Stenting remains a common treatment option for atherosclerotic arteries. The main drawback of early stent platforms was restenosis, which has been combated by drug eluting stents; however, these stents have suffered from a higher incidence of late stage thrombosis. To address current stenting limitations, the major research focuses have been the development of the next generation of drug eluting stents and first generation bioabsorbable stents. The main objective of this dissertation was the design and development of a new class of bioabsorbable stent composed of elastin mimetic protein polymers. The first phase explored different stent design schemes and fabrication strategies. Successfully fabricated stents were then mechanically tested to ensure they possessed sufficient mechanical strength. Additionally, described herein is the potential to modulate the properties of the elastin mimetics through different crosslinking strategies. We have demonstrated that chemical crosslinking allows for the tailoring of the physical, mechanical, drug delivery, and endothelialization properties of these materials. The potential for drug delivery from this elastin mimetic stent was benchmarked as was the potential to endothelialize these stents. Furthermore, we developed the necessary delivery systems to allow for deployment in the rat aorta model.

Tissue engineered human oral mucosa has the potential to fill tissue deficits caused by facial trauma or malignant lesion surgery. It can also help elucidate the biology of oral mucosa and serve as an alternative to in vivo testing of oral care products. The aim of this thesis was to construct a tissue engineered full-thickness human oral mucosa closely mimicking the native tissue. To this end, the feasibility of the concept was tested by co-culturing fibroblasts and epithelial cells isolated from normal human oral mucosa biopsies in a collagen-glycosaminoglycan-chitosan scaffold, developed in our laboratory to construct a skin equivalent. An oral mucosal equivalent closely mimicking the native one was obtained and characterized by histology, immunohistochemistry and transmission electron microscopy. Using the same model, the influence of mesenchymal cells on oral epithelial development was investigated by culturing epithelial cells on lamina propria, corneal stroma and dermal equivalents. They were found to significantly influence the thickness and the ultrastructure of the epithelium. Finally, in order to improve the adhesiveness of conventional scaffolds, an elastin-like recombinamer (ELR) containing the cell adhesion tripeptide, RGD, was used in the production of novel bilayer scaffolds employing lyophilization and electrospinning. These scaffolds were characterized by mercury porosimetry, scanning electron microscopy and mechanical testing. In vitro tests revealed positive contribution of ELR on the proliferation of both fibroblasts and epithelial cells. It was thus possible to construct a viable oral mucosa equivalent using the principles of tissue engineering.

Recombinant synthesis of elastin-mimetic proteins has been employed for several decades, however, long-term biocompatibility and biostability of such proteins was not fully defined. We present virtually crosslinked elastin-mimetic proteins which exhibit exceptional biocompatibility and long-term biostability over a period of at least seven months. This report is the first evidence of a non-chemically or ionically crosslinked system that exhibits long-term in vivo stability. Although, physically crosslinked protein-based materials possess a number of advantages over their chemically crosslinked counterparts, physical crosslinks and the related domains so formed may be deformed or damaged at applied stresses lower than those required to disrupt covalent crosslinks. In this regard, we have synthesized a new class of recombinant elastin-mimetic triblock copolymer capable of both physical and chemical crosslinking. We have demonstrated that chemical crosslinking provides an independent mechanism for control of protein mechanical responses. Specifically, elastic modulus was enhanced and creep strain reduced through the addition of chemical crosslinking sites. A number of reports have described the design of synthetic genes, which encode elastin-like proteins for bacterial expression in Escherichia coli. Although advantages with this expression system exist, significant limitations including the lack of eukaryotic post-translational systems, the tendency to sequester mammalian proteins into inclusion bodies, difficult purification protocols, and endotoxin contamination have been noted. We demonstrate the expression of a recombinant elastin-mimetic protein from P. pastoris. A novel synthetic strategy, monomer library concatamerization, was utilized in designing non-repetitive elastin genes for highly repetitive protein sequences. It is likely that this strategy will be useful for creating large, repetitive genes for a variety of expression systems in order to more closely approach the genetic diversity inherent to native DNA sequences. All told, elastin-based protein polymers are a promising class of material characterized by high degree of biocompatibility, excellent biostability, and a tunable range of mechanical properties from plastic to elastic. A variety of options facilitate the processing of these biopolymers into chemically crosslinked or non-crosslinked gels, films, or nanofibers for any of a number of implant applications including structural components of artificial organs and engineered living tissues, carriers for controlled drug release, or biocompatible surface coatings.

