Connective tissue extracellular matrix (ECM) consists of an interwoven network of contiguous collagen fibers that inform cell activity, direct biological function, and guide tissue homeostasis throughout life. Recently, ECM analogs have emerged as a unique ex vivo culture platform for studying healthy and diseased tissues and in the latter, enabling the screening for and development of therapeutic regimen. Unfortunately, current commonly used platforms, such as tissue-culture polystyrene (TCPS) or the basement membrane matrix, Matrigel, fail to fully recapitulate the physical and biochemical properties of the ECM. Tissue-culture polystyrene is significantly stiffer than typical ECM tissues and lacks the composition and 3-dimensional architecture that is critical for ECM function. Improving upon TCPS’s shortcomings, Matrigel retains a natural ECM structure and is comprised of native biopolymers. However, it is derived from mouse sarcomas, and thus, has significant batch-to-batch variability and often contains growth factors at non-physiologic concentrations. Moreover, despite being biopolymer based, Matrigel has relatively low amounts of type I collagen and high levels of type IV collagen, and as such, compositionally does not match the predominantly type I collagen matrix intrinsic to connective tissues. Thus, it is clear that new and improved models of the ECM are needed for in vitro culture. In pursuit of developing a highly biomimetic ECM analog, the objectives of this work were three-fold— first, to fabricate collagen-based ECM analogs with nanoscale mimicry, second, to systematically optimize crosslinking protocols in order to produce a stable substrate with continuous fibrous architecture, and third, to evaluate the substrate’s biocompatibility and utility as a platform for studying biomineralization. It was hypothesized that an architecturally and chemically relevant fibrous substrate could be prepared from gelatin and provide an optimal ex vivo platform for cell culture and new therapy screening and development. Thus, the ECM analog will be collagen-like, biocompatible, consist of continuous fibers, demonstrate both viscoelastic and elastic behavior, exhibit relevant mechanical properties, and remain stable for at least 14 days at cell culture conditions. To this end, first, a “green” electrospinning method was developed for preparing fibrous meshes from gelatin, which avoids typical electrospinning solvents that present significant health risks and barriers to large scale production. Next, crosslinking methods were developed using the reactive dialdehyde, glutaraldehyde (GTA), and the naturally derived enzyme, transglutaminase (TGase). These methods stabilized the meshes for over 28 days under cell culture conditions without disrupting its biomimetic architectures and chemical properties. In addition, a third approach to mesh fabrication using gelatin methacryloyl (gelMA) was developed to overcome the shortcomings of GTA and TGase crosslinking. With gelMA, the number of crosslinking sites were customized and, by taking advantage of its ability to undergo free radical polymerization, stable fibrous meshes were prepared with reproducible architecture, chemistry, and tunable mechanical properties. Following fabrication, the biocompatibility of the meshes was evaluated through macrophage, stem cell, and differentiated cell cultures. During culture, the macrophages maintained a naïve, non-polarized state, indicating they were not triggered towards an inflammatory response by the meshes. In addition, fibrochondrocytes, a cell critical for maintaining the collagen-based matrices where ligaments attach to bone, remained viable and maintained phenotypic expression on the meshes, as evident by their enhanced proteoglycan and collagen production relative to TCPS cultures. After demonstrating biocompatibility, the gelatin platform was coupled with a synthetic matrix vesicle (SMV) system and successfully acted as a mineralization platform in the presence of human osteoblast-like cells. Additionally, the platform supported mesenchymal stem cell expansion and mineralization when cultured with an alkaline phosphate conjugated SMV. In this work, three unique methods were developed for preparing ECM analogs. These efforts led to the production of a collagen-like mesh with nano- and micro-scale cues, fibrous continuity with little batch-to-batch variability, and proven stability in both dry and wet conditions. Importantly, these meshes did not instigate any inflammatory responses and supported fibrochondrocyte, osteoblast, and stem cell culture. Furthermore, the mesh successfully functioned as a template for biomineralization using both human osteoblast-like cells and stem cells. Collectively these findings demonstrate the potential of a collagen-like ECM analog with physiological relevance for ex vivo cell culture studies; and furthermore, its potential as a high-fidelity platform for studying cell-mediated biomineralization, cell-matrix interactions, and developing new therapeutic approaches for the treatment of connective tissue disorders.

