L'angiogenèse est une étape clef dans le processus de progression tumorale. Elle est caractérisée par une surexpresssion d'un grand nombre de métalloprotéinases matricielles (MMP). Parmis ces MMP, les gélatinases (MMP-2 et MMP-9) sont connues pour jouer un rôle important dans l'angiogenèse tumorale et la croissance de nombreux cancers. Les "Photodynamic Molecular Beacon" (PMB) sont des constructions moléculaires qui peuvent être utilisées dans le traitement de cancers en associant un photosensibilisateur (PS) de type chlorine et un inhibiteur d'états excités, aussi appelé "Quencher", liés par un peptide substrat des gélatinases afin d'inhiber la toxicité du PS dans les cellules non ciblées, et de restaurer sa toxicité uniquement à proximité des gélatinases. Nous avons donc cherché à déterminer le couple PS/quencher permettant la meilleure inhibition de la production d'oxygène singulet, principale source de la toxicité du PS, puis avons synthétisé une famille de PMB ciblant les gélatinases. Différents peptides et bras espaceurs ont été utilisés pour évaluer l'influence de la distance entre le PS et le quencher sur les propriétés photophysiques et l'activation enzymatique du PMB / Angiogenesis is a key step in the tumoral progression process. It is characterized by an over-expression of a number of matrix metalloproteinases (MMP). Among these MMPs, gelatinases (MMP-2 and MMP-9) are known to play a critical role in tumor angiogenesis and the growth of many cancers. Photodynamic Molecular Beacons (PMB) can be designed for cancer treatment by associating a chlorin-like photosensitizer and a black hole quencher linked by a gelatinase substrate peptide with the aim of silencing photosensitizer toxicity in non-targeted cells and restore its toxicity only in surrounding gelatinases. We investigated the PS/quencher pair allowing the best singlet oxygen production inhibition, and then we synthesized a novel family of PMB triggering gélatinases MMP-2 and MMP-9. Different lengths of peptide and spacers were used in order to determinate the influence of the distance between PS and quencher on the PMB photophysical properties and enzymatic activation.

In this thesis, a review is given on the most popular polymers used today for polymer modification of bitumen. Furthermore, the development of a model for phase separation in polymer modified bitumen (PMB) is proposed, that will enable a better control and understanding of PMB phase behaviour, allowing thus to enhanced long-term performance. PMB is hereby considered as a blend and focus is placed on its structure, its equilibrium thermodynamics and its phase separation dynamics. The effects of dynamic asymmetry on phase separation in PMB are analysed with related theories and some image data. Based on the discussion in this thesis, it is concluded that the effects of dynamic asymmetry between bitumen and polymer should be taken into consideration when studying phase separation in PMB. By analysing related literature and image data, it is found that some features of viscoelastic phase separation are shown during the phase separation process in some PMBs. It is therefore possible and useful to develop a viscoelastic model for PMB to describe its phase separation behaviour. In this, the stress-diffusion coupling is expected to play a key role in the model. Finally, recommendations are made towards the future research which is needed to realize the proposed model. / <p>QC 20150409</p>

No description available.

