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Tissue engineers – in order to develop therapies for the treatment of complex neurological injuries and diseases – attempt to recreate elaborate developmental mechanisms in vitro. Neuronal precursor cells are excellent candidates for the study of developmental operations such as cell adhesion, differentiation, and axonal pathfinding. Hyaluronan (HA) is a common polysaccharide that is found extensively throughout the neuronal extracellular matrix (ECM), and can be functionalised and crosslinked to form stable hydrogels that support growing neuronal cells. Hyaluronan hydrogels can be modified chemically and mechanically to mimic the ECM of the developing brain, awarding control over mechanisms such as differentiation and axonal pathfinding. This thesis is concerned with the functionalisation and characterisation of HA hydrogels, ultimately in order to simulate vital properties of the developing brain. Here we show that HA hydrogels can be finely tuned mechanically (by modulating stiffness and viscosity), and chemically, by the conjugation of peptides that mimic the neural cell adhesion molecule (NCAM). NCAM mimics and novel mimics of sialylated NCAM significantly influence the differentiation of NSPCs in 2D and 3D. HA hydrogels successfully support long term culture of neural cells in 3D, and encourage the formation and extension of neurites of several cell types including human, mouse and rat neuronal precursor and stem cells. These results demonstrate for the first time that novel NCAM mimicking peptides can be conjugated to well defined hydrogel matrices that influence intricate developmental behaviours in 3D. Understanding how neural cells form functional networks is essential for the development of clinical approaches that attempt to address the injuries and diseases that affect these systems.

The current study is focused on the characterization of four natural leaf species (quaking, bigtooth and columnar european aspen as well as black locust) possessing a unique dual-scale cuticle structure composed of micro- and nano-scale asperities, which are able to effectively resist wetting (superhydrophobic), characteristic of The Lotus Effect. Scanning Electron Microscopy (SEM) was used to track the growth and evolution of their distinctive nano-scale epicuticular wax (ECW) morphologies over one full growing season. In addition, the stability of their superhydrophobic property was tested in various environments. It was determined that the long-term stability of these surfaces is tentatively linked to various environmental stress factors. Specifically, a combination of high temperature and humidity caused the degradation of nano-scale asperities and loss of the superhydrophobic property. The dual-scale surface structure was found to provide a suitable template for the design of future superhydrophobic engineering materials.

The current study is focused on the characterization of four natural leaf species (quaking, bigtooth and columnar european aspen as well as black locust) possessing a unique dual-scale cuticle structure composed of micro- and nano-scale asperities, which are able to effectively resist wetting (superhydrophobic), characteristic of The Lotus Effect. Scanning Electron Microscopy (SEM) was used to track the growth and evolution of their distinctive nano-scale epicuticular wax (ECW) morphologies over one full growing season. In addition, the stability of their superhydrophobic property was tested in various environments. It was determined that the long-term stability of these surfaces is tentatively linked to various environmental stress factors. Specifically, a combination of high temperature and humidity caused the degradation of nano-scale asperities and loss of the superhydrophobic property. The dual-scale surface structure was found to provide a suitable template for the design of future superhydrophobic engineering materials.