abstract: Gold nanoparticles as potential diagnostic, therapeutic and sensing systems have a long history of use in medicine, and have expanded to a variety of applications. Gold nanoparticles are attractive in biological applications due to their unique optical, chemical and biological properties. Particularly, gold nanorods (GNRs) are increasingly used due to superior optical property in the near infrared (NIR) window. Light absorbed by the nanorod can be dissipated as heat efficiently or re-emitted by the particle. However, the limitations for clinical translation of gold nanorods include low yields, poor stability, depth-restricted imaging, and resistance of cancer cells to hyperthermia, are severe. A novel high-throughput synthesis method was employed to significantly increase in yields of solid and porous gold nanorods/wires. Stable functional nanoassemblies and nanomaterials were generated by interfacing gold nanorods with a variety of polymeric and polypeptide-based coatings, resulting in unique properties of polymer-gold nanorod assemblies and composites. Here the use of these modified gold nanorods in a variety of applications including optical sensors, cancer therapeutics, and nanobiomaterials were described. / Dissertation/Thesis / Ph.D. Chemical Engineering 2012

La Broncho-Pneumopathie Chronique Obstructive (BPCO) est une pathologie pulmonaire complexe entrainant une diminution progressive et irréversible des capacités respiratoires. L'emphysème est la composante majoritaire de la BPCO. Une importante réponse inflammatoire chronique est associée à la BPCO et implique à la fois les cellules de l'immunité innée, comme les macrophages et les polynucléaires neutrophiles (PN), et les cellules de l'immunité adaptative. La BPCO est également caractérisée par une destruction importante du tissu pulmonaire qui est associée à une dégradation de l'élastine et du collagène, deux protéines essentielles de la matrice extra-cellulaire (MEC). Cette dégradation génère des peptides doués de nombreuses activités biologiques. Le travail de thèse présenté ici nous a permis de montrer que l'injection intra-trachéale du peptide VGVAPG, une séquence active des peptides d'élastine (PE) induit un emphysème chez la souris, emphysème caractérisé par une forte infiltration des macrophages et des PN dans les lavages bronchoalvéolaires (LBA) et dans le parenchyme pulmonaire et par une destruction de la MEC au niveau du tissu pulmonaire. L'effet des PE s'exerce par le biais du complexe récepteur à l'élastine (CRE) et est déclenché par l'interaction des PE avec la sous-unité EBP du complexe. Nous avons montré, dans un travail précédent, que la spécificité de l'interaction entre l'EBP et les PE est associée à la présence du motif GXXP dans les peptides et à l'adoption d'une conformation en coude β de type VIII par le peptide. Sur la base de ces résultats, nous avons effectué, à partir du peptide VGVAPG, des mutations systématiques des résidus centraux de GXXP afin de déterminer les peptides les plus favorablement repliés en coude beta de type VIII et nous avons sélectionné les meilleurs candidats pour la fixation à l'EBP. Nous avons focalisé notre attention sur les peptides interagissant avec l'EBP et qui montraient des effets biologiques modérés. La possibilité d'utiliser de tels peptides comme des antagonistes des effets du peptide VGVAPG a été évaluée in vitro et in vivo. / The Chronic Obstructive Pulmonary Disease (COPD) is a complex lung disease causing a progressive and irreversible decrease in respiratory capacity. Emphysema is the major component of COPD. An important chronic inflammatory response is associated with COPD and involves both cells of innate immunity, such as macrophages and neutrophils (PN), and the cells of the adaptive immunity. COPD is characterized by extensive destruction of lung tissue that is associated with degradation of elastin and collagen, two essential proteins of the extracellular matrix (ECM). This degradation generates peptides endowed numerous biological activities. The thesis presented here has allowed us to show that the intratracheal injection of peptide VGVAPG, an active sequence of elastin peptides (PE)-induced emphysema in mice, emphysema characterized by a strong infiltration of macrophages and PN in the bronchoalveolar lavage (BAL) and in the lung parenchyma and the destruction of the ECM in the lung tissue. The effect of PE is exercised through the elastin receptor complex (CRE) and is triggered by the interaction of PE with subunit EBP complex. We have shown in previous work that the specificity of the interaction between EBP and PE is associated with the presence of GXXP motif in peptides and the adoption of a conformation type VIII β turn by peptide. Based on these results, we have made, from the peptide VGVAPG, systematic changes of the central residues GXXP to identify peptides most favorably bent type VIII β turn and we selected the best candidates for fixing to EBP. We focused our attention on the peptides interact with the EBP, which showed moderate biological effects. The possibility of using such peptides as antagonists of the effects of peptide VGVAPG was evaluated in vitro and in vivo.

