Tackling protein interfaces with small molecules capable of modulating protein-protein interactions remains a challenge in structure-based ligand design. Particularly arduous are cases in which the epitopes involved in molecular recognition have a non-structured and discontinuous nature. Here, the basic strategy of translating continuous binding epitopes into mimetic scaffolds cannot be applied, and other innovative approaches are therefore required. We present a structure-based rational approach involving the use of a regular expression syntax inspired in the well established PROSITE to define minimal descriptors of geometric and functional constraints signifying relevant functionalities for recognition in protein interfaces of non-continuous and unstructured nature. These descriptors feed a search engine that explores the currently available three-dimensional chemical space of the Protein Data Bank (PDB) in order to identify in a straightforward manner regular architectures containing the desired functionalities, which could be used as templates to guide the rational design of small natural-like scaffolds mimicking the targeted recognition site. The application of this rescaffolding strategy to the discovery of natural scaffolds incorporating a selection of functionalities of interleukin-10 receptor-1 (IL-10R1), which are relevant for its interaction with interleukin-10 (IL-10) has resulted in the de novo design of a new class of potent IL-10 peptidomimetic ligands.

Tackling protein interfaces with small molecules capable of modulating protein-protein interactions remains a challenge in structure-based ligand design. Particularly arduous are cases in which the epitopes involved in molecular recognition have a non-structured and discontinuous nature. Here, the basic strategy of translating continuous binding epitopes into mimetic scaffolds cannot be applied, and other innovative approaches are therefore required. We present a structure-based rational approach involving the use of a regular expression syntax inspired in the well established PROSITE to define minimal descriptors of geometric and functional constraints signifying relevant functionalities for recognition in protein interfaces of non-continuous and unstructured nature. These descriptors feed a search engine that explores the currently available three-dimensional chemical space of the Protein Data Bank (PDB) in order to identify in a straightforward manner regular architectures containing the desired functionalities, which could be used as templates to guide the rational design of small natural-like scaffolds mimicking the targeted recognition site. The application of this rescaffolding strategy to the discovery of natural scaffolds incorporating a selection of functionalities of interleukin-10 receptor-1 (IL-10R1), which are relevant for its interaction with interleukin-10 (IL-10) has resulted in the de novo design of a new class of potent IL-10 peptidomimetic ligands.

Le glioblastome (GBM) est la forme de cancer du cerveau la plus courante et la plus meurtrière. Sa nature diffusive entraine une impossibilité d’élimination complète par chirurgie. Une récidive de la tumeur chez ≥ 90% des patients peut être provoqué par des cellules GBM résiduelles se trouvant près du bord de la cavité de résection. Un implant pouvant libérer de manière durable la protéine SDF-1α, qui se lie aux récepteur CXCR4 à la surface des cellules GBM, peut être utile pour induire le recrutement des cellules GBM résiduelles, permettre leur élimination sélective et finalement réduire la récurrence de la tumeur. Dans ce travail, le SDF-1α a été initialement encapsulé dans des nanoparticules à base d'acide poly-lactique-co-glycolique (PLGA). Une efficacité d'encapsulation élevée (76%) a pu être obtenue en utilisant un processus simple de séparation de phase. Les nanoparticules chargées de SDF-1α ont ensuite été incorporées dans un scaffold à base de chitosan par électrofilage pour obtenir des implants nanofibreux imitant la structure de la matrice extracellulaire du cerveau. Une étude de libération in vitro a révélé que l'implant pouvait fournir une libération prolongée de SDF-1α jusqu'à 35 jours, utile pour établir un gradient de concentration de SDF-1α dans le cerveau et induire une attraction des cellules GBM. Une étude de biocompatibilité in vivo à 7 jours a révélé des signes d'inflammation locale sans aucun signe visible de détérioration clinique chez les sujets animaux. Une étude à 100 jours visant à confirmer l'innocuité in vivo des implants avant de passer aux études d'efficacité dans un modèle de résection GBM approprié est actuellement en cours. / Glioblastoma (GBM) is the most common and lethal form of brain cancer. The diffusive nature of GBM means the neoplastic tissue can not be removed completely by surgery. Often, residual GBM cells can be found close to the border of the resection cavity and these cells can multiply to cause tumor recurrence in ≥90% of GBM patients. An implant that can sustainably release chemoattractant molecules called stromal cell-derived factor-1α (SDF-1α), which bind selectively to CXCR4 receptors on the surface of GBM cells, may be useful for inducing chemotaxis and recruitment of the residual GBM cells. This may then give access to selective killing of the cells and ultimately reduce tumor recurrence. In this work, SDF-1α was initially encapsulated into poly-lactic-coglycolicacid (PLGA)-based nanoparticles. A high encapsulation efficiency (76%) could be achieved using a simple phase separation process. The SDF-1α-loaded nanoparticles were then incorporated into a chitosan-based scaffold by electrospinning to obtain nanofibrous implants that mimic the brain extracellular matrix structure. In vitro release study revealed that the implant could provide sustainedSDF-1α release for 5 weeks. The gradual SDF-1αrelease will be useful for establishing SDF-1α concentration gradients in the brain, which is critical for the chemotaxis of GBM cells. A 7-day in vivo biocompatibility study revealed evidence of inflammation at the implantation site without any visible signs of clinical deterioration in the animal subjects. A long-term study (100 days) aiming to confirm the in vivo safety of the implants before proceeding to efficacy studies in a suitable GBM resection model is currently underway.

