Dissertations / Theses on the topic 'Biomacromolecules'

This thesis details various techniques and considerations for the purification of complex biomacromolecules.   Initially an α-mannosidase from babaco fruit was purified using anion exchange-, lectin affinity- and size exclusion chromatography.  The enzyme was approximately 260-280 kDa in size with an apparent an unusual octagonal stoichiometry and displayed properties similar to other known plant α-mannosidases.   Mucins were fractionated by ion exchange and size exclusion chromatography to assess the properties that govern the mucin surface coating interactions in biomaterial research.  Commercially available mucins, of bovine and porcine origin, as wells as crude human mucin were tested. All showed to consist of a population of molecules which differ in size, charge and composition.   The third part of the thesis concerns different aspects of plasmid DNA purification processes. A two-step method for analysis of plasmid DNA consisting of size exclusion followed by thiophilic adsorption chromatography was evaluated. It allowed determination of the supercoiled plasmid DNA concentration in all process steps without requirement for extensive sample preparation. This method was shown to be fully comparable in terms of accuracy to capillary gel electrophoresis, considered as the industry standard. Purification of plasmid DNA generally involves bacterial cell alkaline lysis, which creates a solution with flocculate material which needs to be removed prior to further processing. The addition of ammonium hydrogen carbonate to the suspension was evaluated to clarify the solution. The released carbon dioxide and ammonium lifts the flocculate to the surface and allows draining of a clear solution. The method is fully scalable, does not affect the plasmid DNA quality and requires no special equipment. Thiophilic adsorption chromatography was evaluated for simplification of an existing commercial large scale purification process and was shown to increase both product purity and yields of several tested plasmids. Also, implementation of this step significantly reduced overall production process time.

The work presented in this thesis describes the development of novel methodology for the preparation of macromolecular conjugates using proteins and dendrimers as scaffolds. This methodology is based on the discovery that chemical modification of proteins can be achieved in vacuo, in the absence of solvents, viz., water. The results obtained represent the first attempts to use this methodology to achieve protein modifications that are otherwise difficult or impossible under aqueous conditions. While the inspiration for this research stemmed from practical objectives, much of the work undertaken developed into a proof of principle, not by design, but due to the fact that there is no precedence or established theoretical base for the in vacuo chemical modification of proteins. Initially, the scope of the project involved the design and development of a novel detoxication construct for an artificial liver consisting of an enzyme coupled to lipophilic poly(propyleneimine) dendrimers as partitioning agents for the clearing of hydrophobic compounds from aqueous solution. While the lipophilic dendrimers proved to be feasible partitioning agents when tested with some drugs in aqueous solution, attempts to attach a model enzyme to the construct by established methods in solution met with limited success. The search for better, more efficient methods of tethering, immobilizing and cross-linking enzymes lead to the investigation of alternative conjugation methods. p-Nitrophenylchloroformate is a common activating reagent used in organic synthesis and has been used to activate chemical modifiers for proteins. However, this reagent has not been used for direct chemical modification of proteins because of its insolubility in water. The results obtained show that this reagent, when used with the in vacuo procedure, can activate and cross-link proteins through chemical modification of protein carboxyl groups. In the course of these studies it was observed that a covalent dimer was present in ribonuclease A heated in the absence of reagent. This observation lead to an investigation which demonstrated that proton transfers between interacting carboxylate and ammonium groups in vacuo results in the formation of amide bonds. Compared to solution methods, the in vacuo methods developed are experimentally simple, and may also be carried out without the use of reactive chemical reagents. Glycation of protein amino groups with reducing monosaccharides can readily be achieved by the formation of stable ketoamine derivatives using the in vacuo methodology. (Abstract shortened by UMI.)

Plant cell wall polysaccharides are abundant natural polymers making them potential sources for sustainable and biodegradable materials. Interfacial behavior, including adsorption and enzymatic degradation, of several plant cell wall polysaccharides and their derivatives were studied with a quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR) and atomic force microscopy (AFM). Xyloglucan adsorption isotherms were obtained to probe how cellulose-hemicellulose interactions were affected by the type of cellulose substrate and molar mass of xyloglucan. Xyloglucan as small as a heptasaccharide still adsorbed irreversibly onto cellulose. Carboxymethyl cellulose (CMC) adsorption onto cellulose and viscoelastic properties and water contents of the adsorbed CMC layers were obtained from a combination of QCM-D and SPR data. The CMC samples formed hydrated and viscoelastic layers compared to the relatively rigid xyloglucan layer. Pectin model surfaces were prepared by pectin adsorption from citric phosphate buffer onto gold substrates. These pectin model surfaces were used for subsequent interaction studies with xyloglucan and enzymatic degradation behavior. There is a strong correlation between the degree of esterification (DE) and film resistance to degradation with the high DE being the most susceptible to degradation. The adsorption of two mixed linkage glucans (MLG), barley and lichen MLG, onto regenerated cellulose (RC) surfaces in the absence and presence of other matrix polysaccharides was studied. Viscoelastic properties of the resulting layer were compared as a function of the proprotion of '-(1''3) linkages with lichen MLG forming softer gel-like layers on RC. The lichen MLG layers were further used for enzymatic degradation studies with respect to enzyme concentration, temperature, pH and ionic strength. These studies show that polymer adsorption is a promising strategy to modify material surfaces and provides fundamental understanding of interactions and biodegradation of cell wall polysaccharides at solid/liquid interfaces.
Ph. D.

The specific interactions of macromolecules along with the activity of enzymes are central to all aspects of biology. It is well recognized that when the relative concentration or activity of macromolecules is perturbed, it can lead to human diseases. Thus, the development of simple methods for the detection of macromolecules and the activity of enzymes in complex environments is important for understanding biology. Moreover, the development of methods for measuring interactions allows for the testing of inhibitors that can be used as tools or drugs for improving human health. Towards this goal, a promising new method has been developed, which is the focus of this thesis, called split-protein reassembly or protein fragment complementation. In this method, a protein reporter, such as the green fluorescent protein or firefly luciferase, is dissected into two fragments, which are attached to designed adaptor proteins. The designed split-protein systems only produce a measurable signal, either fluorescence or luminescence, when a specific macromolecular interaction or activity is present. In this thesis, I have extended previous research on the direct detection of DNA using split-protein sensors utilizing a red fluorescent protein, dsRED from Discosoma that allows for multiplexed DNA detection. I have designed a new split-luciferase based sensor for detection of poly (ADP-ribose) or PAR, which plays a key role in the response to DNA damage and have applied it for monitoring the activity of poly (ADP-ribose) glycohydrolase that controls PAR levels in the cell. Furthermore, I have significantly expanded upon a three-hybrid split-luciferase system for identifying protein kinase inhibitors. I have designed and tested two orthogonal peptide based chemical inducers of dimerization based on BAD and p53mt conjugates. I have studied these chemically induced dimerization systems in detail in order to begin to provide a theoretical basis for the observed experimental results. Finally, in a less related area, I have developed methods for producing water soluble semiconductor nanoparticles called Quantum Dots (QDs), with potential application in biological imaging. I have developed methods for functionalizing the QDs with orthogonal peptides, which can be potentially used for the assembly of high affinity non-covalent QD targeted proteins.

