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Aqueous Phase Partitioning has a long history of applications to the analytical characterisation of biomolecules. However process applications have attracted the most interest in biotechnology where it has become widely recognized as a cost-effective technique. The main aim of this work was to explore the proposition that partition in Aqueous Two Phase Systems (ATPS) can be used as an analytical tool to detect protein isoforms and to assess the applicability of the method in clinical assays and for quality control in bioprocessing through examination of several analytical problems. The work also examined the development of automated methods of system preparation and sampling techniques to determine the partition coefficient in ATPS. The study demonstrated that the geometrical form of the phase diagram co-existence curve was of crucial importance since this directly affected the accuracy with which systems of defined Tie Line Length and Mass Ratio could be constructed. The TLL %Bias (accuracy) of a theoretical system range in the PEG1000-(NH4)2SO4 system at shorter TLL (12.2) was in the range +80.6% to -100% while at a longer TLL (53.1) the %Bias (accuracy) was reduced to +0.1% to -1.9%. At the same time the MR %Bias (accuracy) at shorter TLL (12.2) was in the range +59.5% to -21.3% while at the longer TLL (53.1) this was reduced to +2.7% to -2.6%. By contrast in the PEG8000-Dextran500 system the TLL %Bias (accuracy) at shorter TLL (13.1) was in the range +3.7% to -4.12%, while at a longer TLL (31.1) the range was +0.74% to -0.67%. The MR %Bias (accuracy) at the shorter TLL (13.1) was in the range +3.6% to -3% while at the longer TLL (31.1) the range was +1.1% to -1.4%. This illustrated that it is more difficult to work with a high degree of accuracy (e.g. %Bias <5%) close to the critical point in PEG-salt systems than in PEG-dextran systems. Two different approaches were taken to examine analytical phase partitioning. In the first approach the structure of the isoforms of a model protein (ovalbumin) were altered enzymatically. Analytical methods involving Strong Anion-Exchange chromatography were developed and applied to the separation of the ovalbumin isoforms. Removal of the phosphorylated groups (dephosphorylation of ovalbumin) was undertaken using alkaline phosphatase and de-glycosylation was attempted using neuraminidase and Endo-glycosidase F. However, both enzymatic approaches to deglycosylation were unsuccessful. Dephosphorylated isoforms were successfully produced and characterised. After partitioning in ATPS a clear difference was demonstrated between the behaviour of the native and dephosphorylated forms of ovalbumin. The mean % recovery in a PEG-salt ATPS was 99.8% (± 3.59) for the naive protein and 75.6% (± 4.03) for the dephosphorylated form. On the other hand, in a PEG3350-Dextran500 system, where solubility was maintained, a significant difference in the partition coefficient (K) of native and dephosphorylated ovalbumin was found. K for native ovalbumin was 0.85 while the partition coefficient of the dephosphorylated ovalbumin was 0.61. Analysis of covariance (ANCOVA) indicated that the regression coefficients of the respective partition isotherms were significantly different (p value < 0.05). In a second approach to examine analytical phase partitioning, chemical modification of a specific target surface amino acid of another model protein (serum albumin) was used to determine the degree of conjugation of the protein and also to determine its oxidative state. The method examined the reactivity of a free surface thiol to a wide range of labels ( (a) 2-methylsulfonyl-5-phenyl -1,3,4 oxidiazole reagent, (b) N-Ethylmaleimide (NEM) reagent, (c) 5, 5’-dithiobis (2-nitrobenzoate)(DTNB) (Ellman’s reagent), (d) N-pyrenylmaleimide (NPM) reagent, (e) Fluorescein-5-maleimide (F-5-M) Reagent). Only DTNB was found to modify the surface free thiol of serum albumin in a highly specific and quantitative manner. In the course of the development of a partitioning assay for surface free thiols of serum albumin significant oxidative properties were found to be associated with poly(ethylene glycol) PEG solutions and several attempts were made to find an oxidatively safe partitioning system by including antioxidants and by removal of contaminants by freeze drying. PEG3350-Dextran500 was found to provide an oxidatively safe environment for the development of a partitioning assay for the determination of albumin free thiols. A phase partitioning assay system capable of quantitatively resolving protein associated free thiols and low molecular weight thiols from a mixture of the two was developed. Correlation coefficients (R2) for the regression of experimentally determined protein free thiols in the presence of different levels of added LMW free thiol on the known addition of protein ranged from 0.77 to 0.83. The results demonstrated that the assay could quantify and distinguish both types of thiol in a simple two-step procedure.

Cette thèse contribue à répondre au problème de la gestion optimale des ressources en eau dans les réseaux de navigation intérieure du point de vue de la théorie du contrôle. Les objectifs principales à atteindre consistent à garantir la navigabilité des réseaux de voies navigables, veiller à la réduction des coûts opérationnels et à la longue durée de vie des équipements. Lors de la conception de lois de contrôle, les caractéristiques des réseaux doivent être prises en compte, à savoir leurs dynamiques complexes, des retards variables et l’absence de pente. Afin de réaliser la gestion optimale, le contrôle efficace des structures hydrauliques doit être assuré. A cette fin, une approche de modélisation orientée contrôle est dérivée. Cependant, la formulation obtenue appartient à la classe des systèmes de descripteurs retardés, pour lesquels la commande prédictive MPC et l’estimation d’état sur horizon glissant MHE peuvent être facilement adaptés à cette formulation, tout en permettant de gérer les contraintes physiques et opérationnelles de manière naturelle. En raison de leur grande dimensionnalité, une mise en œuvre centralisée n’est souvent ni possible ni souhaitable. Compte tenu du fait que les réseaux de navigation intérieure sont des systèmes fortement couplés, une approche distribuée est proposée, incluant un protocole de communication entre agents. Malgré l’optimalité des solutions, toute erreur peut entraîner une gestion inefficace du système. Par conséquent, les dernières contributions de la thèse concernent la conception de stratégies de supervision permettant de détecter et d’isoler les pannes des équipements. Toutes les approches présentées sont appliquées à une étude de cas réaliste basée sur le réseau de voies navigables du nord e la France afin de valider leur efficacité. / This thesis addresses the problem of optimal management of water resources in inland navigation networks from a control theory perspective. The main objectives to be attained consist in guaranteeing the navigability condition of the network, minimizing the operational cost and ensuring a long lifespan of the equipment. However, their complex dynamics, large time delays and negligible bottom slopes complicate their management. In order to achieve the optimal management, the efficient control of the hydraulic structures must be ensured. To this end, a control-oriented modeling approach is derived. The resulting formulation belongs to the class of delayed desciptor systems, for which model predictive control and moving horizon estimation can be easily adapted, as well as being able to deal with physical and operational constraints in a natural manner. However, a centralized implementation is often neither possible nor desirable. As these networks are strongly coupled systems, a distributed approach is followed, featuring a communication protocol among agents. Despite the optimality of the solutions, any malfunction can lead to an inefficient system management. Therefore, the last part of the thesis regards the design of supervisory strategies that allow to detect and isolate faults. All the presented approaches are applied to a realistic case study based on the inland navigation network in the north of France to validate their effectiveness.