Tyrosine (4-hydroxyphenylalanine) is one of the naturally occurring 22 amino acids. The importance of tyrosine is due to the presence of its phenolic side chain. In biological systems, the tyrosyl residue in proteins is found to be sulfated, phosphorylated and nitrated. Upon oxidation with dioxygenases, Tyr residue forms dopaquinone which undergoes a series of reactions ultimately leading to the formation of melanin. Tyr is also a precursor to neurotransmitters (catechol amines namely dopamine, epinephrine and norepinephrine) and thyroid harmones T4 and T3. Tyr residue is also found to be cross linked with other amino acid residues in the active site of certain proteins. Tyr-Tyr cross link has also been associated with neurodegenerative diseases. Tyr residue in proteins has been targeted widely for site selective modifications. A series of chemical modifications like acylation, allylation, ene-type reaction, iodination with radiolabeled iodine, formation of Tyr-Tyr cross link with oxidants and aminoalkylation have been carried out on surface exposed Tyr residues in proteins. Apart from these chemical modifications of Tyr on protein surface, a couple of free Tyr-based scaffolds have also been developed for different applications. Similar to tyrosine-based scaffolds, several phenolate-based scaffolds have also been developed for various applications. Several phenolate-based binuclear metal complexes have been developed as mimics of the active site of metalloenzymes. Moreover, by varying the substituent in the phenolate scaffold, the redox properties of metal bound in these systems can be tuned. The thesis consists of five chapters. The first chapter gives general idea about tyrosine-and phenolate-based scaffolds. The first chapter also gives introduction to zinc(II)-containing enzymes metallo-β-lactamases (mβls) and phosphotriesterase (PTE) and their functional mimics. The importance of copper(II)-containing enzyme, catechol oxidase and its mimics has also been discussed. The significance and formation of o-dityrosine (Tyr-Tyr cross link) has also been briefly discussed. In chapters 2 and 3, a couple of phenolate-based ligands and their corresponding zinc(II)- and copper(II)- complexes have been synthesized and have been checked as mimics of zinc(II)-containing enzymes (mβl and PTE) and copper-containing enzyme catechol oxidase, respectively. In chapter 4, a series of tyrosine-based ligands have been designed and their in situ copper(II) complexes have been tested as mimics of catechol oxidase. In chapter 5, the effect of neighboring amino acid in the formation of Tyr-Tyr cross link has been studied. In chapter 2, a couple of zinc(II) complexes have been synthesized and studied as mimic of zinc(II)-containing enzymes mβl and PTE. Metallo-β-lactamases (mβls) are zinc(II)-containing enzymes which exist in both mono- and binuclear forms. Mβls are capable of hydrolyzing β-lactam ring in antibiotics and make them inactive (Scheme 1(A)). To date, an effective inhibitor for this enzyme is not known. Hence, in order to understand the nature of the enzyme a couple of synthetic mimics are known. However, in most of the synthetic mimics both the metal ions are in symmetrical environment. Therefore, we have attempted to design a few unsymmetrical phenolate- based ligands and their zinc(II) complexes. The unsymmetrical phenolate-based ligands HL1 and HL2 have been synthesized by sequential mannich reaction with formaldehyde and two different amines. Complexes 1 and 2 are obtained from ligands HL1 and HL2, respectively (Figure 1). For comparative purpose, the symmetrical ligands HL3 and HL4, and their zinc(II)-complexes 3 and 4 have been synthesized by reported procedures (Figure 1). The efficiency of the complexes 1-4 towards the hydrolysis of oxacillin has been studied. It has been observed that the binuclear zinc(II) complexes with metal-bound water molecule 1 and 4 are able to hydrolyze oxacillin at much faster rates compared to that of mononuclear complexes 2 and 3. However, between 1 and 4, there is no appreciable change in activity, indicating that the slight change in ligand environment has no significant role. PTE is a binuclear zinc(II)-containing enzyme, capable of hydrolyzing toxic organphosphotriesters to less toxic diesters (Scheme 1(B)). As the binuclear active site of mβl is comparable with that of phosphotriesterase (PTE), PTE activity of complexes 1-4 has been studied. Although the binuclear zinc(II)-complexes 1 and 4 are able to hydrolyze PNPDPP (p-nitrophenyl diphenyl phosphate) initially, these complexes are not able to effect complete hydrolysis. This is due to the inhibition of complexes 1 and 4 by hydrolyzed product, diester. However with mononuclear complexes 2 