Natural polymers from renewable resources have attracted increasing interest as candidates for renewable energy and functional materials. In this work, the interactions between natural polymer thin films and biomacromolecules were studied via surface analysis techniques, such as a quartz crystal microbalance with dissipation monitoring (QCM-D), surface plasmon resonance (SPR) and atomic force microscopy (AFM). Chitinase activity on regenerated chitin (RChitin) films was studied by QCM-D and AFM. The optimal temperature and pH for chitinase activity on surfaces determined by QCM-D and AFM were consistent with bulk solution studies in the literature. Results from QCM-D also indicated that chitinase showed higher activity on fully acetylated chitin than highly deacetylated chitosan. Nanocrystalline chitin (Chitin NC) thin films were prepared by spincoating a nanocrystalline chitin colloidal suspension onto solid surfaces. Solvent exchange experiments via QCM-D with H2O/D2O revealed that Chitin NC films had more water than RChitin films of similar thickness. Results from QCM-D demonstrated that Chitin NC films had high bovine serum albumin loading capacity, and chitinase not only degraded, but also caused swelling of the chitin nanocrystals. Adsorption of human serum albumin (HSA) and fibrinogen (HFN) onto bare gold, regenerated cellulose (RC) and RChitin thin films was studied by SPR and QCM-D. Studies by SPR indicated that HSA and HFN formed close-packed monolayers on gold surfaces and sub-monolayers on polysaccharide surfaces, and the adsorption affinity of HSA for polysaccharide surfaces was greater than that of HFN. Results from QCM-D and SPR showed that the protein layers on polysaccharide surfaces had more associated water than proteins on gold surfaces. The dehydrogenative polymerization of monolignols catalyzed by physically immobilized horseradish peroxidase was investigated using QCM-D and AFM. Results from QCM-D and AFM showed that coniferyl and p-coumaryl alcohol underwent polymerization directly, whereas sinapyl alcohol required the addition of a nucleophile for polymerization. Studies by QCM-D and AFM also indicated that the surface-initiated polymerization was greatly affected by the support surface, monolignol concentration, hydrogen peroxide concentration and temperature. Thin films of dehydrogenative polymer (DHP), kraft (KL), organosolv (OL) and milled wood (MWL) lignins were used to study the enzymatic degradation of lignin mediated by lignin peroxidase (LiP) and manganese peroxidase (MnP). Results from QCM-D showed that the initial rates for degradation catalyzed by LiP increased in the order: KL < OL < MWL < guaiacyl DHP (G-DHP) < p-hydroxyphenyl DHP (H-DHP). In contrast, manganese peroxidase only degraded DHP films with a faster initial rate for G-DHP than H-DHP. Adsorption of hemicelluloses onto KL, OL and MWL thin films was studied by QCM-D and SPR. Results from QCM-D showed that hemicelluloses with different structures displayed very different adsorption behavior. Adsorption isotherms from QCM-D and SPR indicated that xyloglucan possessed stronger affinity for KL and OL films than MWL films. Data from QCM-D and SPR revealed that xyloglucan formed less hydrated layers on lignin surfaces compared to RC surfaces, and the adsorbed xyloglucan layers on different lignin films had similar percentages of coupled water.
Ph. D.

In pharmaceutical R & D, drug stereochemistry, and consequently the rotation of enantiomers, is very important. Because they act as chiral selectors in vivo, biomacromolecules have been extensively used as chiral selectors for the liquid chromatographic (LC) resolution of enantiomers and more latterly have also been employed in the newer separative technique, capillary electrophoresis (CE). However, at the outset of this research programme, this had generally been restricted to common easily accessible biomacromolecules such as plasma-binding proteins. It was clear that it be would be useful therefore to adapt LC and CE in such a way as would allow the use of a much wider range of biomacromolecules. Accordingly the general aim of this study was to develop LC and CE protocols involving biomacromolecules that would give rise to minimum consumption of the biomacromolecule. To study biomacromolecules in free solution CE, a number of experimental variables had to be established for both optimum chiral discrimination and for investigating biomacromolecule-ligand interactions. The typical and widely used biomacromolecule for chiral discrimination, bovine serum albumin (BSA) was used to study the variables of pH from pH 5.4 to 8.4, concentration of BSA form 0 to 60 mM and concentration of organic modifiers in the range 0 – 20 % v/v for chiral selectivity. This involved an investigation into some unusual artefacts such as ghost peaks and stepped baselines, but ultimately the outcome was a successful free solution CE protocol suitable for the rapid evaluation of chiral discrimination of other biomacromolecules. The conditions were: run buffer (30 mM protein, 67 mM phosphate (pH 7.4) – methanol (97.5 : 2.5, v/v)), capillary CElect p150, 40 cm (35 cm to detector) x 50 mm i.d., temperature of ambient or 25 °C and an applied voltage of 10 kV. The ability of other biomacromolecules, such as human serum albumin (HSA), lactoferrin and protamine, to resolve enantiomers was studied using this protocol including looking at the effect of the addition of modifiers to the buffer such as metal ions like manganese and zinc, competing ligands, e.g. warfarin and ibuprofen, and b-cyclodextrin. As well as using CE, miniaturisation of LC was also studied in view of the success of biomacromolecule-affinity chiral LC. Two different, but similar, microbore LC protocols were employed, i.e. using the protein in free solution or as a pseudo stationary phase. For the former, a Lichrosorb DIOL stationary phase, based on hydroxyl groups immobilised on silica, was chosen in order to minimise the adsorption of protein to the stationary phase. Using this protocol it was demonstrated that free solution microbore LC could be easily be carried out, therefore used to evaluate chiral discrimination and that the use of the system to study in vivo interactions was feasible. The creation of a biomacromolecule pseudo stationary phase, as opposed to conventional chiral stationary phases where the protein is permanently bonded to the stationary phases, involves the biomacromolecule being adsorbed within the pores of the stationary phase. In this way the overall biomacromolecule structure should not be grossly distorted. Three stationary phases were evaluated, viz wide-pore Nucleosil silica, Nucleosil C8 and Lichrosorb DIOL, for optimum biomacromolecule loading and minimal biomacromolecule leakage when mobile phase was pumped through the column. The Nucleosil silica with adsorbed BSA proved the most successful, e.g. a of 3.6 and 4.0 for tryptophan and kynurenine respectively, and robust of the stationary phases with respect to demonstrating the chiral discrimination potential for this system. All the miniaturised systems evaluated were successful, to a greater or lesser degree, for the demonstration of chiral selectivity of biomacromolecules. While CE was better for minimisation of the consumption of the biomacromolecule, it was also important that the biomacromolecule LC systems could be operated in reduced dimensions since these systems have perhaps greater potential for exhibiting enantioselectivity and are more appropriate for the ever increasing need for the study of the interaction of ligands with the biomacromolecule in its ‘natural’ form. With the knowledge gained from this research programme it will now be possible to more easily carry out such studies with much smaller amounts of biomacromolecule, and, accordingly be able to work with biomacromolecules which hitherto it has not been possible to study because of limited availability. While some of the protocols have now been superseded by recent developments the system developed still has potential. The use of such small scale systems offers the potential to study chiral selectivity and drug-biomacromolecule binding of rare or expensive biomacromolecules.