No description available.

Extracellular matrix (ECM) is a key component and regulator of many biological tissues. Several cardiovascular pathologies are associated with significant changes in the composition of the matrix. Better understanding of these pathologies and the physiological phenomenon behind their development depends on reliable methods that can measure and characterize ECM content and structure. In this dissertation, infrared spectroscopic methodologies are developed to study the changes in extracellular matrix of cardiovascular tissue in two cardiovascular pathologies; myocardial infarction and abdominal aortic aneurysm. The specific aims of this dissertation were: 1. To develop a Fourier transform infrared imaging spectroscopy (FT-IRIS) methodology for creating distribution maps of collagen in remodeled cardiac tissue sections after myocardial infarction, and to quantitatively compare maps created by FT-IRIS with conventional staining techniques. 2. To develop an FT-IRIS method to assess elastin and collagen composition in the aortic wall. This will be accomplished using ex vivo animal aorta samples, where the primary ECM components of the wall will be systematically enzymatically degraded. 3. To apply the newly developed FTIR imaging methodology to evaluate changes in the primary ECM components (collagen and elastin) in the wall of human AAA tissues. The infrared absorbance band centered at 1338 cm-1, was used to map collagen deposition across heart tissue sections of a rat model of myocardial infarction, and was correlated strongly in the size of the scar (R=0.93) and local intensity of collagen deposition (R=0.86). In enzymatically degraded pig aorta samples, as a model of ECM degradation in abdominal aortic aneurysm (AAA), partial least squares (PLS) models were created to predict collagen and elastin content in aorta based on collected FTIR spectra and biochemically measured values. PLS models based on FT-IRIS spectra were able to predict elastin and collagen content of the samples with strong correlations (R2=0.90 and 0.70 respectively). Elastin content prediction from IFOP spectra was successful through a PLS regression model with high correlation (R2=0.81). The PLS regression coefficient from the FT-IRIS models were used to map collagen and elastin human AAA biopsy tissue sections, creating a similar map of each component compared to histologically stained images. The mean value of collagen deposition in each tissue was calculated for 13 pairs of AAA samples where stress had been calculated using finite element modeling. In most pairs with stress values higher than 5 N/m2, collagen content was lower in the sample with higher stress value. Collagen maturity had a weak negative correlation (R=-0.35) with collagen content in these samples. These results confirm that infrared spectroscopy is a powerful tool that can be applied to replace or complement conventional methods such as histology and biochemical analysis to characterize ECM components in cardiovascular tissues. Furthermore, infrared spectroscopy has the potential for translation to a clinical environment to examine ECM changes in aorta in a minimally invasive fashion using fiber optic technology. / Mechanical Engineering

