The development of small-diameter artificial blood vessels that mimic the properties of natural blood vessels has proven to be a clinical challenge. While autologous vessels are the standard, they can be difficult to obtain and require invasive surgeries. Synthetic materials have been successful in large diameter applications, but they have been unsuccessful in small-caliber environments due to a number of factors including thrombus formation, intimal hyperplasia, and infection. Intimal hyperplasia, of particular interest in this study, involves the build up of smooth muscle cells (SMCs) in the intimal layer of the artery due to abnormal migration and proliferation. This work focuses on the development of a new polymer that has the potential to function as an intimal/medial component of a small-diameter blood vessel. Using recombinant elastin-like polypeptides (ELPs) developed by the Woodhouse laboratory, as well as chondroitin sulphate-specific binding sequences (CSBD1 and CSBD2) determined by the Panitch laboratory, a new elastin-like polypeptide-chondroitin sulphate binding domain (ELP-CSBD) block copolymer has been developed and characterized. The expression of the ELP1-CSBDs was accomplished using E. coli BL21 cells in a bioreactor or shaker flask systems. The polypeptides were purified using dialysis and ion exchange chromatography and expression and purity were characterized using mass spectrometry and amino acid analysis. Both ELP1-CSBDs were successfully expressed using these methods and ELP1-CSBD1 was produced to high purities. ELP1-CSBD1 was able to undergo coacervation in vitro, suggesting that ELP1-CSBD1 is able to self-assemble in a manner similar to native elastin. In the presence of the glycosaminoglycan chondroitin sulphate (CS), the temperature of coacervation of ELP1-CSBD1 is increased, the rate and extent of coacervation is decreased, and aggregates remain in solution even at higher temperatures. The influence of heparin was also explored as previous studies indicated that the CS binding domains were shown to also bind to heparin. Studies completed in the presence of heparin showed that there were no significant changes to the coacervation characteristics of ELP1-CSBD1. It is anticipated that when combined with CS, ELP1-CSBD1 will gel, forming a basis for an intimal/medial layer of a TEBV that will modulate SMC response and increase graft integrity. / Thesis (Master, Chemical Engineering) -- Queen's University, 2013-01-06 21:03:37.788

Les réseaux périneuronaux (PNN) sont des régulateurs clé de la plasticité et de la régénération des neurones au niveau du système nerveux central chez l’adulte. Le PNN est une matrice extracellulaire hautement organisée, qui entoure des populations spécifiques de neurones, enrichie en protéoglycanes à chondroïtine sulfate (CSPG). La chondroïtine sulfate (CS) est un polysaccharide linéaire, appartenant à la famille des glycosaminoglycanes (GAG), qui peut être sulfaté à différentes positions et donner lieu à plusieurs isoformes. Ces isoformes interagissent de manière spécifique avec de nombreuses molécules de signalisation dont la semaphorine 3A (Sema3A). Sema3A est une protéine secrétée, qui interagit avec les CS et s’accumule ainsi dans les PNN. Elle est impliquée dans la guidance des neurones sur lesquels elle agit par chemorepulsion. Les aspects structuraux et fonctionnels de l’ interaction entre Sema3A et CS sont encore mal connus, mais celle-ci pourrait être requise pour renforcer la liaison de la Sema3A avec ses récepteurs et déclencher une voie de signalisation qui aboutit à l’inhibition de la plasticité synaptique. Le but du projet est donc de caractériser biochimiquement l’interface d’interaction Sema3A-CS. Il a pour perspective d’élaborer des molécules interférant avec cette interaction qui pourraient permettre une amélioration de la plasticité neuronale après une maladie neurodégénérative ou une lésion de la moelle épinière.Pour ce faire, la Sema3A est exprimée dans un système hétérologue de cellules eucaryotes pour être purifiée. Deux formes ont été purifiées: une forme complète de 90 kDa qui reste accrochée à la surface cellulaire et une forme clivée de 65 kDa secrétée dans le milieu de culture. La Sema3A-90 interagit d’une manière sélective et avec une très haute affinité avec la CS-E (chondroitine disulfatée en position 4 et 6) et l’héparane sulfate,alors que, la forme clivée n’interagit avec aucun GAG, comme observé par résonance plasmonique de surface (SPR). Quatre sites, situés dans le domaine C-terminal de la protéine, susceptibles d’interagir avec les GAG ont été identifiés et analysés par mutagenèse. Deux d’entre eux sont impliqués dans la reconnaissance des GAG et sont nécessaires à la Sema3A pour inhiber la croissance de neurites sur des cultures de neurones issus de ganglion de la racine dorsale de rats. En parallèle, nos travaux montrent qu’un tetrasaccharide de CS-E est la taille minimale requise pour l’interaction avec la Sema 3A. Enfin, des