Résumé soumis à confidentialité / Not available

Confining light metal hydrides in micro- or mesoporous scaffolds is considered to be a promising way to overcome the existing challenges for these materials, e.g. their application in hydrogen storage. Different techniques exist which allow us to homogeneously fill pores of a host matrix with the respective hydride, thus yielding well defined composite materials. For this report, the ordered mesoporous carbon CMK-3 was taken as a support for LiAlH₄ realized by a solution impregnation method to improve the hydrogen desorption behavior of LiAlH₄ by nanoconfinement effects. It is shown that upon heating, LiAlH₄ is unusually oxidized by coordinated tetrahydrofuran solvent molecules. The important result of the herein described work is the finding of a final composite containing nanoscale aluminum oxide inside the pores of the CMK-3 carbon host instead of a metal or alloy. This newly observed unusual oxidation behavior has major implications when applying these compounds for the targeted synthesis of homogeneous metal–carbon composite materials.

No description available.

Indiana University-Purdue University Indianapolis (IUPUI) / Hydrogels prepared from photopolymerization have been widely used in many biomedical applications. Ultraviolet (200-400 nm) or visible (400-800 nm) light can interact with light-sensitive compounds called photoinitiators to form radical species that trigger photopolylmerization. Since UV light has potential to cause cell damage, visible light-mediated photopolymerization has attracted much attention. The conventional method to fabricate hydrogels under visible light exposure requires usage of co-initiator triethanolamine (TEA) at high concentration (∼200 mM), which reduces cell viability. Therefore, the first objective of this thesis was to develop a new method to form poly(ethylene glycol)-diacrylate (PEGDA) hydrogel without using TEA. Specifically, thiol-containing molecules (e.g. dithiothreitol or cysteine-containing peptides) were used to replace TEA as both co-initiator and crosslinker. Co-monomer 1-vinyl-2-pyrrolidinone (NVP) was used to accelerate gelation kinetics. The gelation rate could be tuned by changing the concentration of eosinY or NVP. Variation of thiol concentration affected degradation rate of hydrogels. Many bioactive motifs have been immobilized into hydrogels to enhance cell attachment and adhesion in previous studies. In this thesis, pendant peptide RGDS was incorporated via two methods with high incorporation efficiency. The stiffness of hydrogels decreased when incorporating RGDS. The second objective of this thesis was to fabricate hydrogels using poly(ethylene glycol)-tetra-acrylate (PEG4A) macromer instead of PEGDA via the same step-and-chain-growth mixed mode mechanism. Formation of hydrogels using PEGDA in this thesis required high concentration of macromer (∼10 wt.%). Since PEG4A had two more functional acrylate groups than PEGDA, hydrogels could be fabricated using lower concentration of PEG4A (∼4 wt.%). The effects of NVP concentration and thiol content on hydrogel properties were similar to those on PEGDA hydrogels. In addition, the functionality and chemistry of thiol could also affect hydrogel properties.

