Les inquiétudes concernant la gestion des déchets sont croissantes dans un contexte environnemental préoccupant. De ce fait, il est apparu urgent de trouver des substituts aux plastiques conventionnels et les biopolymères semblent être une réponse prometteuse. Leurs propriétés leur permettent des usages très divers, de l'emballage alimentaire à la bio-ingénierie tissulaire en passant par la formulation cosmétique ou la pharmaceutique. S'ils jouissent d'une image positive et de l'intérêt croissant des scientifiques comme des industriels, on ne connait encore que peu leur comportement lors du vieillissement. Ce travail de thèse se propose d'étudier d'une part le vieillissement en enceinte climatique à paramètres contrôlés de certains polymères bio-sourcés et biodégradable (HPMC, Chitosan, PLA) et d'autre part, l'influence d'un traitement micro-onde sur des films de chitosan dans le cadre d'une utilisation comme le packaging alimentaire. Au cours du vieillissement, les biopolymères subissent plusieurs modifications structurales telles que des ruptures de chaînes et des phénomènes de cristallisation. Ces modifications altèrent les propriétés mécaniques des films de biopolymères. Par ailleurs, les films de chitosan, exposés au micro-onde, vont présenter un phénomène de jaunissement, révélateur de la production de composés néo-formés issus de la réaction de Maillard et susceptibles de migrer vers l'aliment en cas de contact avec le film de biopolymère / Today's increased environmental concerns and the growing questions about disposal management are contributing to growth in biopolymer industry. These materials are used in various fields such as pharmaceutic, bioengineering or tissue engineering as cell support, food formulation or packaging. In spite of a positive perception of biopolymers to improve environmental issues, biopolymers properties remain unknown, even though their biodegradability is well documented. Here we consider the influence of aging in climatic chamber on different biopolymers (HPMC, Chitosan and PLA) and in to what extent it could modify their structure and properties. Moreover, considering the opportunity for theses materials to be used as food packaging, influence of microwave heating was also tested on chitosan films. The food consumption trends tend, indeed, to the development of ready-to-eat meal that is why it is crucial to investigate theses biopolymers behavior when used in microwave oven in food contact. The aim of this study is to bring new knowledge of theses materials and to guarantee an optimal and safe use as a food contact packaging. The results showed several deep modifications in the biopolymers structure such as chain scissions or crystallization. These modifications involved a decrease in the polymer's properties. Chitosan films, exposed to increasing microwave heating times, showed gradually increasing yellowing. Maillard neoformed compounds are responsible for this color change and could be deleterious for consumer safety because of the possibility of migration in case of food contact

L'objectif de cette thèse était de développer de nouveaux « donneurs de NO » stables en liant du S-nitrosoglutathion (GSNO) à une structure polymérique. Dans une première étape, les polymères ont été liés au glutathion (GSH) : le chitosan-GSH et l'alginate-GSH ont ainsi été préparés par la « méthode des carbodiimides » et dans une deuxième étape, les polymères finaux [SNOC (S-nitrosoglutathione-oligosaccharide-chitosan) et SNA (S-nitrosoglutathione-alginate)] ont été préparés par nitrosation des deux conjugués précédent. La quantité de NO fixée a été déterminée par les méthodes Griess et Saville. L’aptitude des polymères à libérer du NO et à passer la barrière intestinale [SNOC et SNA] a été évaluée dans une chambre d’Ussing. Nous avons obtenu des polymères avec des quantités variables de NO en fonction de la méthode utilisée (159 µmol de NO/g à 525 µmol de NO/g pour le SNOC ; 174 µmol de NO/g à 468 µmol de NO/g pour le SNA). Le SNOC était stable pendant au moins 6h et le SNA pendant au moins 10h. Enfin, nous avons essayé de mettre au point des microparticules de GSH et GSNO par spray drying avec de l’Eudragit ® FS 30D gastro-résistant. La caractérisation des microparticules a été réalisée par microscopie électronique à balayage (SEM), par diffraction X (PXRD) et par spectroscopie infrarouge (FTIR). Les essais de libération in vitro ont été réalisés dans un tampon (pH 1,2, 3, 6, 6,8 et 7,4). Les microparticules étaient chargées négativement avec une taille moyenne allant de 5 à 7 µm. La formulation était