Textiles are very hazardous materials when related to fires, because of the high surface to mass ratio and the open structure, which simplify the contact with heat and oxygen. Fabrics have a great impact on the development of fatal fires because of their easy ignition and high burning rate. For the flame retardancy of fabrics several strategies have been developed throughout the years, and nowadays additives and reagents are widely used for the different kinds of textiles. The most common commercial durable finishes for cotton fabrics contain phosphorous and nitrogen compounds. These products have been predominant in the field of flame retardants for cotton for 50 years, but recently the request for a reduction of environmental impact and of formaldehyde release during manufacturing and utilisation have pushed researchers towards new kinds of finishing. This Ph.D. work aimed at investigating the effectiveness and the possibilities of the use of biomacromolecules as fire retardants for cellulosic textiles (i.e. cotton). To this aim, three biomacromolecules were taken in consideration: whey proteins, caseins and nucleic acids. Whey proteins and caseins, derived from milk, were applied on cotton fabrics and their thermal and thermo-oxidative stability and fire behaviour were assessed through thermogravimetric analysis, cone calorimetry and horizontal flame spread tests. These biomacromolecules were effective in improving the fire retardancy of the treated fabrics, slowing down the combustion rate in flame spread tests and favouring the formation of a coherent carbonaceous residue (char). Then, the fire retardant behaviour of DNA was thoroughly investigated. First, commercially-available DNA from herring sperm and testes, having different molecular weights, were applied on cotton fabrics: the experimental parameters (i.e., molecular weight of the biomacromolecules, pH of the aqueous solution, number of impregnations needed for achieving the final dry add-on on the treated fabrics) were optimized in order to achieve the highest flame retardant effectiveness. The distribution of the nucleic acids on the underlying fabric was studied through SEM analyses. Thermogravimetric analyses, cone calorimetry and horizontal flame spread tests were carried out for assessing the thermal and thermo-oxidative stability and the fire behaviour of the treated fabrics. In detail, the low-molecular-weight DNA solution prepared either at pH 4 or 8 and applied on cotton with multiple impregnation steps was the most effective flame retardant treatment. Pursuing the research, the problem related to the high cost of commercially available DNA was considered: to overcome this drawback, a novel extraction method, starting from exhausted biomasses or agro-food crops, was developed. This method focused on the extraction of high quantities of nucleic acids, exploiting a low environmental impact approach. Overall, the recovered nucleic acids showed a fire behaviour similar to that of commercially-available counterparts. Finally, the washing fastness of the cotton fabrics treated with the biomacromolecules was considered: in fact, all the selected biomacromolecules are waterborne systems, which easily come off the fabrics when subjected to washing cycles, even in hot water only. This issue was taken on by treating cotton fabrics with nucleic acids and chitosan in mixture or as separate layers and also exploiting the layer by layer technique. The washing fastness of the treated fabrics was significantly improved by subjecting them to UV-curing, thus achieving the grafting of chitosan on cotton and, at the same time, entrapping the nucleic acid in the grafted chitosan coating. Notwithstanding the achieved fire retardancy, the fabrics treated with chitosan and nucleic acids also showed an antibacterial activity, due to the presence of chitosan. Furthermore, 30 bilayers of nucleic acids and chitosan provided the fabrics with self-extinction, either before and after a water washing cycle at 55°C. As far as the effectiveness of the treatments is concerned, all the selected biomacromolecules conferred fire retardant features to cotton fabrics. In particular, low-molecular-weight nucleic acids and caseins were the most performing biomacromolecules either in forced combustion or in flame spread tests. Cotton fabrics treated with nucleic acids or caseins were able to achieve self-extinction in horizontal flame spread tests, with a reduction of the burning rate and an increase of the residue left. Similar reductions in the HRR were also observed in cone calorimetry tests. The suggested approach is quite simple and does not involve the use of particular chemicals or expensive equipment; furthermore, the selected biomacromolecules are soluble/dispersible in water. In conclusion, the proposed flame retardants for cotton may represent a new sustainable approach to face the challenges related to the increasing awareness of the health and environmental impact of traditional products and processes.