La thérapie photodynamique (PDT) est une technique utilisée cliniquement pour traiter certaines maladies de la peau, la dégénérescence maculaire liée à l’âge et certains types de cancer. Elle fait intervenir trois composants : une molécule photosensible ou photosensibilisateur (PS), la lumière et l’oxygène. Après administration du PS, celui-ci va se localiser plus ou moins sélectivement dans les zones tumorales où il est alors activé par irradiation lumineuse à une longueur d’onde et une puissance données. Ceci engendre la formation d’espèces réactives de l’oxygène (ROS) très réactives, dont l'oxygène singulet1 O2, qui entraînent la destruction des tissus tumoraux par nécrose ou apoptose. Afin d’améliorer la sélectivité du traitement, différentes pistes sont actuellement exploitées dont l’élaboration de « photodynamic molecular beacons » (PMB). Dans un PMB, le photosensibilisateur (PS) est associé via un peptide à un inhibiteur 1O2, appelé quencher. Ce quencher inhibe la formation d’1O2 tant que le composé n’a pas atteint sa cible. Une fois la zone cancéreuse atteinte, des enzymes spécifiques clivent le peptide, libérant ainsi le PS qui retrouve alors sa capacité à former de l’1O2. Trouver un couple PS/quencher adéquat reste un challenge en PDT. Les propriétés photophysiques particulières des caroténoïdes et leur aptitude à inhiber la production d’1O2 font de ces derniers des quenchers potentiellement utilisables pour l’élaboration de PMBs. Chez les plantes, les caroténoïdes (carotènes et xanthophylles) sont des pigments associés à la photosynthèse, qui ont deux rôles principaux : un rôle de collecteur de lumière et un rôle photoprotecteur en protégeant le(s) système(s) photosynthétique(s) contre les dommages photooxydatifs liés à une exposition trop intense à la lumière. Ceci s’opère, entre autre, via le cycle des xanthophylles. Cette aptitude à capter de l’énergie présente un intérêt potentiel à ne pas négliger dans la perspective de la conception de PMB utilisables en thérapie photodynamique. Dans le cadre de cette thèse en co-tutelle avec la Faculté des Sciences de Bizerte nous avons ciblé les caroténoïdes présents dans trois fruits produits en Tunisie à savoir les kakis (Diospyros kaki L.), les abricots (Prunus armeniaca L.) et les pêches (Prunus persica L.) connus pour leur richesse globale en ces pigments. Divers procédés d’extractions ont été étudiés : (i) L’extraction de type Soxhlet par solvants organiques à pression atmosphérique, utilisée comme référence, (ii) l'extraction accélérée par solvant organique (ASE : Accelerated solvent Extraction) effectuée sous pression, enfin (iii) l'extraction par CO2 supercritique avec l’éthanol comme cosolvant. Pour ces deux derniers procédés, une approche par plan d’expériences (surfaces de réponses) a été utilisée pour identifier les facteurs clé et les conditions optimales d’extractions de divers caroténoïdes (pression, température, débit, % de cosolvant, temps, nombre de cycles). L'analyse par chromatographie liquide à haute performance couplée à la détection UV-Visible et à la spectrométrie de masse a ensuite permis l'identification et la quantification des caroténoïdes présents dans les extraits obtenus, permettant ainsi de comparer les profils caroténoïdiques propres à chaque fruit et les performances de chaque procédé d’extraction. Cette étude ayant révélé un profil caroténoïdique particulièrement intéressant chez le kaki par rapport aux autres fruits, une extraction et une purification des caroténoïdes de ce fruit par chromatographie liquide haute pression préparative a ensuite été effectuée afin de disposer d’une quantité suffisante de chaque caroténoïde, et parfois de leurs isomères conformationnels, en vue de l’étude de leurs propriétés photophysiques (absorption, émission de fluorescence, inhibition d’1O2) et de l’évaluation de leur intérêt potentiel en tant que quencher d’1O2 dans un édifice de type PMB / Photodynamic therapy (PDT) is a clinically used technique for treating skin diseases, age-relatedmacular degeneration but mainly some types of cancer. PDT involves three components: a photosensitive molecule named photosensitizer (PS), light and oxygen. After administration of the PS, this one will be located more or less selectively in tumoral regions where it is activated by light irradiation at appropriate wavelength and power. This leads to the formation of highly reactive and cytotoxic reactive oxygen species (ROS), especially singlet oxygen, resulting in the destruction of the tumor by necrosis or apoptosis. To improve the treatment selectivity, different strategies are being exploited, one of which is the development of "photodynamic molecular beacons" (PMB). In PMB the photosensitizer is linked via a peptide to an inhibitor of 1O2 (quencher). This quencher inhibits the formation of 1O2 as long as the compound has not reached its target, namely cancer cells. In order to inhibit the toxicity of the PS in non-target cells and restore toxicity only close to the biological