analyses réalisées en utilisant une microbalance à cristal de quartz avec dissipation ont montré que la Sema3A pourrait réticuler les chaines de GAGs, participant ainsi à la stabilisation du réseau périneuronal. / Perineuronal nets (PNNs) are the key regulators of neuronal plasticity and regeneration in the mature central nervous system (CNS). They are a unique and highly organised extracellular matrix (ECM) structure, found around sub-population of neurons, composed mainly of chondroitin sulfate proteoglycan (CSPG). Chondroitin sulfate (CS) is a linear polysaccharide belonging to glycosaminoglycans (GAGs) family. The sulphation pattern defines different types of CS, which interact with different signalling proteins including those regulating axonal outgrowth and guidance such as semaphorin 3A (Sema3A). Sema3A is a secreted chemorepulsive protein found accumulated in the PNNs through its interaction with CS. This process is believed to potentiate Sema3A signalling through plexin A1 (PlxnA1) and Neuropilin 1 (Nrp1) and regulate plasticity and regeneration. The aim of the thesis project is to characterise the interface of Sema3A- CS interaction.For this purpose, Sema3A is expressed in eukaryote cells and purified. Interestingly, two major forms were obtained: a full length Sema3A (90 kDa) which remains attached to the cell surface GAGs and a truncated form without the C-ter part (65 kDa) which is released to the culture medium. With the use of surface plasmon resonance (SPR), we observed that full length Sema3A binds selectively to CS-E (4,6-disulfated chondroitin) and heparan sulfate with a high affinity (KD in the sub pM range), while the truncated Sema3A does not bind to any GAG. Four putative GAG binding sequences were identified in the C-ter of Sema3A and mutated using site directed mutagenesis. SPR analysis then revealed that two out of these four sites are required for the binding to CS-E. The importance of these GAG-binding sequences in inhibition of neurites outgrowth of dorsal root ganglion neurons in culture was also reported, indicating thus the importance of GAG-binding in Sema3A signalling. In parallel, the minimal required sequence of Sema3A-binding of CS-E was determined as being a tetrasaccharide. The Sema3A-CS interface was thus characterized. Furthermore, quartz crystal microbalance with dissipation monitoring analysis suggested that Sema3A could crosslink GAG chains. This suggests Sema3A could be involved in stabilising the PNN network and induces mechanical changes on neuronal surface.The detail characterization of Sema3A-CS interaction may enable the design of new strategies aiming at enhancing plasticity and regeneration for neurodegenerative diseases or spinal cord injury.

L’arthrose est un processus menant à la dégénérescence du cartilage articulaire dont l’un des principaux composants est un protéoglycane (PG) : l’aggrécane. Il est constitué de glycosaminoglycanes (GAGs), essentiellement le sulfate de chondroitine (CS), liés de façon covalente à un squelette peptidique par l’intermédiaire d’une zone de liaison tétrasaccharidique commune aux principaux types de GAGs (CS et sulfate d’héparane (HS)). La biosynthèse des PGs met en jeu l’action séquentielle de O-glycosyltransférases qui additionnent spécifiquement chaque unité saccharidique. Lors de cette biosynthèse, encore mal connue, la zone de liaison subit des modifications (sulfatation et phosphorylation) qui peuvent être importantes pour la polymérisation des PGs en faveur des CS ou des HS.L’objectif de ce travail était de synthétiser, pour la première fois, une collection complète de trisaccharides biotinylés diversement monosulfatés ou non de la zone de liaison des PGs ainsi que des tétrasaccharides biotinylés (zone de liaison et amorces de CS) dans le but de déterminer le rôle des différentes sulfatations possibles dans la biosynthèse des PGs par les O-glycosyltransférases. / Osteoarthritis is a process leading to the degeneration of articular cartilage in which one of the major component is the proteoglycan (PG) aggrecan. It is composed of glycosaminoglycans (GAGs), mainly chondroitin sulfate (CS), covalently linked to a peptide backbone through the tetrasaccharide linkage region which is common to the two principal types of GAGs (CS and heparan sulfate (HS)). The PGs biosynthesis involves the sequential action of O-glycosyltransferases that add specifically each saccharide unit. In this biosynthesis, still poorly understood, the linkage region undergoes changes (sulfation and phosphorylation) that may be important for the polymerization of PGs in favor of the CS or the HS.The objective of this work was to synthesize, for the first time, a full collection of biotinylated trisaccharides variously monosulfated or not of the linkage region of PGs and biotinylated tetrasaccharides (linkage region and first aminosugar of CS), in order to determine the role of the possible sulfation within the biosynthetic pathway of PGs by the O-glycosyltransferases.