stable à pH acide mais a montré une libération rapide à pH basique ; elle pourrait donc servir de système de délivrance du NO au niveau intestinal. / The aim of the thesis was to develop novel and stable NO-donors by linking S-nitrosoglutathione (GSNO) to a polymer backbone. In the first step, chitosan-GSH and alginate-GSH conjugates were prepared by a carbodiimide reaction and in the second step SNOC (S-nitrosoglutathione-oligosaccharide-chitosan) and SNA (S-nitrosoglutathione-alginate) were prepared by the nitrosation of both conjugates respectively. The amount of NO was determined by Griess and Saville methods. Stability and ex vivo experiments of SNOC and SNA were performed in an Ussing chamber through rat intestine. We obtained polymers with different amount of NO (i.e. 159 µmol of NO/g to 525 µmol of NO/g for SNOC; 174 µmol of NO/g to 468 µmol of NO/g for SNA) depending upon the procedure of nitrosation. SNOC was stable for at least 6h and SNA for at least 10h. Also, we aimed to develop spray dried microparticles of GSH and GSNO based on Eudragit® FS 30D polymer. The microparticles were characterized by scanning electron microscopy (SEM), X-ray diffraction (PXRD), infrared spectroscopy (FTIR) and in vitro release studies were performed in different pH conditions (pH 1.2, 3, 6, 6.8 and 7.4). The microparticles were negatively charged with mean particle size ranging from 5 to 7 µm. The formulation was stable and was resistant to acidic pH but showed rapid release in basic pH; hence, they can be used as colon specific drug delivery systems for the treatment of Crohn’s disease. We think that these formulations could be used in animal models in the treatment of Crohn’s disease.

<p>The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems <i>in vitro</i> and after lung administration <i>in vivo</i> were established for the first time.</p><p>Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells <i>in vitro</i> and after lung administration <i>in vivo</i>. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for <i>in vivo</i> gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI <i>in vitro</i> and <i>in vivo</i>. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for <i>in vivo</i> gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.</p>

The subject of this thesis was to develop and optimize delivery systems for plasmid DNA (pDNA) based on biocompatible polymers, in particular chitosan, suitable for non-viral gene therapy. At the onset of this thesis, studies had reported conflicting results on the efficiency of chitosan-based gene delivery systems. Therefore, structure-property relationships of chitosans as non-viral gene delivery systems in vitro and after lung administration in vivo were established for the first time. Polymer-pDNA complexes (polyplexes) based on conventional high molecular weight chitosans transfected cells in vitro and after lung administration in vivo. The chitosan polyplexes were, in contrast to polyplexes formed with the "golden standard" polymer polyethylenimine (PEI), essentially non-toxic at escalating doses. However, a very high physical stability of the chitosan-pDNA complexes together with a low buffering capacity of chitosan at the slightly acidic endo/lysosomal pH resulted in a slow onset of the gene expression and also in a lower efficiency of gene expression compared to PEI polyplexes. A slow and biodegradation-dependent release of pDNA from the chitosan polyplexes was concluded to be a rate limiting step for the efficiency of high molecular weight chitosan. The optimized polyplexes of high molecular weight chitosan (around 1,000 monomer units) showed aggregated shapes and gave increased viscosity at concentrations used for in vivo gene delivery. To improve the pharmaceutical properties and the delivery properties of chitosan polyplexes, low molecular weight chitosans were studied. Chitosans of around 18 monomer units retained the ability to protect pDNA against DNase degradation, but were more easily dissociated than those of higher molecular weight and had an efficiency comparable to that of PEI in vitro and in vivo. The pharmaceutical advantages of low molecular weight chitosan polyplexes compared to higher molecular weights are that there is less aggregation and no increased viscosity at the concentrations used for in vivo gene delivery. Coupling of an oligosaccharide targeting ligand to chitosan further increased the efficiency of some oligomer polyplexes. In conclusion, biocompatible chitosan is an interesting alternative to other non-viral gene delivery systems such as PEI.

