to prepare rabbit antisera to the enzyme. These antibodies inhibited the activity of plasminogen activators released by all melanoma cells but had no effect on urokinase. Antibodies to urokinase had no effect on Mel-PA. A survey of human plasminogen activators and their distribution by immunochemical and electrophoretic techniques showed that tissue extracts and body fluids, with the exception of normal urine, contained mixtures of Mel-PA- and urokinase-type enzymes. Urine of patients with some types of renal disease also contained a Mel-PA type enzyme. A study of the distribution of plasminogen activators in tissues and body fluids obtained from a number of animals showed that all mammals examined had two immunochemically distinct plasminogen activators that corresponded, in their distribution, to the urokinase-like and Mel-PA-like enzymes of man. Antibodies to human Mel-PA cross-reacted with the corresponding enzyme in all mammals tested, whereas antibodies to human urokinase were species specific. The seeds of the South African legume, Erythrina latissima, contain a 20 000 dalton protein that functioned as an inhibitor of Mel-PA, plasmin, and trypsin, but had no effect on urokinase. During its reaction with the enzymes the inhibitor was cleaved by Mel-PA and trypsin but not by urokinase. The susceptible bond was straddled by an intrachain disulphide bridge. The inhibitor bound reversibly to Mel-PA and could therefore be used to develop an affinity reagent for a one-step purification procedure for Mel-PA in melanoma cell harvest fluids. Purified preparations of Mel-PA c0uld be shown to comprise both active enzyme (two chain form) and pro-enzyme (one chain form). The one chain form could be converted to the two-chain form by treatment with plasmin. It could also be shown that fibrinogen and fibrin contained a contaminating protease that was capable of converting pro-Mel-PA to Mel-PA. A comparative study of the kinetic behaviour of Mel-PA and urokinase showed numerous differences between the catalytic activities of these two enzymes. Mel-PA was capable of binding to fibrinogen insolubilized on a plastic surface whereas urokinase did not. The presence of fibrinogen enhanced the plasminogen activating activity of Mel-PA but had no effect on urokinase activity

Control Architecture Modeling for Future Power Systems

Uncontrollable power generation, distributed energy resources, controllable demand, etc. are fundamental aspects of energy systems largely based on renewable energy supply. These technologies have in common that they contradict the conventional categories of electric power system operation. As their introduction has proceeded incrementally in the past, operation strategies of the power system could be adapted. For example much more wind power could be integrated than originally anticipated, largely due to the flexibility reserves already present in the power system, and the possibility of interregional electricity exchange. However, at the same time, it seems that the overall system design cannot keep up by simply adapting in response to changes, but that also new strategies have to be designed in anticipation. Changes to the electricity markets have been suggested to adapt to the limited predictability of wind power, and several new control strategies have been proposed, in particular to enable the control of distributed energy resources, including for example, distributed generation or electric vehicles. Market designs addressing the procurement of balancing resources are highly dependent on the operation strategies specifying the resource requirements. How should one decide which control strategy and market configuration is best for a future power system? Most research up to this point has addressed single isolated aspects of this design problem. Those of the ideas that fit with current markets and operation concepts are lucky; they can be evaluated on the present design. But how could they be evaluated on a potential future power system? Approaches are required that support the design and evaluation of power system operation and control in context of future energy scenarios. This work addresses this challenge, not by providing a universal solution, but by providing basic modeling methodology that enables better problem formulation and by suggesting an approach to addressing the general chicken/egg problem of planning and re-design of system operation and control. The dissertation first focuses on the development of models, diagrams, that support the conceptual design of control and operation strategies, where a central theme is the focus on modeling system goals and functions rather than system structure. The perspective is then shifted toward long-term energy scenarios and adaptation of power system operation, considering the integration of energy scenario models with the re-design of operation strategies. The main contributions in the first part are, firstly, by adaptation of an existing functional modeling approach called Multilevel Flow Modeling (MFM) to the power systems domain, identifying the means-ends composition of control levels and development of principles for the consistent modeling of control structures, a formalization of control-as-a-service; secondly, the formal mapping of fluctuating and controllable resources to a multi-scale and multi-stage representation of control and operation structures; and finally the application to some concrete study cases, including a present system balancing, and proposed control structures such as Microgrids and Cells. In the second part, the main contributions are the outline of a formation strategy, integrating the design and model-based evaluation of future power system operation concepts with iterative energy scenario development. Finally, a new modeling framework for development and evaluation of power system operation in context of energy-storage based power system balancing is introduced.<br/

The penetration protease of the cercariae of Schistosoma mansoni

This thesis is concerned with a study of the proteases released by the cercariae of Schistosoma mansoni while penetrating mammalian skin. The proteases present in secretions collected from the preacetabular glands of cercariae were shown to be active against ¹²⁵I-labelled fibrin but not against undenatured ³H-collagen. A sensitive solid phase radioenzyme assay, with ¹²⁵I-fibrin as the substrate, was used to show that the cercarial protease could be totally inhibited by serine protease inhibitors such as diisopropylfluorophosphate or phenylmethylsulfonyl fluoride, but not by the sulfhydryl reagents iodoacetamide or p-chloromercuribenzoate. Typical trypsin inhibitors such as soy bean trypsin inhibitor, trasylol or benzamidine inhibited the enzyme to a lesser degree. The active-site labels, TLCK, TPCK and AcAAAACK of trypsin, chymotrypsin and elastase respectively had no effect. Calcium and magnesium stimulated protease activity at concentrations below 0,5 mM, but inhibited at higher concentrations, whereas EDTA had no effect. The pH optimum of the protease lay between pH 9,0 and 9,5. From these studies, I have concluded that the major cercarial penetration protease is an alkaline serine protease with trypsin-like specificity, but not acting via the same mechanism. A technique was developed for examining cercarial proteases in polyacrylamide gels containing SDS and copolymerized gelatin substrate. Bands of proteolytic activity could be detected by negative staining. This method was used to show that cercarial secretions contained one major protease with a molecular weight of 35 000 and that crude enzyme preparations are readily contaminated with bacterial proteases. Partial purification of the major cercarial protease was achieved by cation exchange chromatography