and 3 no such inhibitions is possible, and are capable of hydrolyzing PNPDPP at comparatively faster rates than 1 and 4. Scheme 1. Function of metallo-β-lactamase and phosphotriesterase. (A) Hydrolysis of β-lactam ring in antibiotics by metallo-β-lactamase. (B) Hydrolysis of organophosphotriesters to diesters by phosphotriesterase. Figure 1. Chemical structures of ligands HL1-HL4 and their corresponding zinc(II)complexes 1-4. In chapter 3, a couple of copper(II) complexes have been synthesized and their catechol oxidase activity has been studied. Catechol oxidase belongs to the class of oxidoreductase and it catalyzes the oxidation of a wide range of o-diphenols to o-quinones through the reduction of molecular oxygen to water (Scheme 2). A four new µ4-oxo-bridged tetranuclear copper(II) complexes (5-8) have been synthesized (Figure 2). The ability of these complexes to catalyze the oxidation of 3,5-DTBC (3,5-Di-tert-butylcatechol) to 3,5-DTBQ (3,5-Di-tert-butylquinone) has been studied. A detailed kinetic study has been carried out which reveals that the complexes with exogenous acetate ligands (5 and 6) are better catechol oxidase mimics compared to complexes with exogenous chloride ligands (7 and 8). This observation is due to the labile nature of acetate compared to chloride, as the displacement of exogenous ligand is essential for the binding of substrate to the catalyst. Based on mass spectral analysis a plausible mechanism has been proposed for the oxidation of 3,5-DTBC by these complexes. Scheme 2. Oxidation of catechol by catechol oxidase. Figure 2. Chemical structures of copper(II) complexes 5-8. In chapter 4, by following the analogy between phenol and tyrosine, a series of binucleating ligands of tyrosine or tyrosyl dipeptides (Figures 3 and 4) have been synthesized by Mannich reaction under mild conditions. The in situ complexation of these fifteen new binucleating ligands (HL5-HL19) with copper(II) chloride has been observed. In situ complexation was followed by UV-visible and mass spectral analysis. These in situ complexes were able to oxidize 3,5-DTBC at slower rate compared to that of the tetranuclear complexes reported in chapter 3. The catecholase activity has also been tested with the addition of base. A slight enhancement in activity of in situ complexes has been observed in the presence of base. Based on mass spectral evidences, a plausible mechanism for the oxidation of catechol by these in situ complexes has been proposed. Figure 3. Binucleating ligands (Mannich bases) of boc-protected tyrosine and tyrosyl dipeptides. Figure 4. Binucleating ligands (Mannich bases) of boc-deprotected tyrosyl dipeptides. In chapter 5 of the thesis, the effect of neighboring amino acid residue in the formation of o,o-dityrosine (Tyr-Tyr cross link) has been studied. o,o’-Dityrosine is a specific marker for oxidative/nitrosative stress. The increase in concentration of dityrosine is associated with several disease states. A detailed study has been carried out in order to find out the effect of neighboring amino acid residues in the rate of formation of dityrosine of several tyrosyl dipeptides. The formation of dityrosine has been carried out with horseradish peroxidase(HRP) and H2O2 (Scheme 3). Except Cys-Tyr, all other tyrosyl dipeptides, form corresponding dityrosine with HRP/ H2O2. With Cys-Tyr, the formation of corresponding disulfide is observed. The appreciably higher rate of dityrosine formation of Phe-Tyr is attributed to the presence of strong hydrophobic environment around the active site of HRP. Among the polar tyrosyl peptides, the positively charged peptides (Arg-Tyr, Lys-Tyr) undergo dityrosine formation at much faster rate compared to that of negatively charged dipepptides (Asp-Tyr, Glu-Tyr). This trend is in accordance with the pKa of neighboring amino acid residues. The positively charged neighboring residues with higher pKa stabilizes ionized tyrosine, hence the rate of dityrosine formation is higher for them. As positively charged neighboring residue enhances the rate of dityrosine formation, the effect of externally added L-Arg has been studied. A coupling of a few biologically relevant tyrosine derivatives has been studied. The derivatives in which one of the ortho-positions of tyrosine is blocked, does not undergo coupling under the experimental conditions employed. Scheme 3. Formation of dityrosine of Ile-Tyr from Ile-Tyr in the presence of H2O2 catalyzed by HRP. (For structural formula and figures pl refer the abstract pdf file)