This dissertation attempts to address the problem of how to prepare protein-based materials with the same level of order and precision at the molecular level similar to the structures we find in nature. It is divided into two parts focusing on feedstock and processing. Part one is devoted to discussing the use of agricultural proteins as a feedstock for material production. Particularly, it focuses on the effect of hydrogen bonding, or lack thereof, between proteins as mediated by hydration or plasticization. The effect of varying plasticizer (glycerol) levels on mechanical properties of a series of proteins is discussed in the context of primary and secondary structure of these proteins. We have found that the extent to which a protein can be plasticized is dependent on its molecular and higher order structure and not simply molecular weight, as it was often assumed in previous studies. The second part of the dissertation focuses on the study of self-assembly as a way to make useful peptide-based materials. There are major efforts underway to study protein self-assembly for various medical and industrial reasons. Despite huge progress, most studies have focused on nanoscale self-assembly but the crossover to the macroscopic scale remains a challenge. We show that peptide self-assembly into macroscopic fibers is possible in vitro under physiological conditions. We characterize the fibers and propose a mechanism by which they form. The macroscopic fibers self-assemble from a combination of β- and α-peptides and are similar to other naturally-occurring systems in which templated self-assembly is used to create functional peptide materials. Finally, the ability to control macroscopic properties of the fiber by varying the ratio of constituent peptides is demonstrated. Owing to the richness of the amino acid building blocks, peptides are highly versatile structural and functional building blocks. The ability to extend and control peptide self-assembly over multiple length scales is a significant leap toward incorporating peptide materials into dynamic systems of higher complexity and functionality. / Ph. D.

Cooking extrusion is becoming an essential processing step in a number of food processes. Modeling of extruder performance is the first step towards the ultimate goal of prediction of product properties and quality based on governing extruder characteristics and operation. The purpose of this study was to develop a numerical model of the cooking extrusion process. This involved. many facets of investigation. A 50% added moisture soy flour dough was selected as the material of study. The material properties for this 50% added moisture dough were then determined. The viscosity of this material was found to be both shear and temperature dependent in addition to exhibiting a yield stress. Both thermal conductivity and heat capacity were determined to be constant over the temperature range of investigation. Finally, although it was discovered that a reaction associated with cooking was present in the system, it was determined that it did not occur under the extrusion processes tested which were to be modelled and hence would not have to be accounted for in the model. These material properties were then incorporated in the three-dimensional finite element program, FIDAP, to model the flow of the l 50% added moisture soy flour dough through an extruder and die assembly. These numerical simulations yielded limited results. Only one case out of the multiple conditions which were attempted converged to a viable solution. As more success was found with a two-dimensional model, it is suggested that the problems of convergence could be due to mesh size and discretization of the three dimensional model as well as the difficult power law index of the material. Suggestions as to methods to overcome these problems are included. / Ph. D.

