Ce mémoire de thèse présente deux nouvelles approches pour l’analyse et la commande des systèmes non-linéaires complexes, comme les systèmes dynamiques à commutation de la classe des convertisseurs d’énergie électrique. Ces systèmes ont plusieurs modes de fonctionnement et ont un point de fonctionnement désiré qui, en général, n’est le point d’équilibre d’aucun des modes. Dans cette classe de systèmes, la commutation d’un mode de fonctionnement à un autre est commandée selon une loi qui doit être synthétisée. Par conséquent, la synthèse de commande implique l’étude des conditions qui permettent à un cycle limite stable de s’établir au voisinage du point de fonctionnement désiré, puis de la trajectoire de commande qui permet de l’atteindre en respectant les contraintes physiques de comportement (courant maximum supporté par les composants,. . .) ou les contraintes de temps (durée minimum entre deux commutations,. . .). Le cycle limite sera qualifié d’hybride car il est composé de plusieurs dynamiques(deux dans ces travaux).La première méthode développée s’appuie sur les propriétés géométriques des champs de vecteurs et est une extension d’une partie des travaux de thèse de Manon au LAGEP. Une condition nécessaire et suffisante d’existence et de stabilité d’un cycle limite hybride composé d’une séquence de deux modes de fonctionnement dans IR2 est présentée. Ce cycle définit la région finale à atteindre par le système depuis son état initial, par une trajectoire déterminée de manière optimale selon un critère donné (durée totale, énergie dépensée, . . .). La méthode proposée est appliquée aux convertisseurs d’énergie Buck et Buck-Boost alimentant une charge résistive. Une extension à IRn a été proposée et démontrée. Elle est illustrée sur un système non-linéaire dans IR3.La deuxième méthode est développée dans IR2 et basée sur la théorie de Lyapunov, bien connue en automatique pour étudier la stabilité des systèmes non-linéaires et concevoir des commandes stabilisantes.Il s’agit de déterminer par une approche géométrique, une fonction de Lyapunov quadratique commune aux deux modes de fonctionnement du système, qui permette d’obtenir un cycle limite hybride stable le plus proche possible du point de fonctionnement désiré et une commande stabilisante directe des interrupteurs / This PhD-thesis presents two new approaches for the analysis and control of complex nonlinearsystems, such as switching dynamic systems of the class of power converters. These systems have severalmodes of operation and a desired operating point, which, in general, is not the equilibrium point of anymode. In this class of systems, switching from one mode to another is controlled by a switching law tobe designed. Therefore, the synthesis of a control law involves the study of the conditions that allow astable limit cycle to settle near the desired operating point, and of the control trajectory to reach thislimit cycle and stabilize on it, meeting the constraints dues to the physical behavior (maximum currentsupported by the components, . . .) or time constraints (minimum duration between two switchings, . . .).The limit cycle is called hybrid because it is composed of several dynamics (two in this work).The first method is based on the geometric properties of vector fields and is an extension of part ofthe PhD-thesis of Manon at LAGEP. A necessary and sufficient condition of existence and stability of ahybrid limit cycle consisting of a sequence of two operating modes, is presented in IR2. This limit cycledefines the final region to be reached by the system from its initial state, along a trajectory determinedoptimally according to a given criterion (total duration, energy expended, . . .). This method is applied tothe Buck and Buck-Boost power converters with a resistive load. An extension to IRn has been proposedand demonstrated. It is illustrated on a nonlinear system in IR3.The second method is developed in IR2, based on the Lyapunov theory, well known in automatic controlfor studying the stability of nonlinear systems and designing stabilizing control methods. The objective isto design, with a geometric approach, a quadratic Lyapunov function common to both modes of operation,which defines a stable hybrid limit cycle closest to the desired operating point and a direct stabilizingcontrol of the switches.

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Cell cycle regulation is an important part for the detection and destruction of mutated and dysregulated cells as it is a natural protection against degenerative diseases and cancer. The ability of the body to detect and destroy these cells is a vital part in maintaining homeostasis in the body. Once cells have circumvented this line of defence, dismantling these cells would become very difficult.  Research into new ways to target and destroy mutated cells are constantly evolving in hopes of being able to control and direct lysis of target cells using therapeutic drugs. One of the possibilities for such a method are Cholesterol Dependent Cytolysins (CDC), specific proteins found in bacteria. These proteins are dependent on the ability to bind to cholesterol in cell-walls to form pores that lyse and effectively destroy cells.   This project aims to study the structure and mechanistic details of pore formation by CDC mitilysin using cryogenic electron microscopy (cryo-EM). Mitilysin was purified by affinity chromatography and its pore formation ability was confirmed by calcein release assay and hemolysis assay. The pore structures of mitilysin were observed by transmission electron microscopy (TEM) using liposomes composed of both 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol as model membranes. Detergent screening directed separation of pores from liposomes; so that they could be visualized by cryo-EM. While these steps were optimized and proven successful, they were time-consuming. An initial 3D-model of pore-structures was rendered, but no molecular characteristics could be determined at the end of the allotted time. The study does lay the ground steps for obtaining the complete structure of mitilysin pores.