target, it is necessary to find an adequate PS/quencher couple. This remains a challenge for PDT. Carotenoids are interesting candidates due to their specific photophysical properties and ability to inhibit 1O2, which makes them potential quenchers for building PMBs. In plants, carotenoids (carotenes and xanthophylls) are pigments involved in the photosynthesis, in which they play two main roles: a light collecting role and a protecting role by preserving the photosynthetic systems against photoxydative damages induced by a too intense light exposure. This protection can for instance occur via the well-known xanthophylls cycle. This capacity to catch energy presents a potential interest that should not be neglected in the framework of the design of PMBs usable in photodynamic therapy. Within the framework as part of this PhD thesis in Cotutelle with the Faculty of Sciences of Bizerte, we focused on carotenoids from three fruits produced in Tunisia: persimmon (Diospyros kaki L.), apricot (Prunus armeniaca L.) and peache (Prunus persica L.), known for their global richness in these natural pigments. Three extraction processes were investigated: (i) the Soxhlet extraction based on the use of organic solvent at atmospheric pressure and used as reference, (ii) the accelerated solvent extraction (ASE) using organic solvent under high pressure, and (iii) the supercritical fluid extraction (SFE) using supercritical CO2 and ethanol as cosolvent. For these two last processes, a design of experiments (Surface Response Design) was used to identify the key factors and optimal extraction conditions of various carotenoids (pressure, temperature, flow, % cosolvent, time, number of cycles). Then, HPLC-PDA coupled with mass spectrometry (MS) enabled the identification and quantification of carotenoids from the extracts. Thus it was possible to compare the profiles in carotenoids content from each fruit as well as the performances of each extraction process. This study showed that the carotenoidic profile in the persimmon was the most interesting as compared to the profiles in the two other fruits. Extraction and purification of the carotenoids from persimmon by preparative high pressure liquid chromatography were then performed in order to have a sufficient amount of each carotenoid and sometimes of their conformational isomers. We finally performed a study of their photophysical properties (absorption, fluorescence emission, 1O2 inhibition) in order to evaluate their potential as 1O2 quencher in molecular construction such as a PMB

This diploma thesis deals with a project of asphalt mixtures with high stiffness modulus using R-material. The theoretical part describes the characteristics of these mixtures in the Czech Republic and in Switzerland. Further this part deals with the use of R-material in the Czech Republic and chosen other countries of the world, it also deals with the ways of recycling roads. The theoretical part also deals with the testing methods used. In the practical part two new asphalt mixtures of the high stiffness modulus type with the addition of 25 % R-material are suggested. As a base for these two mixtures a mixture of the high stiffness modulus type, which was taken from an asphalt mixing plant, was used. Tests were run on the binders and several functional tests (stiffness modulus, fatigue resistance, low temperature qualities) wer done on the suggested asphalt mixtures and also the mixture taken from the mixing plant. Consequently, the results were compared. The conclusion of the thesis is devoted to modelling a construction of a road using the program LayEps, which verified if the suggested mixtures could reduce the thickness of roads.

The diploma thesis is focused on the design and production of two innovative asphalt mixtures of the VMT 22 type into the base asphalt course. The newly produced blends contain modified binders, such as polymer modified bitumen (PMB) and Crumb rubber modified bitumen (CRMB). The theoretical part of the thesis describes the current state of the use of Reclaimed asphalt pavement in the base asphalt course. Furthermore, there are described types of asphalt mixtures for the base asphalt course are used in the Czech Republic and other countries. The conclusion of the theoretical part is devoted to the description of Reclaimed asphalt pavement (RAP), its storage and processing in a hot mix center. In the practical part of this thesis, there is described design and test of two High modulus asphalt mixture VMT 22, into which 25% RAP is dosed cold without rejuvenators. These asphalt mixtures are then subjected to functional tests (Stiffness of asphalt mixture test, Resistance to fatigue test, Low temperature properties of asphalt mixture test). The results of the functional tests are compared with the functional requirements in the Czech regulation TP 151. In the end of this thesis it is verified by means of the LayEps software that a suitable design of the asphalt mixture can significantly extend the road life.