“You just say ‘yes!’ Say something more!” A study of pupils participation, active involvement, motivation and development through student discussion of factual texts about the concepts of climate and sustainable development.  The purpose of this study is to investigate whether student-led text conversations about concepts in the subject of geography, with the help of support structures, can be designed in a way that promotes discussion between pupils. The further aim is to investigate how support structures for increased participation affect the dialogue in text conversations for pupils in upper primary school. The research questions in this study were: How is pupils' participation in text conversations affected by different support structures? How are pupils' participation in text conversations affected by different support structures? How is pupils' dialogue in text conversations affected by different support structures? How do pupils experience structured text conversations as a form of work? As this is a practical study based on the model of a lesson study, where the aim is to increase participation and promote dialogue between a group of pupils, a hermeneutic approach is used in the analysis. Hermeneutics is about interpreting meaning and actions by subjectively trying to put yourself in the place of a text author or participant. This is done by moving in the analysis between partial perspectives and holistic perspectives. In this study, conversation analysis has been used as an analysis tool. It focuses on detailed transcriptions of conversations and studies various aspects of the interaction, including overlaps, turn-taking and conversational structure. By analysing these elements, one can identify social norms, conventions and strategies used by participants to create meaningful interactions. The result of the study can be interpreted as that with a high level of participation and active involvement from the pupils, the dialogue between the pupils was convulsive and bordering on monologic, however, in a dialogically well-functioning text conversation, instead, the participation and active involvement were low.