Humans have two plasminogen activators (PAs), tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), which generate plasmin to breakdown fibrin and other barriers to cell migration. Both PAs are used as pharmaceuticals but their efficacies are limited by their rapid clearance from the circulation, predominantly by parenchymal cells of the liver. At the commencement of the work presented here, the hepatic receptors responsible for mediating the catabolism of the PAs were little understood. tPA degradation by hepatic cell lines was known to depend on the formation of binary complexes with the major PA inhibitor, plasminogen activator inhibitor type-1 (PAI-1). Initial studies presented here established that uPA was catabolised in a fashion similar to tPA by the hepatoma cell line, HepG2. Other laboratories around this time found that the major receptor mediating the binding and endocytosis of the PAs is Low Density Lipoprotein Receptor-related Protein (LRP1). LRP1 is a giant 600 kDa protein that binds a range of structurally and functionally diverse ligands including, activated α2 macroglobulin, apolipoproteins, β amyloid precursor protein, and a number of serpin-enzymes complexes, including PA??PAI-1 complexes. Further studies for the work presented here centred on this receptor. By using radiolabelled binding assays, ligand blots, and Western blots on cultured cells, the major findings are that: (1) basal LRP1 expression on HepG2 is low compared to a clone termed, HepG2a16, but appears to increase in long term culture; (2) a soluble form of LRP1, which retains ligand-binding capacity, is present in human circulation; (3) soluble LRP1 is also present in cerebral spinal fluid where its role in neurological disorders such as Alzheimer??s disease is a developing area of interest; and (4) the release of LRP1 is a mechanism conserved in evolution, possibly as distantly as molluscs. The discovery, identification, and characterisation of soluble LRP1 introduces this protein in the human circulation, and presents a possible further level of regulation for its associated receptor system.

Viruses are entities which are made of a few genes and are reliant on obligate parasitism to propagate. Due to the obligate connection to their hosts, virus evolution is constrained to the type of host. Viruses however do transmit to evolutionary distinct hosts; in these cases, the phylogenetic relationship of the hosts usually are close. In some instances, RNA-viruses have made host jumps between evolutionary distant hosts, such as the host jump from invertebrates to vertebrates, and fungi to arthropod. Partitiviruses are double stranded RNA viruses which mainly infect fungi and plants. The defining characteristic of these double stranded RNA viruses are the double layered capsids which are formed by a single open reading frame (ORF). The capsid proteins form icosahedral virus particles which are in the magnitude of 30-40 nm. Metagenomic studies have discovered partitiviruses originating from an insect in the Odanata family, a finding which contradicts the fungal host specificity of partitiviruses. The finding of the Hubei.PLV 11 thus implies the existence of a partitiviruses containing structural elements in their capsids which could be involved in the infection of arthropods. Thus, this virus could be used as a model for a structural comparison with its fungi infecting relatives with hopes to identify common viral structural factors necessary for the infection of arthropods. For this purpose, the Hubei.PLV ORF was cloned and then transfected into insect Spodoptera frugiperda (Sf-9) cells using a baculovirus expression system, “bac-to-bac” expression system. The FLAG-tagged capsid proteins were expressed by the Sf-9 cells to be approximately 60 kDa. After ultra-centrifugation in a sucrose gradient, some spontaneous assembly into the expected ~40 nm icosahedral virus-like particles were observed using low resolution scanning electron microscopy. The observed particles were also confirmed by a dynamic light scattering experiment (DLS) and a higher resolution cryo-EM microscope. Thus, the bac-to-bac expression system can be used to produce VLPs from this genus of viruses, and this metagenomically derived virus genome. However, for future success in defining a high-resolution model of this virus, it is recommended that the Sf-9 culture volume is sufficiently high for enough particle production which is necessary for a high-resolution map. The other virus, the Southern pygmy squid Flavilike virus (SpSFV) has been suggested to be the oldest relative of the land based flaviviruses. The SpSFV was found to be the most divergent of the flaviviruses, and to infect invertebrates. Solving for the structure of the SpSFV and comparing it to vertebrate infecting flaviviruses could therefore lead to the identification of factors necessary for the adaptation to vertebrates and thus the humoral immunity by flaviviruses. The soluble E-protein was expressed using the bac-to-bac expression system. The protein was indicated to be multiglycosylated and approximately 50 kDa which is in line with other strains in the genus. Affinity chromatography did not elute this protein, likely due to the His-tag not being spatially available. Cation exchange could elute some protein, but not much from the small ~30 mL culture. To conclude, VLP assembly was confirmed by the Hubei.PLV, thus, solving for the structure is a distinct possibility when a larger Sf-9 culture is used to produce the VLPs. For the SpSFV soluble E-protein, the protein is secreted into the supernatant of the Sf-9 cultures, making purification a possibility. For this, a large Sf-9 culture can be used to produce this protein and then purify it with a cat-ion exchange chromatography.

The theoretical part of work is focused on the issue of biomass which can be used for energy purposes, inparticular agricultural waste, as well as can serve as a substrate for biogas station. It also deals with proteomics, its goals and approaches, separation methods. The aim of this work was to measure each sample of enzyme activity of biomass, which are used as a raw materials for biogas plants and their proteomic identification.

F1FO-ATPsynthase is a key enzyme in energy metabolism of the cell. Its deficit is caused usually by mutations in two structural genes MT-ATP6 and MT-ATP8 encoded by the mitochondrial DNA or in nuclear genes ATPAF2 and TMEM70 encoding the biogenesis factors and structural gene ATP5E. Deficiency of the F1FO-ATPsynthase leads to progressive and serious phenotype affecting organs with high energy demands. The first symptoms usually occurs in neonatal age and prognosis of the disease is fatal. Mutations in these genes result in both qualitative and quantitative defects of the F1FO-ATPsynthase. The study of molecular bases of mitochondrial disorders including F1FO-ATPsynthase deficiency uses large number of biochemical and molecular-genetic methods to determine a proper diagnosis which is essential for the symptomatic therapy and genetic counselling in affected families. The aim of the diploma thesis was to characterise the F1FO-ATPsynthase deficiency in isolated mitochondria from the lines of cultured cells by the determination oligomycin- sensitive ATP-hydrolytic activity of the F1FO-ATPsynthase, enzymatic activities of the respiratory chain complexes and to analyse changes in the steady-state levels of the representative subunits and whole complex of the F1FO-ATPsynthase in comparison with controls. 3...