The use of bio-mimetic receptor systems have been considered in a quest for affinities and specificities that are on a par with natural receptors for rapid in-situ analysis based on coupled-sensor techniques. The work describes the experimental optimisation and characterisation of hydrogel-based molecularly imprinted polymers (MIPs) for a range of proteins and pathogens, including plant viruses. A variety of acrylamide-based functional monomers, along with several integrated metal complexes for signal transduction have been exploited in the fabrication of both bulk and surface imprinting of MIPs. Spectrophotochemical, electrochemical and quartz crystal microbalance (QCM) analytical techniques were utilised for quantifying imprinting effects by assessing specific and/or non-specific binding. Bulk imprinting exhibited high selectivities (rebinding efficiencies ≈ 80%) and imprinting factors of ≈ 14 (MIP/NIP ratio) across varied templates. MIP-coupled QCM sensors illustrated binding and elution of target proteins through distinct frequency and impedance transitions at 3 mg mL-1. QCM surface imprinting via electrochemically-induced polymerisation (ECIP) was less successful. Whereas electrochemical ECIP methods using glassy carbon electrodes (GCEs) illustrated good compatibility, higher sensitivities, and a limit of detection (LOD) of 16 μg mL-1 and a limit of quantification (LOQ) of 56 μg mL-1 for BHb. Pattern recognition techniques using multivariate analysis were also implemented to reduce complex data sets. Principle component analysis (PCA) and linear discriminant analysis (LDA) techniques illustrated unique binding pattern profiles depending on the sample matrix analysed, significance (ρ) ≤ 0.0005. The latter ECIP methods were also transposed onto disposable screen-printed carbon electrodes (SPCEs) based on the introduction of redox tracers (both externally and within the MIP matrix). SPCEs offer an attractive opportunity for the development of miniaturised low cost electrochemical sensors. However, several complications arose and little sensitivity was observed in terms of MIP binding. Once fully developed, the benefits of sensitivity, specificity and stability of MIPs coupled with discriminatory sensor techniques, as described here, could be crucial to the future impact of portable diagnostics for personal healthcare and use by health professionals. This technology also presents major potential benefits to environmental and food monitoring as it could provide an inexpensive, fast, and efficient diagnostic method for highly sensitive analytical procedures.

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemistry, 2005.
Includes bibliographical references.
Nuclear magnetic resonance (NMR) spectroscopy is unique in the content of structural as well as dynamic information that it can provide at atomic resolution. The aim of this PhD-thesis was to contribute to the understanding of biochemical processes by means of NMR-spectroscopic techniques, targeting specific problems as well as contributing to the general understanding and providing new, widely applicable methods. The main focus was on the structural as well as dynamic study of ribonucleic acids (RNA). A new structural NMR method was developed aimed at the determination of the glycosidic torsion angle [chi] in RNA, which defines the relative orientation of the nucleobases in respect to the ribose moiety (Duchardt et al., 2004). [Chi] was derived from the angle dependence of carbon-hydrogen dipole-dipole, nitrogen chemical shift anisotropy cross-correlated relaxation rates (gamma-rates). Method development comprised the design of a novel NMR experiment, the [gamma](HCN), as well as the introduction of [gamma] versus [chi] parameterization curves. The novel method provides an accuracy of around 10 degrees or better, comparable to the precision of conventional angle determination techniques. In contrast to conventional methods, the [gamma](HCN) is sensitive to molecular size and will therefore proof beneficial in the investigation of larger RNAs by NMR Apart from this methodological contribution to RNA structure determination, the dynamic properties of the abundant YNMG RNA tetraloop motif (with Y=C or U; N= any base; M=C or A) were studied in a residue specific manner by means of ¹³C NMR relaxation measurements. The dynamics of the extraordinarily stable cUUCGg motif were compared to the less stable uCACGg hairpin, which forms the stem-loop D (SLD) in the regulatory 5'-cloverleaf of coxsackievirus 3B.
(cont.) Measurements were carried out at 25⁰C, at which both motifs are stable, as well as at close to the melting point of SLD (43⁰0). Ribose and base moiety specific amplitudes and time-scales of motion were extracted from R₁, R₁-[rho] and the heteronuclear nOe of C₆ and C₁. in pyrimidines and C₈ and C₁. in purines by application of the model-free formalism (Lipari and Szabo, 1982). The application of the model-free formalism to C₁, and C₆ which possess an additional adjacent carbon spin, was examined for the uniformly isotope labeled RNA hairpins investigated in this study. In addition, the relaxation data analysis was optimized for the ¹³C chemical shift anisotropy based on chemical shift tensors available to date (Stueber and Grant, 2002;Fiala et al., 2000). While at room temperature, the dynamics closely follow the structural features of both hairpins, residues in the loop closing as well as in the adjacent base-pair exhibit highly increased flexibility at temperatures close to the melting point of SLD. In contrast, loop dynamics remain unperturbed. In SLD, the residues close to the loop are conserved among the family of rhino- and enteroviruses, indicating a sequence based mechanism of decoupling loop structure and stability in order to adjust to the twofold requirement of a defined structure for protein recognition and low stability to ensure efficient melting within the genomic transcription process. In addition to investigations on RNA, structural studies were also conducted on the [zeta]-chain of the T-cell receptor (TCR) in order to contribute to the elucidation of the early events in T-cell activation. According to earlier studies (Aivazian and Stem, 2000), the extracellular encounter of the T-cell receptor with an antigen is transmitted into the T-cell via a lipid induced structural transition of the TCR [zeta]-chain cytoplasmic domain.
by Elke Duchardt.
Ph.D.

This Ph.D was aimed to elucidate the physicochemical parameters that rule the penetration of different classes of glycosaminoglycans (GAGs) through human skin, with particular focus on hyaluronan (HA) and heparins. The research activity involved: 1) the investigation of the influence of the main structural features (sequence, molecular weight (Mw), polarity, and conformational behavior) on the percutaneous absorption of HA, heparin (UFH) and low molecular weight heparins (LMWHs); 2) the fine-tuning of a suitable enhancement strategy to improve the permeation of UFH through human epidermis. The overall results confirmed that Mw represents a fundamental factor in determining the permeability properties also of such polysaccharides. Indeed, in in vitro studies performed by using HA having different chain lengths, the diffusion through the skin decreased increasing the polymer’s molecular mass. Similarly, LMWHs sodium salts permeated to a larger extent with respect to UFH and their permeated amounts varied with Mw according to an exponential relationship. However, the key parameter driving the permeation of such macromolecules through the skin resulted to be the polymer flexibility. In fact, the introduction of sulfate groups on HA chains, causing the disruption of the helical motif, led to less ordered structures, which were able to cross the skin to higher extent compared to unsubstituted HA, despite their increased polarity. Accordingly, the presence of divalent cations in LMWHs solution, limiting the chain flexibility through the formation of intramolecular chelates, negatively affected the skin permeability properties of calcium nadroparin. Furthermore, both classes of polysaccharides in study demonstrated to interact with stratum corneum components, in particular keratins, as confirmed by confocal miscroscopy images (HA) and ATR-FTIR spectra (UFH). Since the physicochemical features of these polysaccharides (hydrophilicity, negative charge and high Mw) precluded a significant diffusion through the human epidermis, the phage display technique was exploited to screen a skin penetrating peptide which could enhance the skin permeation of such molecules, allowing their transdermal delivery. The in vitro screening allowed to identify a neutral peptide, DRTTLTN, which resulted to be able to increase the UFH flux of 34-fold after chemical conjugation. These results demonstrated the suitability of skin penetrating peptides to improve the skin delivery of polysaccharide macromolecules.