Com o aumento do número de animais inseminados por IATF (inseminação artificial em tempo fixo), a diminuição de custos e o incremento de índices reprodutivos têm sido os objetivos de vários grupos de pesquisa. Nos protocolos de IATF utilizados, empregam-se dispositivos de liberação sustentada de progesterona (P4), que são, em sua maioria, constituídos de um esqueleto de nylon, recoberto com uma camada de silicone com P4 . Visando a diminuição dos custos de produção e impacto ambiental, foi desenvolvido um dispositivo à base de biopolímeros. Neste estudo, foram comparados dispositivos confeccionados com uma blenda constituída de Poli-hidroxi-butirato (PHB) e Poli-&epsion;-caprolactona (PCL), com o Dispositivo Interno Bovino (DIB&reg;), utilizado como controle . No teste in vitro, utilizaram-se dois tipos de dispositivo à base de biopolímeros, com uma área superficial de 147 cm2: DISP8 (46% PHB, 46% PCL e 8% P4; n=4), DISP10 (45% PHB, 45% PCL, 10% P4; n=4) e o DIB&reg; (1 g de P4, área de 120 cm2; n=3). Os testes in vitro foram conduzidos segundo especificações da USP 23, em um dissolutor de comprimidos, empregando-se uma mistura de álcool/água (60/40) como meio de difusão. Amostras do meio foram colhidas aos 2 min., 2, 4, 8, 12, 24, 48, 60, 72, 84 e 96 h. Os teores de P4 foram dosados por espectrofotometria, em 244 nm de comprimento de onda. Realizaram-se as comparações 3 a 3 dos coeficientes angulares das retas obtidas pela regressão das concentrações acumuladas de P4, em função da raiz quadrada do tempo. As médias e respectivos erros-padrão dos coeficientes angulares foram de 677,39 ± 16.13 µg/cm2/t1/2 para o DIB&reg;, 566,17 ± 3.68 µg/cm2/t1/2 para o Disp8 e 774,63 ± 45.26 µg/cm2/t1/2 para o Disp10. Houve diferença entre o DISP8 e o DISP10 (p< 0,05), mas ambos não diferiram do DIB. Para análise das quantidades liberadas por dia de teste in vitro, consideraram-se 4 períodos: 0-24, 24-48, 48-72 e 72-96 h. Houve interação entre tratamento e tempo (p=0,0024). Nas primeiras 24 horas, o DISP8 liberou significativamente menos P4 do que o DISP10 e o DIB&reg;, cujas liberações não diferiram entre si. No intervalo entre 24 e 48h, o DISP 10 liberou significativamente mais P4 do que o DIB&reg;. O DISP8 liberou uma quantidade de P4 intermediária e não se diferenciou significativamente nem do DIB&reg; e nem do DISP10. No intervalo entre 48 e 72h a quantidade de P4 liberada pelo DISP10 foi significativamente maior do que a do DIB&reg; e a do DISP8, que não diferiram entre si. No intervalo entre 72 e 96h, o DISP10 liberou significativamente mais P4 do que o DIB&reg; e o DISP8 liberou uma quantidade intermediária que não diferiu do DIB&reg; nem do DISP10. No teste in vivo, seis vacas mestiças ovariectomizadas receberam DIB (n=4) ou DISP8 (n=8), em um delineamento alternado, com seqüência não balanceada (cross-over), adicionado de medidas repetidas no tempo, referentes aos 16 dias de colheita das amostras de sangue. As amostras foram analisadas por rádio-imuno-ensaio, em fase sólida, utilizando-se &quotkit&quot comercial da DPC (Diagnostics Products Corporation). As concentrações de pico foram atingidas 4 h após a colocação dos dispositivos e este foi o único momento em que as concentrações plasmáticas de P4 diferiram segundo dispositivo (11,45±1,96 vs 9,23±1,15 ng/mL, respectivamente para DIB e DISP8; p= 0,027). No dia 8 do ensaio, as concentrações plasmáticas de P4 proporcionadas pelo DIB e pelo DISP8 foram respectivamente 2,44±0,09 e 1,89±0,13 ng/mL (p=0.58). Ambos dispositivos mantiveram teores de P4 superiores a 1 ng/mL, durante os 16 dias do teste in vivo. Concluiu-se que os dispositivos confeccionados com a blenda dos biopolímeros PHB/PCL, podem