Endotoxin or Lipopolysaccharide (LPS) is the invariant structural component of gram negative bacterial outer membranes and is the chief causative factor of Sepsis or endotoxic shock. Sepsis is a syndrome that has very high mortality rates even in this age of excellent therapeutics and critical patient care. The treatment for sepsis till date remains nonspecific and supportive due to lack of effective anti-endotoxic drugs. Sepsis is initiated when the circulating bacteria shed LPS from their cell envelopes. Shed LPS aggregates are recognized by LPS binding proteins and receptors, which activate the host's immune system. Uncontrolled and excessive stimulation of the host's immune system precipitates endotoxic shock which in advanced cases involving multiple system organ failure inevitably lead to patient's death. Many strategies have been tested out to combat this deadly affliction. One of the attractive clinical modalities in sepsis treatment is the use of peptides as LPS sequestering anti-endotoxic drugs. A classical peptide antibiotic of this class is Polymyxin B (PMB) a cyclic cationic acylated molecule, that recognizes LPS with a very high affinity. This thesis describes kinetics and thermodynamics of PMB-LPS interactions and applies these parameters over a framework of different models so as to gain insights into the structure-function relationships that govern the interactions of this peptide with endotoxin(s). Classical biophysical techniques like fluorescence, circular dichroism spectroscopy, stopped flow kinetics, titration calorirnetry (ITC) and the relatively new technique of Surface Plasmon Resonance (SPR) have been employed to dissect out the mechanism of the range of non-covalent forces that are involved in peptide-endotoxin recognition. Certain proteins that exhibit LPS binding activity have also been studied to gains insight about their mode of action. Implications of these studies for designing peptides that have better anti-endotoxic properties are also highlighted. The first chapter introduces and highlights the clinical features of sepsis. It also attempts to shed light on the LPS mediated signal transduction pathway that leads to endotoxic shock. This chapter also briefly explains the roles of many LPS receptors that are present in the human system and their specific roles in the signal transduction pathways. The second part of this chapter deals with the role of cationic peptides as anti-endotoxic drugs. Certain key functional aspects of these peptides, which impart in them, the desirable property of LPS recognition have also been discussed The second chapter describes the kinetic studies undertaken to unravel the exact mechanism of LPS-PMB interaction. The studies reveal that PMB recognizes LPS in a biphasic manner, with the second, unimolecular isomerization step of the reaction being the rate-limiting step. The initial reaction is shown to be influenced by the presence of salt in the reaction medium. The dissociation phase of this interaction also shows a biphasic pattern. These data allow us to speculate upon the exact mechanism by which PMB is able to recognize LPS. The studies also shed light on some structural aspects that govern and confer such high LPS binding activity to PMB. Based on these a model has been proposed to explain this recognition (C.J. Thomas et al, 1998). The second chapter discuses the mode of action of various PMB analogs. These analogs have been chosen in terms of their mode of action as well as their structural similarly to PMB. The affinities of these analogs to LPS and lipid A were quantified using the Surface plasmon resonance (SPR) method. SPR, a technique that relies on the quantification of change in mass during a binary binding process occurring between an immobilized entity and a flowing ligand, is a rapid and sensitive method to measure biologically relevant interactions. SPR studies provide us with the binding constants and thermodynamic parameters that allow evaluation of the affinities of these peptides towards LPS (C.J.Thomas and A.Surolia, 1999). The third chapter discusses a hitherto unknown mode by which PMB acts on a LPS lamellae. The results of this study wherein the binding affinities of PMB and its analogs were performed on monolayers and tethered liposomes, show that PMB is able to remove specifically LPS or lipid A from monolayers or bilayer assemblies such as tethered liposomes. The exact mode of action of PMB is deciphered in the light of these new studies, which allow us to posit on the observed efficacy of PMB in neutralizing the endotoxin as compared to peptides with nearly similar affinities for LPS (C.J Thomas et al 1999). In the fourth chapter a series of 23 residue peptides, based on the sequence corresponding to the anti-sense strand of magainin gene have been synthesized. Magainin an amphiphilic helical peptide obtained from frog skins plays a vital role in the innate immune defense mechanisms of these organisms. It also exhibits LPS binding activity that makes it an attractive target as an anti-endotoxic drug. Biochemical and biophysical characterization of these peptides reveal that they