Made available in DSpace on 2015-03-26T13:46:36Z (GMT). No. of bitstreams: 1 texto completo.pdf: 832157 bytes, checksum: b5d2369f96a82a9d4af7de68979a2dad (MD5) Previous issue date: 2006-04-11 / This work was developed in order to evaluate microscoply the influence of gallium arsenate (Ga-As) diodo laser associated with chondroitin sulfate and chitosan on the healing process of the articular surface of the humeral head of dogs following experimental osteochondroplasty. Sixteen adult dogs were randomly allocated into four groups of four animals. All animals had the left humeral head injured by a surgical procedure (osteochondroplasty) and those from the TG were submitted to Ga-As diode laser sessions and received orally chondroitin sulfate and chitosan. In order to analyze microscopically the surgical injury the animals of the subgroups (C1, C2, C3, C4, T1, T2, T3 and T4) were killed on the 7th, 21st, 35th and 60th days after the surgery, respectively. The histological assessment revealed an increased vascularization, repairing potential and local osteogenesis, increase of the activity synthesis and keeping the cartilage matrix and better healing of the defect with fibrocartilage tissue of TG compared to those from CG, such findings were attributed to the physiotherapeutic effects of the Ga-As diode laser and chondroitin sulfate and chitosan. / O objetivo deste estudo foi avaliar, microscopicamente, a influência do laser a diodo de Arsenieto de Gálio (As-Ga) associado ao sulfato de condroitina e quitosana no processo de reparação da superfície articular da cabeça umeral de cães após defeito osteocondral experimental. Foram utilizados 16 cães adultos, distribuídos aleatoriamente em quatro subgrupos de quatro animais. Em todos os animais foi induzido um defeito cirúrgico na articulação escápulo-umeral esquerda sendo, posteriormente, submetido à aplicação de laser As-Ga (904nm) e administração oral de sulfato de condroitina e quitosana. Para realizar as análises microscópica do defeito cirúrgico, os animais de cada subgrupo (T1, T2, T3 e T4) foram eutanasiados aos sete, 21, 35 e 60 dias após a cirurgia, respectivamente. Foi evidenciado aumento da vascularização da área de reparação, potencialização da reparação e da osteogênese local, aumento da atividade de síntese e manutenção da matriz cartilaginosa e reparação do defeito por tecido fibrocartilaginoso. Os resultados foram atribuídos aos efeitos fisioterapêuticos do laser associado ao sulfato de condroitina e quitosana administrado via oral.

This master thesis deals with the optimization of the procedure of modification of hyaluronic acid structure for the use in the artificial synovial liquids. Based on the literature research, the amino acid alanine was used for the modification of carboxylic group in the glucuronic acid. The main subject of study is the improvement of the stability and mechanical properties of synovial liquid. DLS microrheology, macrorheology, thermogravimetric analysis (TGA), multi-angle light scattering with flow-field flow fractionation (AF4-MALS) and infrared spectroscopy (FTIR) were used for characterization. The theoretical part of this theses submits review of the musculoskeletal system, role of hyaluronic acid in metabolism and summary of synovial liquid. The experimental part focuses on the measurement of the stability and mechanical properties of three artificial samples (first with no modification, second with modified hyaluronic acid and third with modified hyaluronic acid and chondroitin sulphate). These samples were compared with real horse synovial fluid and artificial viscosupplement Orthovisc®.