liberar P4 de maneira sustentada, tanto quanto os que empregam silicone em sua fabricação. / With the increase in the number of animals inseminated by TAI (timed artificial insemination) the decrease of costs and the improvement of reproductive performance are the goals of several research groups. Progesterone devices which are majority constituted of a nylon skeleton covered with a silicone layer with P4, are used in TAI protocols. Aiming the decrease of the production costs and the environmental impact, was a new device developed with biopolymers matrix. In this study, were compared a blend of Poli-hidroxi-butirate (PHB) and poli-?-caprolactone (PCL) device with the Bovine Internal Device (DIB&reg;). In the in vitro test, two kinds of the new device with a superficial area of 147 cm2: DISP8 (46% PHB, 46% PCL and 8% P4; n=4), DISP10 (45% PHB, 45% PCL, 10% P4; n=4) and DIB&reg; (1 g of P4, area of 120 cm2; n=3). The in vitro tests were led following USP 23 specifications in a drug dissolutor in alcohol/water (60/40) solution as a diffusion medium. Samples were collected at 2 min., 2, 4, 8, 12, 24, 48, 60, 72, 84 and 96 hours. Progesterone concentrations were estimated by spectrophotometry at UV 244 nm wavelenght. Comparisons 3 by 3 of the angular coefficients of the straight lines obtained by the regression of the accumulated P4 concentrations in function of the square root of time were performed. The averages and respective standard-errors of the angular coefficients were 677,39 ± 16.13 µg/cm2/t1/2 to DIB&reg;, 566,17 ± 3.68 µg/cm2/t1/2 to Disp8 and 774,63 ± 45.26 µg/cm2/t1/2 to Disp10. Difference were observed between DISP8 and DISP10 (p< 0.05) but both did not differ to DIB&reg;. Four periods were considered for analysis of the amounts released per day of in vitro test: 0-24, 24-48, 48-72 and 72-96 hours. Interaction between treatment and time was observed (p=0.0024). In the first 24 hours DISP8 released significantly less P4 than DISP10 and DIB&reg; but the lathers did not differ from each other. In the interval between 24 and 48 hours DISP10 released significantly more P4 than DIB&reg;. The device DISP8 released an intermediary concentration of P4 and was similar to DIB&reg; and to DISP10. In the interval between 48 and 72 hours the progesterone concentration released by DISP10 was significantly higher than DIB&reg; and DISP8 but lathers did not differ from each other. In the interval between 72 and 96 hours DISP10 released significantly more P4 than DIB&reg; while DISP8 released an intermediary amount which did not differ to DIB&reg; or to DISP10. In the test in vivo test, six ovariectomized beef cows received the devices DIB&reg; (n=4) or DISP8 (n=8) in an alternated delineation in a nom balanced sequence (cross-over) added of repeated measures in time, related to the 16 days of blood samples collects. Progesterone concentration from these samples were measured by radioimmunoassay in solid phase using the commercial kit from DPC (Diagnostics Products Corporation). The peak of the progesterone concentrations were observed 4 hours after the devices insertion and this was the unique moment in which plasmatic progesterone concentrations differed depending on device (11,45 ± 1,96 vs 9,23 ± 1,15 ng/mL, respectively for DIB and DISP8; p= 0,027). After 8 days from the device insertion plasmatic progesterone concentrations provided by DIB and DISP8 were respectively 2,44 ± 0,09 and 1,89 ± 0,13 ng/mL (p=0.58). Both devices kept the progesterone concentration higher than 1 ng/mL during the 16 days of the in vivo test. In conclusion, the devices made with biopolymers blends of PHB/PCL can release sustained P4 comparable with silicone matrix.