The acoustic microscope is an efficient non-invasive tool that can explore the acoustic properties and the related mechanical microstructure of a wide diversity of materials, including biomedical and biological samples, which are, nowadays, among the most intriguing targets for investigations. In the presented work, an acoustic microscope with vector contrast is used to image and characterize the acousto-mechanical properties of chitosan, an abundant natural derivative of chitin known to be a biodegradable, nontoxic and versatile biopolymer that suits many biomedical applications such as its usage in tissue engineering. The work also presents key measurements for the study of the acousto-mechanical properties that are subject to variations during the life span of red blood cells (RBCs). The characteristic signature of fixed cells from groups of three different ages, fractionated according to mass density, is obtained from the acoustic microscope images. The analysis of these data enabled the quantitative comparisons between the acousto-mechanical properties (velocity and attenuation of ultrasound propagating in the cells, mass density, and bulk modulus of compression). Comparison of the contrasts in the acoustic micrographs for the cells of the different age groups is exploited to generate a model that determines the age of the individual cells in a sample of red blood cells collected from a healthy person. The dependence of the parameters of the cells including density, velocity and attenuation of longitudinal polarized ultrasonic waves travelling in the cells on the age of the cell is also presented. The output signal in dependence on the thickness of the sample, the so called V(d), represented as polar graph was exploited as the method of analysis of the data extracted from the acoustic micrographs imaged with ultrasound of a center frequency of 1.2 GHz. This procedure allows for the extraction of the quantitative information from a single image in magnitude and phase contrast and allows for height profiling with so called super resolution, relating to resolution below the diffraction limit, based on the developed modeling, beside of other advantages concerning the acoustic characterization of biomedical and biological samples. This method and the applications are presented and discussed together with the developed or adapted modeling.

<p>Nicaragua is a country in which the toxic metal contamination of freshwater resources has become an increasingly important problem in certain regions posing a threat to the environment as well as to human health. Among the metals found in the waters of Nicaragua, arsenic is one of the most problematic since its long time consumption is connected to serious health problems such as cancer and neurological disorders. The arsenic contamination of water recourses in Nicaragua is mostly attributable natural factors, even though anthropogenic activities including gold mining may be a contributing factor.</p><p>In this work the biopolymer Chitosan was studied as a potential adsorption material for the removal of arsenic from aqueous solutions for water treatment design purposes.</p><p>The Chitosan used in this study was extracted from shrimp shells with an overall yield of 40% and a deacetylation grade of 59%. The maximum adsorption capacity was determined to 20.9 mg As/g at a controlled pH of 5.5 using the Langmuir isotherm. The adsorption was found to be strongly pH dependant with a fourfold increase in adsorption capacity when pH was well under the pKa of Chitosan. The pH dependence indicates that ionic exchange was the most important mechanism. No difference in adsorption capacity with respect to the initial pH of the solution was detected in the pH range 3-7. This was attributed to the ability of Chitosan to act as a weak base in water solutions.</p><p>The arsenic was desorbed from Chitosan using NaOH, (NH4) 2SO 4 and NaCl, with a 1M NaOH solution being the most efficient displaying a concentration ratio of 1.08. The NaOH and (NH4) 2SO 4 solutions displayed a steep desorption curvature with a large fraction of the arsenic being easily desorbed. The arsenic was, however, not completely desorbed from the Chitosan implying that the adsorption capacity would decrease for the coming cycles. Being a biopolymer the Chitosan is quite easily degraded in acid and alkali solutions, which might be a limiting step for the process applicability.</p>