Le mélanome est un cancer agressif dont la progression est facilitée par la dégradation de la matrice extracellulaire, à la fois par les fibroblastes et les cellules tumorales. Cette dégradation génère des peptides d’élastine, notamment responsables de la prolifération des cellules saines et cancéreuses. Le peptide d’élastine (VGVAPG)3 accélère la reprise et le déroulement du cycle cellulaire de fibroblastes normaux et de cellules de mélanomes préalablement synchronisés (expression de pKi-67, détection de la phase S et quantification d’ADN). L’architecture nucléaire associée à la reprise de la synthèse des ARNm concerne les compartiments nucléaires PML-NBs et domaines SC35, partenaires indissociables de la transcription et de l’épissage, qui sont étudiés, après immunomarquages, en microscopie confocale suivie d’une reconstruction 3D. Les compartiment PML-NBs et domaines SC35 se réorganisent en fonction d’une part des phases du cycle cellulaire et d’autre part de l’activité transcriptionnelle, passant d’une séquestration du SC35 dans les PML-NBs à une interpénétration des deux compartiments. L’analyse quantitative de ces compartiments complète les résultats architecturaux en 3D. Le peptide se fixe sur le complexe récepteur de l’élastine, et induit de l’activation de la voie MEK ½ ERK ½. Un antagoniste de cette fixation (lactose) ainsi que l’inhibition de la voie ERK ½ (UO126) conduisent à l’abolition des effets dus au peptide tant pour le cycle cellulaire que pour l’organisation des PML-NBs et domaines SC35, confirmant ainsi l’implication de ces voies. / Melanoma is an aggressive cancer for which invasion is facilitated by degradation of the extracellular matrix, both by normal fibroplasts and tumor cells. This degradation generates elastin peptides, in particular responsible for the proliferation of normal and tumor cells. The elastin peptide (VGVAPG)3. accelerates recovery and progression in cell cycle of normal fibroblasts and melanoma cells previously synchronized (expression of pKi-67, S-phase detection and quantification of DNA). The nuclear architecture associated with the recovery of the synthesis of mRNA on the PML-NBs nuclear compartments and SC35 domains, inseparable partners of transcription and splicing, which are studied after immunostaining by confocal microscopy followed by 3D reconstruction. Compartments PML-NBs and SC35domains are reorganized according of the phases of the cell cycle and also the transcriptional activity, from SC35 sequestration in PML-NBs to an interpenetration of the two compartments. The quantitative analysis of these compartments consolidates the 3D architectural results. The peptide binds to the elastin receptor complex and induceds the activation of the MEK 1/2 ERK 1/2. An antagonist of the fixation (lactose) and inhibition of ERK 1/2 (UO126) lead to the abolition of effects due to the peptide for both the cell cycle and the organized of PML-NBs and SC35 domains, confirming the involvement of these pathways.

La BPCO est une affection de l’appareil respiratoire associée à une réponse inflammatoire chronique. La BPCO associe une bronchite chronique, avec obstruction des voies aériennes, et un emphysème caractérisé par la destruction du parenchyme pulmonaire. Si l’exposition au tabac est le facteur de risque principal de la BPCO, plusieurs observations cliniques sont en faveur du rôle du vieillissement dans la pathogénèse de la maladie. La dégradation des fibres élastiques du poumon en peptides solubles d’élastine (PE) est une caractéristique constante de la BPCO, et le vieillissement physiologique du poumon est associé à une augmentation de son activité élastinolytique. Nous avons montré dans un travail précédent que l’instillation de PE à des souris induit un emphysème aigu. La relation existant entre l’élastolyse et le vieillissement conforte l’hypothèse que le vieillissement pourrait représenter un risque majeur de la BPCO par le biais de production de PE. Le travail de thèse présenté dans ce manuscrit a porté sur l’étude comparative des paramètres histologiques, inflammatoires et immunitaires liés à l’emphysème induit par les PE dans des souris jeunes et des souris âgées. Les résultats obtenus montrent que l’emphysème est d’apparition plus précoce chez les souris âgées et que cette précocité s’accompagne d’une augmentation de la production de PE et de clones T CD28- mémoires spécifiques des PE et du processus d’immunosénescence. / Chronic obstructive pulmonary disease (COPD) is a progressive and irreversible inflammatory state of the lung. COPD includes chronic bronchitis, with obstruction of small airways, and emphysema, characterized by the destruction of lung parenchyma. Although cigarette smoke exposure is the best known risk factor for COPD development, several clinical observations support the hypothesis that aging also play a key role in the pathogenesis of COPD. Degradation of lung elastin fibers, generating soluble elastin peptides (EP), is a feature of COPD development, and normal physiological aging of the lung is associated with increased elastolysis. We previously demonstrated in mice that endotracheal instillation of EP resulted in an acute emphysema establishment. The positive association between lung elastin breakdown and aging is consistent with the hypothesis that aging is a major risk factor of COPD through EP production. The work done during this thesis focused on the comparative study of histological, inflammatory and immune parameters related to PE-induced emphysema in young and elderly mice. The results we obtained show that emphysema development is earlier in the elderly mice and that this earliness is associated with an increase of EP-specific CD28- senescent memory T cell clones.