Η παρούσα διατριβή εξειδίκευσης αποτελεί το προοίμιο μιας μακρόπνοης εμπλοκής του εργαστηρίου υλοποίησής της στη μελέτη ενδεχόμενης μετανάστευσης ουσιών που χρησιμοποιούνται ως ενισχυτικά φραγής ή/και ως χημικοί αισθητήρες σε βιοπολυμερικές συσκευασίες τροφίμων και αποδέσμευσής τους σε προσομοιωτές τροφίμων. Στο πλαίσιο αυτό, η εργασία αυτή αποτελεί μια προσπάθεια ανάδειξης της μεθόδου επιφανειακής ενίσχυσης της σκέδασης Raman (Surafce Enhanced Raman Scattering) ως κατάλληλης για τον ποσοτικό προσδιορισμό μικρού μοριακού βάρους ενώσεων που ενδεχομένως αποδεσμεύονται σε υδατικά διαλύματα ή/και συγκεκριμένους προσομοιωτές τροφίμων. Η μελέτη εστιάστηκε στην ελεγχόμενη αποδέσμευση φαρμακευτικών ουσιών από μια βιοπολυμερική μήτρα κυρίως κατά το πρώιμο στάδιο της μελέτης και τις παραμέτρους που επηρεάζουν το φαινόμενο αυτό. Βασικός στόχος της μελέτης ήταν η κατά το δυνατό μείωση του ορίου ανίχνευσης με SERS της αποδεσμευόμενης ουσίας με την εμβάπτιση της βιοπολυμερικής μήτρας που την εμπεριέχει σε πρότυπα υδατικά διαλύματα. Πραγματοποιήθηκε μια διεξοδική μελέτη των υποστρωμάτων που χρησιμοποιούνται στο SERS και συγκεκριμένα του νανοκολλοειδούς αργύρου (Ag). Για τον σκοπό αυτό, πραγματοποιήθηκαν πειράματα τα οποία έδειξαν την εξάρτηση της έντασης SERS από τη συσσωμάτωση των υποστρωμάτων Ag συναρτήσει του χρόνου και του παράγοντα συσσωμάτωσης, NaCl. Η εφαρμογή του SERS σε μελέτες ουσιών εξαιρετικά χαμηλών συγκεντρώσεων αναδεικνύεται ως ένα πολύ ενδιαφέρον πεδίο έρευνας. Επίσης, ως πρότυπο πείραμα, παρουσιάζεται μεθοδολογία μελέτης με την τεχνική SERS της αποδέσμευσης του αντικαρκινικού φαρμάκου Μitoxantrone (ΜΤΧ) από εμπορικά ράμματα Maxon. Για το σκοπό αυτό, παρασκευάστηκαν πολυμερικά υμένια με εγκλωβισμένη τη δραστική ουσία και η μελέτη της αποδέσμευσης της σε νερό και PBS (phosphate buffered saline) πραγματοποιήθηκε με SERS και UV-Vis, αντίστοιχα. Η φασματοσκοπία UV-Vis χρησιμοποιήθηκε συμπληρωματικά. Στηριζόμενοι στη μεθοδολογία που αναπτύξαμε εξάγαμε ποσοτικά αποτελέσματα από τρία διαφορετικά εργαστηριακά δείγματα, τα οποία προήλθαν από ανάμιξη εμπορικών ραμμάτων Maxon με 1% κ.β. MTX: (α) στην άμορφη φάση έπειτα από ταχεία ψύξη του τήγματος, (β) στην ημικρυσταλλική φάση με σχετικά χαμηλό ποσοστό κρυσταλλικότητας, που λάβαμε έπειτα από ανόπτηση της άμορφης φάσης για περιορισμένο χρόνο στη θερμοκρασία κρυστάλλωσης και (γ) σε μια επίσης ημικρυσταλλική φάση με αρκετά μεγάλο ποσοστό κρυστάλλωσης (όσης και τα εμπορικά ράμματα). Τα αποτελέσματα δείχνουν πως υπάρχει συσχέτιση μεταξύ κρυσταλλικότητας και αποδέσμευσης του φαρμάκου, με τα μικρότερα ποσά αποδέσμευσης στην περίπτωση του άμορφου δείγματος. Αυτό που παρατηρήθηκε στα πρώιμα στάδια της αποδέσμευσης από τις μετρήσεις SERS φαίνεται να επαληθεύεται από αντίστοιχα αποτελέσματα σε μεταγενέστερα στάδια αποδέσμευσης που λάβαμε με εφαρμογή της συμβατικής τεχνικής απορρόφησης ορατού – υπεριώδους (UV-Vis). Ωστόσο, οι ποσοτικές μετρήσεις με τη χρήση του SERS σε πολύ μικρές συγκεντρώσεις έδειξαν μεγαλύτερη ανιχνευτική ευαισθησία σε σχέση με αυτές που πραγματοποιήθηκαν με την απορρόφηση UV-Vis. Συμπερασματικά, το SERS δείχνει ικανό στον ποσοτικό προσδιορισμό ενεργών ουσιών που αποδεσμεύονται από βιοσυμβατά πολυμερικά συστήματα μεταφοράς δραστικών ουσιών σε πολύ μικρές συγκεντρώσεις. / This thesis of specialization is the precursor of a long-term involvement of the laboratory of Applied Molecular Spectroscopy of FORTH/ICE-HT in the implementation of the study of the migration of substances used as barrier and/or as chemical sensors in biopolymer based food packaging and their release into food simulants. In this context, this work attempts to highlight the method of surface enhanced Raman scattering (SERS) as appropriate for quantifying low molecular weight compounds that may be released in aqueous solutions and/or specific food simulants. The study focused on the controlled release of pharmaceuticals from a biopolymeric matrix mainly during the early stage of the study and the parameters affecting this phenomenon. The main objective of the study was to reduce SERS detection limit of the released substance by emerging the substance-incorporated biopolymeric