PhD (Department of Biotechnology, Faculty of Applied and Computer Sciences), Vaal University of Technology. / Tulbaghia violacea Harv. has found extensive uses in traditional medicine for the treatment of numerous ailments among which are tuberculosis, oesophageal cancer, diabetes and cardiovascular diseases. Current reports show that cardiovascular diseases are now the primary cause of mortality worldwide. Thus, the potential of T. violacea plant extracts against cardiovascular diseases should be explored. The objectives of this study were, (i) to conduct qualitative and quantitative preliminary phytochemical screening of T. violacea aqueous leaf extracts, (ii) to conduct Gas chromatography–mass spectrometry (GC-MS) analysis for screening of compounds present in the plant extract, (iii) to evaluate the antioxidant activity of the T. violacea crude extracts using the DPPH:1.1-diphenyl-2-picrylhydrazyl and ABTS: 2,2-azino-bis 3-ethylebenzthiazoline-6-sulfonic acid assays, (iv) to evaluate the antimicrobial activity of the T. violacea crude extracts using disk diffusion and Minimum inhibitory concentration/Minimum bactericidal concentration (MIC/MBC), (v) to evaluate the antithrombogenic properties of T. violacea crude extracts on polystyrene, (vi) to fabricate polycaprolactone (PCL) and PCL-T. violacea incorporated scaffolds, (vii) to evaluate the antithrombogenic properties of T. violacea crude extracts on the fabricated PCL and PCL-T. violacea fabricated scaffolds and, (viii) to evaluate the growth and differentiation of adipose derived stem cells (ADSCs) on the fabricated scaffolds. The qualitative and quantitative phytochemical screening was conducted using standard procedures. Folin-Ciocalteu method was used to evaluate both total phenolic content (TPC) and total tannin content (TTC), the Aluminium chloride method was used for total flavonoid content (TFC) and GC-MS was used to screen for compounds present in the plant extract. The antioxidant activity was evaluated using DPPH and ABTS and the antimicrobial activity was evaluated using disc diffusion and MIC/MBC assays. The antithrombogenic properties of the T. violacea aqueous leaf extracts was then evaluated using platelet activation and whole blood clotting kinetics on polystyrene discs which have been reported to induce platelet activation. The experiment was performed in the absence and presence of 100 and 1000 μg/ml T. violacea plant extracts for both the platelet activation study which used blood plasma and the whole blood clotting kinetics assay which used fresh whole blood. Platelet adhesion was evaluated using fluorescence microscopy and a scanning electron microscope (SEM) was used to evaluate their morphology. Three scaffolds designated as PCL, 10% Tvio and 15% Tvio were fabricated which consisted of a 10% PCL powder and 10% as well as 15% T. violacea aqueous plant extract with respect to the PCL powder weight. The scaffolds were then characterized using Fourier-transform infrared spectroscopy (FTIR) and Energy-dispersive x-ray spectroscopy (EDS). The scaffolds were then evaluated for their antithrombogenic properties in the presence and absence of 100 and 1000 μg/ml T. violacea plant extracts. Platelet adhesion was evaluated using a fluorescent microscope and the morphology was evaluated using SEM. For the cell study, adipose derived stem cells (ADSCs) were cultured on the designed scaffolds and evaluated for their toxicity, viability, adhesion, proliferation, morphology and differentiation into osteoblasts over a period of 3 weeks. Lactate dehydrogenase (LDH) assay was used for toxicity studies, alamar blue assay was used for viability, fluorescence microscopy was used to evaluate cellular adhesion and proliferation while the alkaline phosphate (ALP) assay was used to evaluate differentiation of the cells into osteoblasts. Cell morphology was evaluated using SEM. Phytochemical screening of the prepared T. violacea aqueous extract revealed the presence of terpenoids, flavonoids, cardiac glycosides, saponins, protein, phenols, tannins, carbohydrates and amino acids. This is the first study that has identified the presence of carbohydrates and amino acids in T. violacea aqueous leaf extracts. Different concentrations of 0.1, 1.0 and 10 mg/ml of plant extract were used to conduct the quantitative phytochemical screening assays. There was a concentration dependent increase in the amount of phenols, tannins and flavonoids as the concentration of the plant extracts increased. This was the first study that evaluated the total tannic content of T. violacea plant extracts. The amount of total phenols was higher than that of flavonoids and tannins at every concentration range studied followed by the total flavonoids and lastly total tannins. The GC-MS analysis showed the presence of 33 compounds among which were 2,4 – Dithiapentate - 2,2-dioxide, Cannabidiol, 2,4,5,7 –Tetrathiaoctane and 2,4,5,7 - Tetrathiaoctane 2-dioxide. The presence of sulphur compounds support the characteristic garlic-like smell as well as some of the biological activities of T. violacea plant extracts. The antioxidant activities based on DPPH (0.49 mg/ml) and ABTS (0.24 mg/ml) suggest that T. violacea can be used as potential antioxidant agents. For the antimicrobial activity using disc diffusion, the extracts exhibited appreciable antibacterial activities against Bacillus subtilis, Serratia marcescens, Staphylococcus aureus and S. epidermidis. The highest zone of inhibition was observed for S. epidermidis at 19.50 ± 0.87 mm. The MIC results revealed that the plant extract of T. violacea was moderately active against B. subtilis, S. aureus, S. epidermidis, E. coli, and S. marcescens with MIC value of 2.5 mg/ml. However, the antimicrobial effect of the extract on S. epidermidis was bactericidal when compared to the bacteriostatic effect on the other active microorganisms. The antithrombogenic results on the polystyrene discs showed a significant reduction