have the tendency to perturb both the inner and the outer membranes of E.coli. The peptides are amphiphilic and have helical structure in a membrane bound environment. Three of the peptides tested have high affinities for lipid A that approach the values shown by PMB. The kinetic parameters obtained by stopped flow and SPR studies in conjunction with the therrnodynamic parameters obtained using ITC studies allow us to highlight the key structural features that need to be exhibited by peptides that are designed to be LPS recognizers. The studies also project the fact that ionic forces play an important role in the initial recognition of LPS by these peptides. Fortification of the might of these ionic charges increases affinity for LPS where as the hydrophobic residues that interact at the next phase of binding are more amenable to disruptions in contiguity. These factors are discussed using the helical wheel diagram that shows the clear amphiphilicity displayed by these peptides. (C.J Thomas et al Manuscript under preparation, 2000) Chapter six discusses the mode of action of certain LPS binding proteins. Limulus anti endotoxic factor (LALF) plays a vital role in the innate immune based defense systems of the horseshoe crab. Galectin-3 is a metal ion independent, galactosc binding Icctin of human origin with unknown functions. Both these phylogcntically-unrclatcd proteins exhibit LPS/lipid A recognizing properties. ITC and SPR studies have been used to determine the binding constants displayed by these proteins for lipid A. LALF bind to lipid A with very high affinity than compared to Galectin-3 and is also able to take away selectively lipid A from both monolayers and tethered liposomes. Galectin-3 does not show this property of LALF, which might account for its lowered affinities. Also structurally LALF has amphiphilic nature that confers high lipid A binding activity, which is clearly lacking in Galectin-3. These studies in conjunction with the knowledge gained from the study of LPS-PMB interaction stress on the importance of amphiphilicity in LPS recognition. (C.J Thomas et al Manuscript under preparation, 2000). The final chapter is a general discussion that attempts to collate all these kinetic and thermodynamic observations in the pursuit of designing small easily manipulatable peptides that exhibit high LPS binding activity. These studies are aimed to act as rough guidelines to the design of LPS sequestering peptides that might have better therapeutic and pharmacokinetic properties. The appendix to the main body of work presented in thesis are two pieces of work pertaining to the elucidation the kinetics and mechanism of sugar lectin interactions, when sugars are presented as glycolipids in monolayers or bilaycrs liposomes. Mode of the presentation of sugars at cell-surfaces in the form of glycolipids as ligands influence their recognition by macromolecular receptors like lectins. Appendix 1 is a study of the mode of action of Ulex europeus I lectin binding to H-fucolipid containing tethered liposomes, by SPR. Fucosylated sugars are often used as key markers in histochemical analysis of malignant cancerous tissues. Ulex lectin plays a vital role as a marker for identification of these tissues. The kinetics and thermodynamic parameters that are obtained in this study throw some light on the mode of recognition of glycolipid receptor by Ulex europeus I lectin (C.J Thomas and A. Surolia 2000). Appendix 2 is a study, that attempts to quantify the initial kinetic parameters that correlate the recognition of glycolipid receptors with their inclination at the membrane surface and the influence of charge on them by soyabean agglutinin (SBA), Abrus agglutinin I and II. Studies on the soyabean agglutinin-globoside interaction highlights the divalent cation mediated reorientation of these receptors on their accessibility and recognition to the agglutinin. The divalent cations are speculated to orient the oligosaccharide head groups in a spatial geometry that allows a heightened kinetics of their interaction by SBA. These studies reveal that the reorganization of the binding pocket of a lectin can also have a profound influence on ihc rates of recognition of a glycospingolipid ligand by a lectin as exemplified by Abrus agglutinin II- GM1 interactions (C.J Thomas ct al, Manuscript under preparation).

Diploma thesis deals with the influence of various dosing of Reclaimed asphalt pavement (RAP) using modified bitumen on the properties of asphalt concrete (ACO 11+) of cemented by polymer modified bitumen, while a rejuvenating agents are added into asphalt mixtures. The theoretical part of the thesis summarizes the basic knowledge of pavement recycling, polymer modified bitumens and their reuse in asphalt mixtures. The practical part deals with laboratory tests of ACO 11+ mixtures with the RAP proportion of 0 % to 50 %. The asphalt mixtures compared are evaluated by means of the Thermal Stress Restrained Specimen Test (TSRST) and Stiffness test. The results obtained within the diploma thesis can approximate the issue of recycling of asphalt mixtures with polymer modified bitumens.