matrix in standard aqueous solutions. In this context, we developed methods to maximize SERS enhancement and consequently reduce the limit of detection of an active substance, Mitoxantrone (MTX). This was achieved by a thorough study of the substrates used in SERS, namely nanocolloidal silver (Ag) suspensions. For this purpose, we contacted experiments which show the dependence of the SERS intensity on the aggregation of Ag substrates as a function of both time and the aggregating agent, NaCl. Also, as a standard experiment, present SERS methodology was applied in the study of the release of the anticancer drug Mitoxantrone (MTX) from commercially available sutures, Maxon. For this purpose, polymeric films prepared with the encapsulated active substance were immersed either in water or/and in PBS (phosphate buffered saline) and the release of MTX was probed by both SERS and UV-Vis. Based on the developed methodology we obtained quantitative results from three different laboratory samples produced by mixing commercial Maxon sutures with 1 wt% MTX: (a) an almost completely amorphous mixture produced by quenching from the melt, (b) a semi-crystalline one possessing low crystallinity that was produced by annealing the amorphous sample at the temperature close to the crystallization one and (c) a semi-crystalline one possessing high crystallinity similar to the commercial product. The results indicate a correlation between crystallinity and drug release rate; the more amorphous the sample is the less quantity of the drug is released. SERS was able to probe the active agent at the early state of release; UV-Vis has supported these results at a later state of the release process. In conclusion, SERS may enable low concentration quantitative assessment of controlled release of drugs from biopolymer-based delivery systems.

Aus der Einleitung: "Im Tissue Engineering und in der Medizintechnik gewinnt das Rapid Prototyping (RP), das zu den additiven Fertigungsverfahren zählt, zunehmend an Bedeutung (Zhang, et al. 2015) (Li, et al. 2014). Für die Verarbeitung von thermoplastischen Biopolymeren ist das Fused Deposition Modeling (FDM, schematische Darstellung in Abbildung 1) von zentralem Stellenwert. ..."

L'objectif de ce travail de thèse est de fournir une étude systématique et détaillée des propriétés de mélanges de PLA et de PHA préparés à l'état fondu. En particulier, le comportement rhéologique, la miscibilité, la morphologie, les propriétés mécaniques et l'influence du vieillissement physique sur les propriétés en traction des mélanges PLA/PHBV réalisés en mélangeur interne et par extrusion bi-vis sur toute la gamme de composition ont été étudiés.La sensibilité du PHBV à la dégradation thermique a été mise en évidence, ainsi que l'importance des interfaces entre le PLA et le PHBV sur le comportement rhéologique aux basses fréquences. Les analyses de calorimétrie différentielle à balayage ont montré l'immiscibilité des deux polymères. Deux types de morphologies distinctes (nodulaire et co-continue) selon la composition de mélange ont été observées par microscopie électronique à balayage et microscopie optique en réflexion. La résistance au choc des mélanges PLA/PHBV avec et sans modifiant choc a été étudiée et les propriétés en traction de différents mélanges de PLA avec du PHBV, du Lotader GMA ou du PBAT ont été caractérisées. L'ajout d'une faible quantité de phase dispersée dans la matrice de PLA, initialement fragile, permet d'augmenter considérablement son allongement à la rupture. Cependant, les propriétés ductiles obtenues diminuent avec le temps écoulé entre la mise en forme des échantillons et leur test mécanique. Une étude de l'influence du vieillissement physique sur les propriétés des mélanges à base de PLA a donc été réalisée et un modèle qualitatif a été proposé. / The objective of this PhD work is to provide a systematic and detailed study of the properties of PLA and PHA blends prepared by melt mixing. In particular, the rheological behaviour, the miscibility, the morphology, the mechanical properties and the influence of physical aging on the tensile properties of PLA/PHBV blends