Longtemps considérée comme un simple support mécanique, la matrice extracellulaire (MEC) est un élément majeur dans le maintien de l’homéostasie. Ainsi l’élastine, principal constituant de la MEC des gros vaisseaux élastiques, est dégradée au cours du vieillissement, produisant ainsi des peptides d’élastine bioactifs (PE). Plusieurs études ont démontré l'implication des PE en physiopathologies tels que l’invasion tumorale, l’athérosclérose ou l’insulino-résistance (IRes). Ces effets s’expliquent par l’activation du complexe récepteur de l’élastine (CRE), composé par : une sous-unité extracellulaire liant les PE (EBP, elastin binding protein), la cathepsine A (dont le rôle reste inconnu), et la neuraminidase 1 (induisant la signalisation intracellulaire). L'IRes décrite, pourrait être associée à l’activité de désialylation de la neuraminidase-1 sur les chaines de N-glycosylation (Ng-c) du récepteur de l’insuline (RI). Sur la base de cette hypothèse, notre objectif a donc été de confirmer ce mécanisme et ses conséquences in silico (sur le RI), in vitro (pré-adipocytes 3T3-L1) et in vivo (aorte de souris). Nous montrons ainsi in vitro que les PE provoquent un dysfonctionnement de l’autophosphorylation du RI se répercutant sur plusieurs processus cellulaires comme l’entrée du glucose ou encore la différenciation adipocytaire. In silico, nous montrons pour la première fois le rôle des acides sialiques sur le comportement des Ng-c d'une part et sur le RI d'autre part. Enfin, in vivo, cette interaction CRE / IR engendre une hypertension artérielle par une diminution de la vasorelaxation des cellules endothéliales. / Often considered as a simple mechanical support, the extracellular matrix (ECM) is a major element of homeostasis regulation. Thus, elastin, the main constituent of large elastic vessels, is degraded during aging, producing bioactive elastin-derived-peptides (EDP). Several studies have demonstrated the EDP effects in physiopathologies such as tumor invasion, atherosclerosis, or insulin resistance (IRes) development. Those effects are explained by the activation of the elastin receptor complex (CRE), composed of: an extracellular subunit binding EDP (EBP, elastin binding protein), cathepsin A (its role is still unknown) and the sialidase neuraminidase-1 (Neu-1, involved in signaling pathway induction). Interestingly, the lab suggested that IRes may be induced by the desialylation of the N-glycan chains (Ng-c) on the insulin receptor (IR). The aim of this study was to confirm this hypothesis by demonstrating the consequence of desialylation on the IR in silico, on a 3T3-L1 pre-adipocyte cell in vitro, and on vascular complications in vivo. We show that EDP induce in vitro an impairment of IR autophosphorylation, affecting glucose uptake and adipocyte differentiation. In silico approach demonstrates the role of sialic acids on the behavior of Ng-c in the one hand and in other hand of IR. Finally, the IRes induced by ERC-IR interaction increase the vascular complication such as arterial hypertension by endothelial cell impairment. To conclude, Ng-c alteration would likely be responsible for structural changes in the IR at the origin of insulin resistance.