Oily wastewater produced from petroleum and petrochemical refining processes is one of the gravest environmental threats. Oil waste ending up in sewers and dumps each year is equal to 25 times the amount of crude oil spilled in the Exxon Valdez accident (1989). Oil/Water separation covers a broad spectrum of industrial process operations. There are many techniques employed depending on each situation. The byproduct of water recovery from oily wastewater is a sludge rich in oil, surfactants and particles (oily sludge). The oily sludge still contains significant amounts of waters, which need to be recovered prior to its disposal. The use of polyelectrolytes for the flocculation of the emulsified oil and its separation from the aqueous phase is usually one of the steps of the wastewater as well as oily sludge treatment process. The efficiency of polyelectrolytes as floculants is quite often evaluated via trial and error and the appropriate polymer is selected according to the case. Even in scientific investigations it is rather common to use industrial oily sludge samples. The industrial oily sludge is characterized and treated by polyelectrolytes. Nevertheless industrial oily sludge is quite complicated and variable to be approximated by a model. For the systematic study of polyelectrolytes efficiency a stable, realistic and well-defined oily sludge model is necessary. In the present work an oily sludge model was successfully developed and characterized. The model consists on water, oil (kerosene), surfactant (sodium dodecyl sulphate, SDS) and clay particles (Blauton). The emulsifying efficiency of the surfactant and the clay were studied independently. The interactions between the surfactant and the clay including adsorption of the former on the later and cation exchange reactions were investigated. The four components were finally combined to form a series of emulsions varying the relative amounts of the emulsifiers for the highest stability to be encountered. Having concluded on the composition of the oily sludge model the efficiency of various polyelectrolytes was evaluated. Commercial natural (chitosans), synthetic (PolyDADMACs) polyelectrolytes and oilbreaks as well as lab-scale semi-synthetic polymers (modified chitosans) were tested. The flocculation efficiency was determined based on the amount and quality of water that was recovered as well as on the floc stability, size and sedimentation speed. The recovered water was characterized according to the environmental protection agency (EPA). The analysis included measurements on: total organic carbon (TOC), chemical oxygen demand (COD), biochemical oxygen demand (BOD5), total suspended solids (TSS) and pH. Selection of the appropriate flocculant also depends on the type of flocs formed in combination to the treatment following the flocculation. When filtration or centrifugation is used as a post-flocculation process the appropriate polymers are those that form large and porous flocs, as the case of the modified chitosans AG95 and AG97. Clarification devices on the other hand require dense flocs as these produced by the use of PolyDADMACs, commercial chitosans, oilbreaks and modified chitosans GA35, GA41 and AG79. Regarding the water quality, some of the polymers used that have low values of COD, TOC and BOD5, may not need the secondary treatment (biological) prior to discharge, such as P187K (PolyDADMAC) and GA41 (modified chitosan). The others require a biological treatment for the regulation limits to be reached. The pH values of modified chitosans (except of AG97), lab-scale PolyDADMACs and an oilbreak (OCAA) are all in range of the regulation limits. The applicability of biopolymers as flocculants for oil sludge dewatering is a relatively new field of investigation. As a consequence of the growing demand for environmentally friendly technologies as well as renewable resources the interest on natural flocculants has increased. The aminopolysaccharide chitosan