prepared in an internal mixer and by twin screw extrusion over the entire composition range were studied.The sensibility of PHBV to thermal degradation was highlighted, as well as the importance of the interfaces between PLA and PHBV on the rheological behaviour at low frequencies. The differential scanning calorimetry showed that the two polymers are immiscible. Two different types of morphologies (nodular and co-continuous) depending on blend composition have been observed by scanning electron microscopy and optical microscopy in reflection. The impact resistance of the PLA/PHBV blends with or without impact modifier was studied and the tensile properties of different blends containing PLA blended with PHBV, Lotader GMA, or PBAT were characterised. The addition of a small amount of dispersed phase in the initially brittle PLA matrix increases considerably its elongation at break. However, the obtained ductile properties decrease as the time between preparation of samples and their mechanical testing increases. A study of the influence of physical aging on the properties of PLA based blends was conducted and a qualitative model was proposed.

La chitine est l’un des biopolymères les plus abondants dans la biomasse. Son principal dérivé industriel est le chitosane. Ces deux polysaccharides présentent un intérêt croissant en raison de leurs nombreuses propriétés physico-chimiques et biologiques remarquables. Leur utilisation en tant que matériaux est potentiellement intéressante dans de nombreux domaines tels que la pharmacie, la médecine, l’industrie alimentaire et l'agriculture. Malgré de nombreuses avancées dans les méthodes chimiques de production de la chitine et du chitosane, l'utilisation de solutions concentrées d'acides et de bases alcalines présente le désavantage de générer de grandes quantités d’effluents toxiques. Récemment, des méthodes d'extraction de la chitine par biotechnologie ont été proposées comme une alternative aux méthodes chimiques. Dans ce contexte, l'objectif principal de ce travail a été de développer un procédé biotechnologique d’extraction de la chitine à partir de carapaces de crustacés à l’aide de bactéries lactiques et d’enzymes. A cette fin, les facteurs influençant les réactions mises en jeu au cours de l’extraction, telles que la déminéralisation, la déprotéinisation et la N-désacétylation ont été étudiés en considérant les caractéristiques physico-chimiques des chitines et chitosanes ainsi obtenus. Ces caractéristiques sont principalement les masses molaires moyennes et le degré de N-acétylation. Ce travail s’est également intéressé à la valorisation des sous-produits d’extraction (protéines, pigments…) et aux applications potentielles des différents chitosanes préparés, notamment comme agents fongistatiques. / The chitin is one of the most abundant biopolymers in biomass. Its main industrial derivative is the chitosan. These two polysaccharides present an increasing interest thanks to their various interesting physicochemical and biological properties. Their potential applications concern diverse fields as the pharmacy, medicine, food industry and agriculture. Despite numerous advances in methods for the chemical production of chitin and chitosan, the use of concentrated solutions of acids and alkaline bases presents the disadvantage to generate large amounts of toxic wastes. Recently, biotechnological methods of chitin extractions have been proposed as an alternative to chemical methods. In this context, the main objective of this work was to develop a biotechnological process for the extraction of chitin from crustacean shells using lactic acid bacteria and enzymes. For this purpose, factors influencing reactions involved in the extraction, i.e. the demineralization, deproteinization and N-deacetylation, were studied considering the physicochemical characteristics (molecular weight and degree of N-acetylation) of chitin and chitosan produced. The recovery of extraction side products such as proteins and pigments has also been considered in this project, as well as the evaluation of various chitosans prepared by this process as fungistatic agents.