En janvier 2008, une éclosion de réactions anaphylactoïdes (RA) potentiellement mortelles associées à l’injection intraveineuse d’héparine manufacturées en Chine et contaminée par le chondroïtine sulfate hypersulfaté (CSHS) a forcé le rappel de ces dernières par la U.S. Food and Drug Administration. Ces RA ont rapidement été attribuées à la libération de la bradykinine (BK) suite à l’activation du système de contact par le CSHS. Cependant, aucune évidence expérimentale définitive n’est à ce jour venue appuyer directement cette hypothèse. En se basant sur le nombre de morts déclaré et associé à la contamination (>150 morts au niveau mondial) ainsi qu’aux données épidémiologiques, qui stipulent que 25% des patients ayant développés une RA aux États-Unis étaient essentiellement des insuffisant rénaux en dialyse traités au moyen d’un inhibiteur de l’enzyme de conversion de l’angiotensine (iECA), nous avons émis l’hypothèse suivante : les RA causées par l’injection intraveineuse d’héparine contaminée au CSHS sont de nature multifactorielle et complexe. Le but de notre travail est donc, dans un premier temps, d’évaluer le pouvoir kininoformateur du CSHS en présence d’un iECA et de le comparer à celui du sulfate de dextran, un activateur de référence du système de contact. Comme les RA associées à l’injection intraveineuse d’héparine contaminée par le CSHS se produisent généralement dans les premières minutes des séances de dialyse, nous allons étudier l’effet de la dilution du plasma sur la quantité de BK libérée en présence ou en absence d’un iECA. Nous allons également mesurer les profils cinétiques de la libération de la BK sur un plasma stimulé par différents lots d’héparine contaminée, et associée à des RA, et nous comparerons cette cinétique avec celles d’une héparine de référence complémentée ou non avec différentes concentrations de CSHS synthétique. Enfin, nous allons caractériser le profil de libération de la BK et de son métabolite actif, la des-Arg9-BK, dans le plasma de patients dialysé ayant présenté une RA associée à une membrane de dialyse chargée négativement. L’application de méthodes expérimentales développées dans notre laboratoire nous a permis de montrer, pour la première fois, que l’héparine contaminée au CSHS a la capacité de libérer la BK à des concentrations susceptibles d’expliquer le rôle de ce peptide inflammatoire dans la physiopathologie des RA causées par l’injection intraveineuse d’héparine d’origine chinoise contaminée au CSHS. / In January 2008, fatal anaphylactoid reaction (AR) has been associated to oversulfated chondroitin sulphate (OSCS) contaminated heparin. Although attributed to bradykinin (BK) released during contact system activation by OSCS, no definitive evidence exists until now for a BK release during incubation of contaminated heparin with human plasma. While looking at the number of death associated with OSCS (>150 worldwide) and at the epidemiologic fact, who state that 25% of the cases of AR associated to OSCS in United-States were treated with an angiotensin converting enzyme inhibitor (ACEi), we hypothesis that: AR associated with bolus injection of OSCS contaminated heparin are bind to a complex and multi-factorial aspect. The first objective of our study is to measure the kinetics of BK release in human plasma incubated with OSCS in presence of an ACEi and to compare it to the kinetics profile of the reference activator dextran sulfate. As the AR associated with OSCS contaminated heparin occurred mainly in the first minutes of dialysis session, we also studied the effect of the plasma dilution on the amount of BK released when treated or not with an ACEi. We also quantify the BK forming capacity of different batches of OSCS contaminated heparin responsible for AR and we compare this effect with reference heparin spiked or not with increasing concentrations of synthetic OSCS. Finally, we measure the kinetics of BK and des- Arg9-BK, its active metabolite, release in human plasma collected from patients who developed an AR associated to negatively charged dialysis membrane. The application of experimental method developed in our laboratory show, for the first time, that OSCS contaminated heparin incubated with human plasma has the capacity to liberate BK at a concentration that could explain the role of this inflammatory peptide in the pathophysiology of AR associated with OSCS contaminated Chinese heparins.