and its modified derivatives have outstanding properties such as biocompability, biodegradability, hydrophilicity, adsorption, flocculating ability and antibacterial properties. These natural polymers derived from the sea-food industry waste products would be very useful as residue oil adsorbents in any oily wastewater and can be among the most promising candidates as a replacement of the synthetic flocculants. / Ölhaltiges Abwasser, das bei Erdöl- und petrolchemischen Raffinierungsprozessen entsteht, ist eine der größten Umweltgefahren. Dieses Altöl landet jedes Jahr in der Kanalisation und in Deponien. Es handelt sich dabei um die 25-fache Menge an Rohöl, die beim Unfall der Exxon Valdez (1989) ausgeflossen ist. Die Öl/Wasser-Trennung überspannt ein breites Spektrum industrieller Prozesse. Es gibt viele Techniken, die abhängig von jeder Situation eingesetzt werden. Das Nebenprodukt bei der Abtrennung von Wasser aus ölhaltigen Abwässern ist ein Schlamm (Ölschlamm), der reich an Öl, Tensiden und Partikeln ist. Der Ölschlamm enthält noch bedeutende Mengen an Wasser, die vor ihrer Entsorgung verwertet werden müssen. Die Verwendung von Polyelektrolyten zur Ausflockung des emulgierten Öls und seine Trennung von der wässrigen Phase ist in der Regel einer der Schritte zur Behandlung von Abwässern sowie ölhaltigen Schlämmen. Die Effizienz von Polyelektrolyten als Flockungsmittel wird ganz häufig über Versuch und Fehler bewertet, und das passende Polymer wird entsprechend dem jeweiligen Fall ausgewählt. Sogar in wissenschaftlichen Untersuchungen ist es eher üblich, industrielle Ölschlamm-Proben zu verwenden. Der industrielle Ölschlamm wird charakterisiert und mit Polyelektrolyten behandelt. Dennoch ist der industrielle Ölschlamm ziemlich kompliziert und variabel und muss durch ein Modell angenähert werden. Für die systematische Untersuchung der Effizienz von Polyelektrolyten als Flockungsmittel ist ein stabiles, realistisches und klar definiertes Ölschlamm-Modell notwendig. In der vorliegenden Arbeit wurde ein Ölschlamm-Modell erfolgreich entwickelt und charakterisiert. Das Modell besteht aus Wasser, Öl (Kerosin), Tensid (Natriumdodecylsulfat, SDS) und Tonteilchen (Blauton). Die Emulgiereffizienz des Tensids und des Tons wurden unabhängig voneinander untersucht. Die Wechselwirkungen zwischen dem Tensid und dem Ton, die sowohl die Adsorption des Ersteren auf dem Letzteren einschließen als auch einen Kationenaustausch, wurden untersucht. Die vier Komponenten des Ölschlamm-Modells wurden schließlich kombiniert und es wurde eine Reihe von Emulsionen hergestellt, bei denen die relativen Mengen der Emulsionsmittel verändert wurden, um eine möglichst hohe Stabilität zu erreichen. Nachdem ein geeignetes Ölschlamm-Modell zur Verfügung stand, wurde die Effizienz der verschiedenen Polyelektrolyte als Flockungsmittel bewertet. Kommerzielle natürliche (Chitosane), synthetische (PolyDADMACs) Polyelektrolyte und Oilbreaks sowie Labor-semi-synthetische Polymere (modifizierte Chitosane) wurden getestet. Die Flockungseffizienz wurde sowohl basierend auf der Menge und Qualität des Wassers, das zurückgewonnen wurde, als auch bezogen auf die Flockenstabilität, die Flockengröße und die Sedimentationsgeschwindigkeit bestimmt. Das zurückgewonnene Wasser wurde entsprechend der Vorschriften der Behörde für Umweltschutz (EPA) charakterisiert. Die Analysen enthielten die Bestimmung des gesamten organischen Kohlenstoffs (TOC), des chemischen Sauerstoffbedarf (CSB), des biochemischen Sauerstoffbedarfs (BSB5), des Gesamtgehalts an suspendierten Partikeln (TSS) und die Bestimmung des pH. Die Auswahl geeigneter Flockungsmittel hängt auch von der Art der gebildeten Flocken, in Kombination mit der Behandlung, die den Flockungsprozess folgt, ab. Wenn als Postflockungsprozess Filtration oder Zentrifugation folgen, sollten Polymere verwendet werden, die große und poröse Flöckchen bilden, wie im Fall der modifizierten Chitosane AG95 und AG97. Andererseits verlangen Geräte zur Klärung von Abwässern dichte Flocken, wie solche, die beim Einsatz von PolyDADMACs, kommerziellen Chitosanen, Oilbreaks und den modifizierten Chitosanen GA35, GA41 und AG79 entstehen. In Bezug auf die Wasserqualität erhält