Compared to conventional fibers, carbon nanotubes possess several significant properties, which make them as an excellent alternative reinforcement in multi-functional material industry. In this study, the possibility of dispersion of the multi-wall carbon nanotube (MWCNTs) in a thermoset bio-based resin (synthesized based on end-functionalized glycerol-lactic acid oligomers, GLA, at university of Borås) was investigated. Furthermore, the addition of the MWCNTs as reinforcement to improve the mechanical and thermal properties of was investigated. The nanocomposites were prepared in three different concentrations of MWCNTs, 0.3 wt.%, 1.0 wt.%, and 2.0 wt.%, and each sample was prepared using three different dispersion methods such as the high speed mixer(HSM), the ultra-sonication (US), and a combined method of HSM &amp; US. The mechanical and thermal properties were analyzed by flexural test, thermogravimetric analysis (TGA), and differential scanning calorimetry (DSC). The results confirm that the nanotubes can be dispersed in GLA but the cured nanocomposite didn’t exhibit any considerable improvement in their thermal properties. Considering to the mechanical properties, the addition of 0.3 wt. % MWCNTs to the GLA increased the flexural strength a little but increasing the nanotubes to 1.0 wt. % decreases the flexural strength to almost 50%. This is mainly due to increase in the brittleness of the produced nanocomposites. Both the distribution methods dispersed the nanomaterials in the matrix initially but they are not efficient enough to stop the re-agglomeration which leads to undesired curing dynamics and low efficiency. Thus, these dispersion methods need to be optimized for improvement of nanocomposites’ properties.

RNA damage occurring under oxidative stress has been shown to cause RNA dysfunction and must be detrimental to cells and organisms. We propose that damaged RNA can be removed by specific RNA surveillance activities. In this work, we investigated the role of polynucleotide phosphorylase (PNPase), a 3'->5' exoribonuclease, in protecting the cells against oxidative stress and eliminating oxidatively-damaged RNA. Previously, it was reported that E. coli PNPase has a higher affinity to poly(8-oxoG:A). We further confirmed that E. coli PNPase can specifically bind to an oxidized RNA with a high affinity. An E. coli strain deficient in PNPase (pnp) is hypersensitive to hydrogen peroxide (H2O2). Importantly, the level of H2O2-induced RNA damage, measured by the content of 8-hydroxyguanosine, increases significantly in the pnp mutant cells. Consistent with the notion that PNPase plays a direct role in these processes, introduction of the pnp gene encoding E. coli PNPase can restore the viability and RNA oxidation level of the pnp mutant cells in response to H2O2 treatment. Interestingly, degradosome-association is not required for PNPase to protect cell against oxidative stress. PNPase is evolutionary conserved in most of organisms of all domains of life. The human polynucleotide phosphorylase (hPNPase) localizes mainly in mitochondria and plays pleiotropic roles in cell differentiation and has been previously shown to bind 8- oxoG-RNA with a high affinity. Here we show that similar to E. coli PNPase, hPNPase plays an indispensable role in protecting HeLa cells against oxidative stress. The viability in HeLa cell and 8-oxoG levels in RNA are inversely correlated in response to H2O2- treatment. After removal of oxidative challenge, the elevated level of 8-oxoG in RNA decreases, suggesting the existence of surveillance mechanism(s) for cleaning up oxidized RNA. / We have shown that hPNPase may be responsible for the surveillance of oxidized RNA in mammalian cells.Overexpresion of hPNPase reduces RNA oxidation and increases HeLa cell viability against H2O2 insult. Conversely, hPNPase knockdown decreases the viability and increases 8-oxoG level in HeLa cells exposed to H2O2. Taken together, our results suggest that RNA oxidation is a challenging problem for living organisms, and PNPase may play an important role in protecting both prokaryotic and eukaryotic cells by limiting damage to RNA under oxidative stress. / by Jinhua Wu. / Thesis (Ph.D.)--Florida Atlantic University, 2008. / Includes bibliography. / Electronic reproduction. Boca Raton, Fla., 2008. Mode of access: World Wide Web.