Abstract The present project has been developed on two distinct lines of work related to the use of biomacromolecules, in particular polysaccharides, to give special purpose hybrid materials. The first is related to preparation of calcite based materials that can potentially replace cement, the second concerns the synthesis of iron oxide nanoparticles for biomedical applications. I) Calcite-based biocomposite material with special mechanical properties The production of building materials is essentially related to cement production. Although cement is a material characterized by the ability to quickly hydrate and to convert into a highly mechanical stress and weather agent resistant material, its production is very ancient and polluting. In fact, the raw material consists of cement minerals which are treated at very high temperatures (1450 °C) with consequent development of CO2 which is a widely diffused greenhouse gas. Moreover, heat treatment is very expensive in terms of electricity and fossil fuel consumption. For these reasons the development of new building materials, involving mild condition chemical processes and renewable raw materials, is highly desirable for human development. Object of this investigation is the innovative preparation and evaluation of materials based on calcium carbonate and biopolymers as alternative to cement. In this formulation calcium carbonate will be used without any heat treatment, avoiding the energy demanding and pollutant decarboxylation process. Our purpose is to find a formulation in which a natural polymer, interacting with calcium carbonate granules induces a mechanical reinforcement in the composite material, that will be measured by mechanical compression stresses. The work consisted in finding the right ingredients of the formulation, which should have a consistency similar to that of mortar. A number of polysaccharides, anionic and neutral, were tested for their capacity to interact with calcite lattice. In particular carboxylated polysaccharides with high density charge such as: carboxymethylcellulose with substitution degree (DS) 2, sodium alginate, and chondroitin sulfate, were chosen. Interestingly chondroitin sulfate resulted as promising agent in improving the mechanical properties of calcium carbonate based material, in addition this hybrid material was able to raise the mechanical resistance of a concrete. 4 II) Biomacromolecules coated iron oxide nanoparticles: synthesis and properties In the first year of PhD course I’ve worked on hybrid materials for biomedical application: nanoparticles are nowadays a new important object of studies of medicinal chemistry and different scientists collaborate in order to mix their skills: biologists, engineers , and chemists. The goal was to synthesize nanoparticles with an appropriate size for the human tissue and with small size dispersion, with a shell made by two components biological matrix: a polysaccharide and a protein, these should ensure tissue biocompatibility and biological activity. In detail, the aim of the project is to prepare bovine serum albumin (BSA) and hyaluronic acid (HA) coated SPIONs (BSA-HA@Fe3O4), to characterize them, load them with drugs and chemosensitizers; the hybrid nanoparticles will be evaluated in their performance in selective drug delivery for cancer cells, and in diagnostic localization of tumors in vivo. The adduct between nanoparticles composed by magnetite (Fe3O4) or maghemite (γ-Fe2O3) and biomacromolecules, hyaluronic acid (HA) and albumin (BSA), were obtained using dopamine (DA) to covalently bind these macromolecules to the surface of the nanoparticle iron oxide core. The prepared product is an iron oxide nanoparticle surrounded by HA and BSA macromolecules as ligands, which act stabilizing the superstructure, anti-cancer targeting and drug delivery.