in the number of platelets that adhered on the polystyrene surfaces treated with plasma mixed with 100 μg/ml of plant extract when compared to the untreated control and the 1000 μg/ml treatment. For the 1000 μg/ml treatment, there was a significant increase in the number of platelets that adhered to polystyrene surfaces. These results were confirmed by the fluorescence and SEM results which showed a higher platelet count for the 1000 μg/ml treatment when compared to the other groups. The whole blood clotting kinetics study showed delayed blood clotting with the 100 μg/ml treatment over a period of 60 min when compared to the untreated control and the 1000 μg/ml treatment. These results correspond with the lower platelet adhesion observation and thus confirm the anticlotting properties of T. violacea aqueous leaf extracts at lower concentrations. The mean diameter of the scaffolds was recorded on the SEM as 275.60 ± 60.65 nm, 193 ± 30 nm and 537 ± 138 nm for the PCL, 10% Tvio and 15% Tvio scaffolds, respectively. The FTIR spectrum revealed the presence of amide groups as well hydroxyl O–H stretching groups which were the characteristic groups for the presence of T. violacea plant extracts in the polycaprolactone. The EDS results showed the presence of potassium, chlorine and sulphur compounds which were only present in the T. violacea scaffolds in addition to the carbon, oxygen and silicon observed in the PCL scaffold. The fabricated scaffolds were then used to evaluate platelet adhesion and activation on blood plasma in the absence and presence of 100 and 1000 μg/ml T. violacea aqueous leaf extracts. The results showed that the 10% Tvio scaffold was more effective in inhibiting platelet adhesion and activation at every treatment group especially when plasma was used in the absence of T. violacea plant extracts. A similar observation to the polystyrene study was observed were addition of 1000 μg/ml of plant extract resulted in the highest number of activated platelets. The study suggests the potential of the 10% Tvio scaffold in the prevention of platelet adhesion and aggregation. The in vitro cell adhesion, proliferation and differentiation of adipose derived stem cells (ADSCs) on the fabricated T. violacea loaded PCL nanofibers was then evaluated. The LDH assay illustrated less activity on the 10% Tvio scaffold when compared to PCL and 15% Tvio scaffolds however, none of the scaffolds were considered as toxic. The alamar blue assay was used for viability after 4 and 7 days of culture. The results showed a significant increase in cell viability for all scaffolds from day 4 to day 7 with the 10% Tvio scaffold having the highest overall cell viability for both day 4 and day 7 of cell cultures. Immunofluorescence staining was then used to count the number of cells using DAPI (4′,6-diamidino-2-phenylindole) stained images and illustrated that the T. violacea incorporated scaffolds supported better cell growth compared to the PCL scaffold. Cell morphology on the T. violacea scaffolds was denser and spread out into cellular extensions when compared to the PCL scaffold after 7 days of cell culture, supporting the higher number of adhered cells from the fluorescence results. For the long term cell study after week 1 and 3, the ALP results showed a significant difference in ALP activity between week 1 and week 3 for all scaffolds. The highest ALP activity was observed for the 15% Tvio scaffolds which is a marker for initial phase of bone matrix deposition. The designed T. violacea scaffolds supported better cell growth compared to the PCL scaffold and their morphology was more spread out and covered the entire surface of the scaffolds after week 3. Lastly, the cell count and osteocalcin differentiation was more prominent on 10% Tvio scaffold indicating higher levels of the protein marker for bone formation. Thus, supporting the use of the 10% Tvio scaffold for long-term cell studies. In conclusion, the results of this study indicated that the aqueous extract of T. violacea is rich is phytochemicals and also possess a broad range of pharmaceutically important compounds which may be attributed to the high antioxidant and antimicrobial activities identified. The results from this study suggest that T. violacea aqueous extracts have antithrombogenic properties at lower concentrations. Scaffolds fabricated with the incorporation of T. violacea plant extract also confirm the potential antiplatelet activity of the fabricated 10% Tvio scaffold. The results also suggest the potential of the fabricated 10% Tvio scaffold to enhance cell adhesion, proliferation and differentiation over long-term cell studies. It can thus be recommended that T. violacea may be useful for tissue engineering applications and bone repair with prospects of preventing cardiovascular diseases associated with bone defects. This research study has provided the foundation for clinical evaluation and outlined the potential effects of T. violacea aqueous leaf extracts as a clinical drug.

In this thesis, several types of scaffolds composed of different materials and designed structures and functions were fabricated by 3D plotting under mild conditions (room temperature and without using any organic solvent). Broad biomaterials including inorganic (such as calcium phosphate cement and mesoporous bioglass), organic (such as alginate and gelatin) and composite materials were prepared into printable pastes to plot as 3D scaffolds for bone tissue engineering. Organic/inorganic biphasic and bipartite structure, core/shell alginate/nano-hydroxyapatite and hollow fiber structure were designed and realized. Scaffolds with multi functions including suitable mechanical properties, sustained drug/protein delivery and in vitro vascularization were achievable. 3D plotting provided great achievements in the field of tissue engineering by preparing advanced scaffolds, as well as by plotting cell/matrix constructs, and even complex tissues and organs.