En janvier 2008, une éclosion de réactions anaphylactoïdes (RA) potentiellement mortelles associées à l’injection intraveineuse d’héparine manufacturées en Chine et contaminée par le chondroïtine sulfate hypersulfaté (CSHS) a forcé le rappel de ces dernières par la U.S. Food and Drug Administration. Ces RA ont rapidement été attribuées à la libération de la bradykinine (BK) suite à l’activation du système de contact par le CSHS. Cependant, aucune évidence expérimentale définitive n’est à ce jour venue appuyer directement cette hypothèse. En se basant sur le nombre de morts déclaré et associé à la contamination (>150 morts au niveau mondial) ainsi qu’aux données épidémiologiques, qui stipulent que 25% des patients ayant développés une RA aux États-Unis étaient essentiellement des insuffisant rénaux en dialyse traités au moyen d’un inhibiteur de l’enzyme de conversion de l’angiotensine (iECA), nous avons émis l’hypothèse suivante : les RA causées par l’injection intraveineuse d’héparine contaminée au CSHS sont de nature multifactorielle et complexe. Le but de notre travail est donc, dans un premier temps, d’évaluer le pouvoir kininoformateur du CSHS en présence d’un iECA et de le comparer à celui du sulfate de dextran, un activateur de référence du système de contact. Comme les RA associées à l’injection intraveineuse d’héparine contaminée par le CSHS se produisent généralement dans les premières minutes des séances de dialyse, nous allons étudier l’effet de la dilution du plasma sur la quantité de BK libérée en présence ou en absence d’un iECA. Nous allons également mesurer les profils cinétiques de la libération de la BK sur un plasma stimulé par différents lots d’héparine contaminée, et associée à des RA, et nous comparerons cette cinétique avec celles d’une héparine de référence complémentée ou non avec différentes concentrations de CSHS synthétique. Enfin, nous allons caractériser le profil de libération de la BK et de son métabolite actif, la des-Arg9-BK, dans le plasma de patients dialysé ayant présenté une RA associée à une membrane de dialyse chargée négativement. L’application de méthodes expérimentales développées dans notre laboratoire nous a permis de montrer, pour la première fois, que l’héparine contaminée au CSHS a la capacité de libérer la BK à des concentrations susceptibles d’expliquer le rôle de ce peptide inflammatoire dans la physiopathologie des RA causées par l’injection intraveineuse d’héparine d’origine chinoise contaminée au CSHS. / In January 2008, fatal anaphylactoid reaction (AR) has been associated to oversulfated chondroitin sulphate (OSCS) contaminated heparin. Although attributed to bradykinin (BK) released during contact system activation by OSCS, no definitive evidence exists until now for a BK release during incubation of contaminated heparin with human plasma. While looking at the number of death associated with OSCS (>150 worldwide) and at the epidemiologic fact, who state that 25% of the cases of AR associated to OSCS in United-States were treated with an angiotensin converting enzyme inhibitor (ACEi), we hypothesis that: AR associated with bolus injection of OSCS contaminated heparin are bind to a complex and multi-factorial aspect. The first objective of our study is to measure the kinetics of BK release in human plasma incubated with OSCS in presence of an ACEi and to compare it to the kinetics profile of the reference activator dextran sulfate. As the AR associated with OSCS contaminated heparin occurred mainly in the first minutes of dialysis session, we also studied the effect of the plasma dilution on the amount of BK released when treated or not with an ACEi. We also quantify the BK forming capacity of different batches of OSCS contaminated heparin responsible for AR and we compare this effect with reference heparin spiked or not with increasing concentrations of synthetic OSCS. Finally, we measure the kinetics of BK and des- Arg9-BK, its active metabolite, release in human plasma collected from patients who developed an AR associated to negatively charged dialysis membrane. The application of experimental method developed in our laboratory show, for the first time, that OSCS contaminated heparin incubated with human plasma has the capacity to liberate BK at a concentration that could explain the role of this inflammatory peptide in the pathophysiology of AR associated with OSCS contaminated Chinese heparins.