A estabilização de suspensões de alumina é essencial para a fabricação de vários produtos, especialmente na indústria cerâmica e com o controle das propriedades que se pode atingir usando polímeros como agentes estabilizantes. Na busca de processos industriais que preservem o meio ambiente, polímeros sintéticos podem ser substituídos por biopolímeros, com a vantagem adicional de encontrar usos nobres para rejeitos, como quitina, da qual se obtêm a quitosana e ligninas. Nesta tese, os derivados carboximetilados de celulose, a carboximetilcelulose (CMC) e quitosana, a carboximetilquitosana (CMQ) foram preparados e caracterizados por Espectroscopia de Ressonância Magnética Nuclear de Próton (1HRMN), Espectroscopia na Região do Infravermelho (FTIR), Termogravimetria (TG), Calorimetria Diferencial Exploratória (DSC), Difração de raios X, Cromatografia de Exclusão por Tamanho (SEC). O derivado obtido da lignina, a carboximetil-lignina, (CML) foi caracterizado por Espectroscopia na Região do Infravermelho (FTIR), Termogravimetria (TG), Calorimetria Diferencial Exploratória (DSC). Os três derivados obtidos foram usados como agentes estabilizantes para suspensões aquosas de alumina. A reação de carboximetilação ocorreu em meio heterogêneo, gerando derivados solúveis em água. O grau de substituição (GS) para as carboximetilceluloses foi determinado por 1HRMN, obtendo-se valores de 0,7; 1,3 e 1,8 para as três amostras analisadas nesse trabalho. Para as carboximetilquitosanas, o GS foi determinado por FTIR e análise elementar sendo obtidos valores de 0,6 e 0,8 para duas amostras de CMQ. O valor de GS de 0,5 para carboximetil-lignina foi obtido por titulação potenciomêtrica. Os derivados CMC, CMQ e CML permitiram a estabilização de suspensões de alumina, como demonstrado em medidas de tamanho de partículas, potencial zeta e viscosidade. De relevância especial foi a estabilização em altos valores de pHs, incluindo o ponto de carga zero, pcz, no qual a atração entre as partículas de alumina, de cargas opostas, é máxima, levando a aglomeração de partículas em suspensões sem agentes de estabilização. A distribuição de tamanhos de partícula também foi afetada positivamente com a incorporação dos derivados. De maneira geral, os resultados com os derivados foram promissores em termos de potencial zeta e tamanho de partícula no pcz da alumina, o intervalo de interesse deste trabalho. O destaque é para o derivado de celulose, a CMC GS 1,3 que obteve a melhor performance dentre todos do derivados levando aos menores valores de viscosidade para a suspensão, salientando que esta foi preparada com alto teor de sólidos, nas mesmas condições de suspensões utilizadas em processamentos cerâmicos,. Foi importante também a estabilização ao longo do tempo das suspensões contendo CMC, CMQ e CML, pois o tamanho médio de partícula permaneceu invariável por períodos de até 2 horas, tempo suficiente para processos de moldagem de cerâmicos como extrusão, injeção, tap-casting. O uso de derivados carboximetilados de lignina, quitosana e celulose, como agentes estabilizantes de suspensões de alumina, abre caminho para novas aplicações de produtos obtidos a partir de fontes naturais e renováveis, em substituição aos tradicionalmente usados, oriundos de fontes fósseis.
The stabilization of alumina suspensions is crucial for the fabrication of various products, especially in the ceramic industry, with fine control of materials properties being reached using polymers as stabilizing agents. In this context, in the search for environmentally-friendly industrial processes, synthetic polymers may be replaced with biopolymers, with the added advantage of providing noble uses for waste materials such as those deriving from lignins and chitosans. In this thesis, carboxymethylated cellulose (CMC) and chitosan (CMQ) were prepared and characterized with proton nuclear magnetic resonance (1HNMR), Fourier transform infrared spectroscopy (FTIR), thermogravimetry (TG), differential scanning calorimetry (DSC), X-ray diffraction and size exclusion chromatography (SEC). Carboxymethylated lignin (CML) was characterized with Fourier Transform infrared (FTIR) spectroscopy, thermogravimetry (TG) and DSC. These three derivatives were used as stabilizing agents in aqueous solutions of alumina. The carboxymethylation reaction was carried out in a heterogeneous medium yielding water-soluble derivatives. GS values of 0.7, 1.3 and 1.8 for CMC were obtained with 1HNMR measurements, while values of 0.6 and 0.8 for CMQ were determined using FTIR and elemental analysis. For CML, GS = 0.5 was found using potentiometric titration. The success of the carboxymethylation was confirmed via 1HNMR measurements. The effectiveness of the derivatives CMC, CMQ and CML as stabilizing agents was proven by measuring the size distribution of particles, viscosity and zeta potential of alumina suspensions. Of particular relevance was the stabilization at high pHs, including the point of zero charge (pcz) for which attraction between oppositely charged particles is maximum, where alumina particles normally agglomerate in the absence of stabilizing agents. The particle size distributions were also affected positively by incorporation of the derivatives. Overall, the data presented indicated that CML was responsible for optimized results for the zeta potential and mean particle size at the pzc of alumina suspensions. The CMC with GS = 1.3 exhibited the best performance with the lowest viscosity values at the pcz, even in dispersions with high contents of solid materials which are the conditions prevailing in ceramic processes. Also worth mentioning was the stability over time of the alumina suspensions incorporating CMQ, CML and CMC, with the average particle size remaining the same for 1-2 hours, which is a sufficient period of time for ceramic molding processes such as extrusion, injection and tap-casting. The use of carboxymethylated derivatives of lignin, chitosan and cellulose as stabilizing agents opens the way for the development of new products from natural and renewable sources, to replace those materials traditionally used which are obtained from fossil sources.

This PhD project aims to the optimization of the food packaging materials currently available on the market. The purpose is to develop high performance coatings able to improve the original characteristics of the plastic substrate beneath, as a consequence of the coating deposition. In particular, to fulfil the increasing request for replacing synthetic polymers already used in the nowadays packaging applications as well as to reduce the overall amount of plastics, biomacromolecules, possibly combined with inorganic compounds, will be used to generate a new class of “bio-nanocomposite-hybrid-coatings”. This will lead to a “greener” packaging structure with the same overall performances of the conventional ones. Some films properties will be considered, in particular the oxygen barrier performances at high relative humidity values, the surface properties and the optical properties. The overall project has been divided in three parts. In the first part, selected bio-macromolecules have been used as individual coatings onto polyethylene terephthalate (PET) and polylactid acid (PLA) films and the changes induced by the coating application were investigated by different techniques. Rising from the contact angle investigation, a new potential antifog coating has been disclosed, while the barrier analysis allowed highlighting the great potential of pullulan as oxygen barrier coating even at high relative humidity values. A comparison with some commercial synthetic coatings has also been performed. Ensuing from the obtained results, the second part of the project focused on the coating barrier performance, with special focus on the nanotechnology approach adopted. Specifically, both a bottom-up and a top-down approach were used. With regards to the former, a metal alkoxides was properly treated and combined with a biopolymer (pullulan) to design a final bio-nanostructure to be applied on PET, and the barrier performance was analyzed; concerning the latter, an inorganic clay was exfoliated through an ultrasonication process and mixed with pullulan; the Design of Experiment technique was then used to optimize the formulation in terms of permeability coefficient (P’O2) at the following condition: 23°C and 70% of relative humidity. The best coating formulation allowed decreasing P’O2 of ~84% with respect to the bare substrate (PET), and of ~77% with respect to the film composed by PET coated with pure pullulan (i.e without clays). In addition, microscopy analyses revealed some features of these bionanocomposites and supported the indication about a good exfoliation process. The third and last part of the PhD project, that is still an ongoing project, concerns the simultaneous combination of the aforementioned approaches (i.e. bottom-up and top-down). In principle, this would allow exploiting benefits arising from each individual approach, as well as exploiting any potential synergism.

The interactions and binding of various ligands to biomacromoleculcs e.g. DNA and proteins finds widespread application in the design and development of novel pharmaceuticals. DNA has been identified as the target molecule for a number of drugs and carcinogens and the supramolecular synthetic approach has led to the discovery of a range of bimetallo iron cylinders that bind to DNA inducing remarkable structural effects. The cylinders arc chiral and the enantiomers were separated on cellulose packed in paper or in a column. The optimum mobile phase for efficient separation was found to be 90% acctonitrilc: 10% 0.02 M NaCl. The (M)-enantiomers of the parent cylinder have been found to bind to DNA in the major groove. I Hydrophobic methyl groups were added at various positions on the ligand backbone. UV/visible absorbance, circular and linear dichroism were used to investigate any interactions of the metal complex with DNA with the aim of investigating any sequence preference or selectivity upon binding. Competitive binding studies and molecular dynamics simulations were used to probe the binding geometries of the enantiomers of the parent cylinder and two methylated cylinders to DNA as the exact site of interaction of the (P)-enantiomers of the parent cylinder was unclear. It was concluded that the methylated bimetallo iron cylinders bind to DNA and provide major groove recognition and may show some sequence preference. Circular dichroism was used to structurally characterise a range of buanosine-rich oligonucleotides (GRO's) and to investigate their interactions with a nucleolar protein - nucicolin. Biological/anti-proliferative activity has been related to the ability of the oligonucleotide to bind to this protein. It was found that nucleolin does bind to a biologically active GRO in the presence of K+ and induces a structural change in it.