<p> There is a current interest in exploring novel microstructural architectures that take advantage of the response of independent phases. Current guidelines in materials design are not just based on changing the properties of the different phases but also on modifying its base architecture. Hence, the mechanical behavior of composite materials can be adjusted by designing microstructures that alternate stiff and flexible constituents, combined with well-designed architectures. One source of inspiration to achieve these designs is Nature, where biologically mineralized composites can be taken as an example for the design of next-generation structural materials due to their low density, high-strength, and toughness currently unmatched by engineering technologies.</p><p><br></p> <p>The present work focuses on the modeling of biologically inspired composites, where the source of inspiration is the dactyl club of the Stomatopod. Particularly, we built computational models for different regions of the dactyl club, namely: periodic and impact regions. Thus, this research aimed to analyze the effect of microstructure present in the impact and periodic regions in the impact resistance associated with the materials present in the appendage of stomatopods. The main contributions of this work are twofold. First, we built a model that helped to study wave propagation in the periodic region. This helped to identify possible bandgaps and their influence on the wave propagation through the material. Later on, we extended what we learned from this material to study the bandgap tuning in bioinspired composites. Second, we helped to unveil new microstructural features in the impact region of the dactyl club. Specifically, the sinusoidally helicoidal composite and bicontinuous particulate layer. For these, structural features we developed finite element models to understand their mechanical behavior.</p><p><br></p> <p>The results in this work help to elucidate some new microstructures and present some guidelines in the design of architectured materials. By combining the current synthesis and advanced manufacturing methods with design elements from these biological structures we can realize potential blueprints for a new generation of advanced materials with a broad range of applications. Some of the possible applications include impact- and vibration-resistant coatings for buildings, body armors, aircraft, and automobiles, as well as in abrasion- and impact-resistant wind turbines.</p><br>

Cellulose is the most abundant biopolymer on Earth as it is found in every plant cell wall; therefore, it represents one of the most promising natural resources for the fabrication of sustainable materials. In plants, cellulose is mainly used for structural integrity, however, some species organise cellulose in helicoidal nano-architectures generating strong iridescent colours. Recent research has shown that cellulose nanocrystals, CNCs, isolated from natural fibres, can spontaneously self-assemble into architectures that resemble the one producing colouration in plants. Therefore, CNCs are an ideal candidate for the development of new photonic materials that can find use to substitute conventional pigments, which are often harmful to humans and to the environment. However, various obstacles still prevent a widespread use of cellulose-based photonic structures. For instance, while the CNC films can display a wide range of colours, a precise control of the optical appearance is still difficult to achieve. The intrinsic low thermal stability and brittleness of cellulose-based films strongly limit their use as photonic pigments at the industrial scale. Moreover, it is challenging to integrate them into composites to obtain further functionality while preserving their optical response. In this thesis, I present a series of research contributions that make progress towards addressing these challenges. First, I use an external magnetic field to tune the CNC films scattering response. Then, I demonstrate how it is possible to tailor the optical appearance and the mechanical properties of the films as well as to enhance their functionality, by combining CNCs with other polymers. Finally, I study the thermal properties of CNC films to improve the retention of the helicoidal arrangement at high temperatures and to explore the potential use of this material in industrial fabrication processes, such as hot-melt extrusion.

Adhezní schopnosti gekona byly předmětem mnoha studií a inspirací pro vytvoření mnoha napodobenin. Tato práce navrhuje vlastní verzi umělých gekoních struktur ve tvaru mikroskopických pilířů, které by vykazovaly adhezní vlastnosti srovnatelné s tlapkou gekona. Vyrobeny byli struktury z polymeru Parylen C pomocí fotolitografie a technik na leptání křemíku. Dalším cílem bylo různými metodami pro modifikaci povrchu a charakterizaci vytvořených struktur, které určí adhezní síly těchto povrchů, před a po modifikacích.