man mit einigen der verwendeten Polymere so niedrige Werte von CSB, TOC und BSB5, dass wie im Falle von P187K (PolyDADMAC) und GA41 (modifiziertes Chitosan) keine biologische Sekundärbehandlung notwendig ist. Im Falle der anderen Polymere ist eine biologische Behandlung nötig, um die vorgeschriebenen Grenzen zu erreichen. Die pH-Werte der modifizierten Chitosane (außer der AG97), der im Labor hergestellten PolyDADMACs und des Oilbreak OCAA sind alle in Bereich der vorgeschriebenen Grenzen. Die Anwendbarkeit von Biopolymeren als Flockungsmittel für die Ölschlammentwässerung ist ein relativ neuer Forschungsbereich. Als Folge der wachsenden Nachfrage nach umweltfreundlichen Technologien sowie erneuerbarer Ressourcen hat das Interesse an natürlichen Flockungsmitteln zugenommen. Das Aminopolysaccharidchitosan und dessen modifizierte Produkte haben hervorragende Eigenschaften wie Biokompatibilität, Biodegradierbarkeit, Hydrophilie, Adsorption, die Fähigkeit zur Flockung und antibakterielle Eigenschaften. Diese natürlichen Polymere, die aus Meeresfrüchte-Industrieabfallprodukten gewonnen werden, sollten als Restöl-Adsorbentien bei der Aufarbeitung jedes ölhaltigen Abwassers sehr nützlich sein und können zu den vielversprechendsten Kandidaten als Ersatz der synthetischen Flockungsmittel werden.

Degradation of peroral administered drugs by various enzymes in the gastrointestinal tract has proven to be troublesome for the absorption' and bioavailability of protein and peptide drugs. Mucoadhesive polymers such as poly(acrylates) have proven to inhibit protease enzymes responsible for initiating digestion of peptide drugs. Enzyme inhibitors have unique chemical properties enabling it to interact with enzymes to form complexes with such enzymes prohibiting it from functioning properly. Anionic carboxymethylated chitosan derivatives such as N,N-dicarboxymethyl chitosan and N, O-carboxymethyl chitosan display unique structural similarities to enzyme inhibitors being anionic polymers that may interact with bi-valent cations... / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

Degradation of peroral administered drugs by various enzymes in the gastrointestinal tract has proven to be troublesome for the absorption' and bioavailability of protein and peptide drugs. Mucoadhesive polymers such as poly(acrylates) have proven to inhibit protease enzymes responsible for initiating digestion of peptide drugs. Enzyme inhibitors have unique chemical properties enabling it to interact with enzymes to form complexes with such enzymes prohibiting it from functioning properly. Anionic carboxymethylated chitosan derivatives such as N,N-dicarboxymethyl chitosan and N, O-carboxymethyl chitosan display unique structural similarities to enzyme inhibitors being anionic polymers that may interact with bi-valent cations... / Thesis (M.Sc. (Pharm.))--North-West University, Potchefstroom Campus, 2004.

Chitosan-glucan complex is fungal origin copolymer that finds application in medicine and cosmetics. Traditionally mycelium of Aspergillus and Penicillium is considered as industrial chitosan-glucan complex source, though utilization of Micromycetes in biotechnological productions is sometimes undesirable. The aim of the work was to study the possibility of Basidiomycete Schizophyllum commune submerged cultivation for industrial scale chitosan-glucan complex production use. Within the work there was studied effect of cultivation conditions (type and concentration of carbon sources in nutrient medium, ratio of carbon source to nitrogen source, medium initial pH and aeration intensity) on Sch. commune #127 mycelium growth, chitosan-glucan complex formation and exopolysaccharide synthesis. As the result, the method for chitosan-glucan complex production increase and exopolysaccharide synthesis suppression was suggested. Chitosan-glucan complex from Sch. commune #127 submerged mycelium was separated by successive alkali and acid treatments. Effects of alkali concentration and application technique, and type of acid on physical and chemical properties of chitosan-glucan complex were described. Analytical methods for in process control and final product characteristics were suggested.