A hidrólise enzimática de celulose representa, em relação à hidrólise ácida, uma alternativa limpa para produção de etanol. No entanto, existem duas dificuldades: o alto custo das enzimas e recalcitrância das regiões cristalinas da celulose. Para o primeiro problema, propomos a imobilização de celulase, um complexo enzimático que sinergicamente promove a degradação da celulose em glicose e celobiose, sobre wafers de silício. Apesar da atividade enzimática de celulase adsorvida ser em geral menor que a de celulase livre, a imobilização de celulases provou ser vantajosa, pois permite até seis reusos, mantendo um nível de atividade apenas 20% inferior ao original. Quanto à questão da recalcitrância das regiões cristalinas da celulose, utilizamos diferentes pré-tratamentos de celulose, a fim de reduzir a sua cristalinidade e o seu grau de polimerização, além de também modificar a estrutura supramolecular da celulose e a quantidade de poros que esta apresenta, avaliando todos estes parâmetros quantitativamente frente à atividade enzimática livre e imobilizada. A sacarificação enzimática de celulase livre e imobilizada foi determinada na hidrólise de celulose microcristalina (Avicel), e dois tipos de celulose nativa, algodão e eucalipto. Avicel foi pré-tratada a partir da (i) dissolução e degradação em ácido fosfórico, (ii) dissolução em acetato de 1-etil-3-metil-imidazólio (EMIMAc), e (iii) da hidrólise por endoglucanases adsorvidas, uma enzima do complexo enzimático celulase. Celulose de eucalipto e algodão foram mercerizadas a fim de se retirar contaminantes. A hidrólise com celulase livre levou a taxas de conversão de celulose à glicose que não apresentaram correlação com o índice de cristalinidade, nem com o grau de polimerização, mas apresentaram correlação direta com a capacidade de absorção de água, também chamada de constante de capilaridade. As taxas de conversão obtidas na presença de celulase adsorvida apresentaram correlação inversa com a constante de capilaridade, evidenciando que o mecanismo de hidrólise neste caso é fortemente dependente da camada de hidratação da celulose.
Enzymatic hydrolysis of cellulose represents, in relation to acid hydrolysis, a clean alternative for production of ethanol. However, there are two difficulties: the high cost of enzymes and the recalcitrance of the crystalline regions of cellulose. For the first problem, we propose the immobilization of cellulase, an enzymatic complex which synergistically promotes the degradation of cellulose to glucose and cellobiose, onto Si wafers. Although the enzymatic activity of immobilized cellulase is generally lower than that of free cellulase, immobilization has proven to be advantageous since it allows up to six reuses maintaining the activity level at 80% of the original one. Concerning the recalcitrance of the crystalline regions of cellulose, we used different cellulose pretreatments in order to reduce its crystallinity and degree of polymerization, and to modify the cellulose supramolecular structure along with the amount of pores. All these parameters were quantitatively correlated with the activity of free and immobilized cellulase. The enzymatic activity of free and immobilized enzyme was determined by the hydrolysis of microcrystalline cellulose (Avicel), and two types of native cellulose, cotton and eucalyptus. Avicel was pretreated in three different ways: (i) dissolution and degradation in phosphoric acid, (ii) dissolution in 1-ethyl-3-methyl-imidazolium acetate (EMIMAc), and (iii) hydrolysis by immobilized endoglucanase, an enzyme that is part of the cellulase enzyme complex. Eucalyptus and cotton pulp were mercerized in order to remove contaminants. Hydrolysis with free cellulase yielded cellulose to glucose conversions that were neither correlated with the crystallinity index nor with the degree of polymerization, but were directly correlated with the cellulose ability to absorb water (capillary constant). The conversion rates obtained in the presence of immobilized cellulase correlated inversely with the capillary constant values, indicating that hydrolysis mechanism in this case is strongly dependent on the hydration layer of cellulose

All biological processes are fundamentally inter-molecular interactions. In order to understand, and hence control, biomolecular structure and function, methods are required that probe biological systems at the molecular level, ideally with those molecules being in their native environment. The research summarized herein has at its core the development and application of ultra violet (UV)-visible spectrophotometric techniquies for this prupose, in particular circular dichrosim (CD) and linear dichrosim (LD) but also absorbance, fluorescence and resonance light scattering. The spectroscopy is complemented by fundamental theoretical work on molecular structure and reactivity that forms the basis for designing molecules to bind to biomolecules for a particular structural or functional effect. A brief summary of the contributions of the listed publications to our understanding of 'Molecular aspects of biololecule structure and function' is given below under five headings: Circular dichroism theory Molecular geometry and reactivity Small molecule-macromolecule interactions: spectroscopic probes of inter-molecular geometries Molecular design for nucleic acid structure and control Spectroscopic probes of biomolecule structure: instrumentation and application In general terms these correspond to successive phases of the research programme, however, all areas have been present since the first publications in 1983 and can be traced weaving through all subsequent activity.

All biological processes are fundamentally inter-molecular interactions. In order to understand, and hence control, biomolecular structure and function, methods are required that probe biological systems at the molecular level, ideally with those molecules being in their native environment. The research summarized herein has at its core the development and application of ultra violet (UV)-visible spectrophotometric techniquies for this prupose, in particular circular dichrosim (CD) and linear dichrosim (LD) but also absorbance, fluorescence and resonance light scattering. The spectroscopy is complemented by fundamental theoretical work on molecular structure and reactivity that forms the basis for designing molecules to bind to biomolecules for a particular structural or functional effect. A brief summary of the contributions of the listed publications to our understanding of 'Molecular aspects of biololecule structure and function' is given below under five headings: Circular dichroism theory Molecular geometry and reactivity Small molecule-macromolecule interactions: spectroscopic probes of inter-molecular geometries Molecular design for nucleic acid structure and control Spectroscopic probes of biomolecule structure: instrumentation and application In general terms these correspond to successive phases of the research programme, however, all areas have been present since the first publications in 1983 and can be traced weaving through all subsequent activity.