The present thesis focuses on the fabrication of bio-stimuli responsive micro- and nano-carriers for drug delivery applications. In particular, the objective of this work is to investigate the possibility of using polypeptide drug protamine and glycosaminoglycan drug, chondroitin sulphate as stimuli responsive components in the design of bioresponsive carriers. These biopolymers are biocompatible, biodegradable and clinically used for various applications. Two designs that incorporate these stimuli responsive components have been studied in this thesis. The first design involves hollow micro and nanocapsules that have been fabricated by incorporating the stimuli responsive biopolymers as wall components. Upon exposure to biological triggers, these hollow capsules disintegrate releasing the encapsulated drug. The second design consists of mesoporous silica nanoparticles-biopolymer hybrids. The mesoporous silica nanoparticles act as a gated scaffold that carries the drug molecules. The mesopores of these drug loaded nanoparticles are then blocked with the bioresponsive polymers. Upon exposure to the bio-triggers which consist of enzymes over-expressed in conditions such as cancer and inflammation, these “molecular gates” disintegrate allowing the drug trapped in the mesoporous silica nanoparticles to escape into the surroundings. The thesis has been divided into five chapters: Chapter 1 is an introduction to bio-responsive drug delivery. The broad classification of stimuli used in responsive drug delivery systems are visited. A brief discussion on the various types of bio-stimuli that can be utilized in designing bio-responsive systems is also included in this chapter. Chapter 2 defines the aims and scope of the thesis which is followed by an overview of the various design parameters involved in the fabrication of systems presented in this work. The major stimuli responsive components and the architectures incorporating these elements are discussed in detail here. A literature review of the various carrier designs involved in the study is provided , with special emphasis on stimuli responsive drug delivery. Chapter 3 gives an overview of the various materials and methods involved in this work. A summary of the various characterisation techniques used in the thesis is also included along with the details of the experiments that has been carried out. Chapter 4 provides an overview of the results and discussions of the thesis. The chapter has been divided into six sections: Chapter 4.1 deals with the fabrication of a hollow microcapsule system incorporated with protamine as the stimuli responsive element for bio-responsive drug delivery. The hollow microcapsules that were fabricated by Layer by Layer assembly of protamine and heparin display pH responsive variations in permeability and disintegrate in the presence of the enzyme trypsin that degrades protamine. The biologically triggered enzyme responsive drug release from these microcapsules is also demonstrated using enzymes secreted by colorectal cancer cells. Chapter 4.2 presents nanocapsules fabricated from protamine and heparin. The pH and enzyme responsive drug release of this systems is evaluated in vitro. A wall crosslinking strategy has been tested to control the rate of drug release under physiological pH conditions in the absence of the trigger. The cellular interactions of these nanocapsules loaded with an anticancer drug, doxorubicin was studied using cancer cell lines. Bioavailability studies of doxorubicin encapsulated in these nanocapsules were performed using a BALB/c mice model. Chapter 4.3 discusses the fabrication of a hollow microcapsule system that can disintegrate in response to dual biological stimuli. These carriers have been fabricated by incorporating protamine and chondroitin sulphate as the wall components. Due to the incorporation of two separate stimuli responsive components in the walls, these capsules are expected to be sensitive to the enzymes trypsin or hyaluronidase I. Chapter 4.4 deals with the fabrication of dual enzyme responsive hollow nanocapsule which can be targeted to deliver anticancer agents specifically inside cancer cells. The enzyme responsive elements integrated in the hollow nanocapsule walls can undergo degradation in presence of either of the enzymes trypsin or hyaluronidase I leading to the release of encapsulated drug molecules. The drug release from these nanocapsules which were crosslinked and functionalised with folic acid, is evaluated under varying conditions. The cellular uptake and intracellular drug delivery by these nanocapsules were evaluated in cervical cancer cell lines. Chapter 4.5 introduces a mesoporous silica nanoparticle − protamine hybrid system. The system consists of a mesoporous silica nanoparticle support whose mesopores are capped with protamine which effectively blocks the outward diffusion of the drug molecules from the mesopores of the mesoporous silica nanoparticles. Upon exposure to the enzyme trigger, the protamine cap disintegrates opening up the molecular gates and releasing the entrapped drug molecules. The drug release from this system is evaluated in different release conditions in the presence and absence of the enzyme trigger. The ability of these particles to deliver hydrophobic anticancer drugs and induce cell death in colorectal cancer cells has also been demonstrated. Chapter 4.6 discusses the fabrication of another mesoporous silica nanoparticles based bio-responsive drug delivery system consisting of mesoporous silica and chondroitin sulphate hybrid nanoparticles. The ability of the system to modulate drug release in response to hyaluronidase I is demonstrated. By utilizing a cervical cancer cell line, we have demonstrated the cellular uptake and intracellular delivery of hydrophobic drugs encapsulated in these particles. Interestingly, the system showed ability to enhance the anticancer activity of hydrophobic drug curcumin in these cancer cells. Chapter 5 gives a summary of the general conclusions drawn from the thesis work.