The cellular response to a biomaterial, such as a dental implant, is mainly governed by the surface properties, and can thus be altered by the introduction of a surface coating. In this thesis the buildup of a biomacromolecule-based coating formed by layerby-layer (LbL) deposition of the charged polypeptides poly(L-lysine) (PLL) and poly(L-glutamic acid) (PGA) has been studied. In an attempt to make these coatings bioactive and useful for bone-anchored implants, an amelogenin protein mixture (EMD), has been immobilized in these thin polyelectrolyte multilayer (PEM) films. Multilayers were also built by LbL deposition of the natural biomacromolecules collagen (Col) and hyaluronic acid (HA). Multilayer films of these two extra-cellular biomacromolecules should be of interest for use as a scaffold for tissue engineering.

The buildup of the multilayer films has been followed in situ, using ellipsometry, quartz crystal microbalance with dissipation (QCM-D), and dual polarization interferometry (DPI). The studied PLL/PGA multilayers were found to be highly hydrated, and to exhibit a two-regime buildup behavior, with an initial “slow-growing” regime, and a second “fast-growing” regime with a linear growth in film thickness and more than linear growth in mass. A net diffusion of polypeptides into the film during the buildup led to an increase in density of the films for each layer adsorbed. A change in density was also observed in the Col/HA film, where HA penetrated and diffused into the porous fibrous Col network.

The formed PLL/PGA films were further found to be rather stable during drying, and post-buildup changes in temperature and pH, not losing any mass as long as the temperature was not raised too rapidly. The film thickness responded to changes in the ambient media and collapsed reversibly when dried. A swelling/de-swelling behavior of the film was also observed for changes in the temperature and pH.

The EMD protein adsorbed to silica surfaces as nanospheres, and could by itself form multilayers. The adsorption of EMD onto PLL/PGA multilayer films increased at lower pH (5.0), and EMD could be immobilized in several layers by alternate deposition of EMD and PGA.

An understanding of the interactions between polyelectrolytes and proteins is vital to determine structure and functionality of materials constructed of these two components. Possible applications for the protein-polyelectrolyte composites are ranging from materials used to deliver drugs to the methods of protein stabilisation for storage of therapeutics, biosemsors, and encapsulation of medicines for triggered release. The binding of globular proteins to the polyelectrolyte chains can prevent undesired protein aggregation and may help to extend the shelf-life of the protein-containing food. The aim of this project is to study the mechanism of non-covalent binding between proteins and polyelectrolytes, responsiveness of the proteinpolyelectrolyte composites to external stimuli such as changing pH, presence of salt of different types and concentrations or influence of enzyme on the integrity of protein-polyelectrolyte multilayer film. Our study was focused on the effects of different mono- and multivalent salts on binding affinitybetween a negatively charged polyelectrolyte - poly(styrene sulfonate) PSS and bovine serumalbumin BSA or myoglobin. The complex formation between these polymers was examinedusing the static light scattering (SLS), turbidimetric and potentiometric titrations, differentialscanning calorimetry (DSC) and theoretical studies based on molecular dynamics simulations. We established that the inter- and intramolecular interactions between proteins and polyelectrolytes are primarily driven by the electrostatic forces at the conditions when thepolymers are in low ionic strength solutions and attractive or repulsive relations are based upon the charge density and its distribution. When proteins are interacting with polyelectrolytes in solutions of high ionic strength the electrostatic interactions are screened by the salt originated co-ions. In these conditions there is a competition between salting-out effect on proteins leading to protein aggregation or protein-polyelectrolyte complex formation, which can prevent undesired protein-protein association. The forces driving the attractive interactions at high ionic strength are of non-electrostatic origin, these are mainly hydrophobic forces. The computer simulation study shows that more flexible polyanionic chains are stronger binders to the positive patches on protein surface than these of a more rigid backbone. Also a total energy of binding depends on a sum of electrostatic and non-electrostatic energies. The formation of multilayers composed of a protein and a polyelectrolyte, where componentswere: poly-L-lysine – a positively charged homopolypeptide and polygalacturonic acid - apolysaccharide was examined using a quartz crystal microbalance with dissipation monitoring. A 10 and 11 layer film, deposited on the charged surface, exhibited the linear growth pattern for first 5 layers and exponential growth for a flowing 5 (or 6) layers. The influence of pectinase enzyme on digesting the polygalacturonic acid component of the multilayer was most effective for 1 AU/mL concentration of pectinase. After the enzyme was applied the multilayer film was fully disintegrated within the period of 20 minutes for pectinase at 1 AU/mL and the time of disintegration was extended to 120 minutes for pectinase at 0.1 AU/mL.Silk fibroin aqueous solutions were tested rheologically for their structural properties involvingthe existence of fibroin aggregates. We examined the process of ageing of fibroin solutions and solid-liquid transformations taking place within the fluid. The transitions between viscous and elastic behaviour of the fibroin’s semi-dilute solutions were initiated by strain, shear frequency and temperature. We highlighted that the irreversible change in secondary structure of the silk fibroin in aqueous solutions are taking place after the 48 hour period of time since the preparation of protein fluids. We recommend that further processing of silk fibroin such aselectrospinning should be completed within the 48 hour after dissolution.

Dissertation (PhD)--Stellenbosch University, 2004
ENGLISH ABSTRACT: Over the past decade peptide bola-amphiphiles have been the subject of much attention because of their role as potential models of functionalised membranes and as new generation surfactants. In the quest for new surfactants a peptidomimetic-based approach was used to design a library of novel 'hybrid' bola-amphiphilic peptide surfactants derived from sapecin B and a model symmetrical oligo-glycine bola-amphiphile. The library was divided into different series, each one purpose-built; first, to investigate hierarchal supramolecular architecture and, second, to investigate potential antimicrobial activity. The bola-amphiphiles were synthesised using Fmoc-polyamide based solid phase peptide synthesis and purified via high performance liquid chromatography. The peptide hybrids were characterised using electro spray mass spectrometry, nuclear magnetic resonance, different modes of electron microscopy, Fourier-transform infrared spectroscopy and, in some cases, further studies were done using circular dichroism and bioactivity tests. The model bola-amphiphile suberamide(GGh was synthesised using peptide fragment condensation based on solid phase peptide synthesis. The synthesis is bi-directional (N~C and C~N) and versatile, making it possible to synthesis new dicarboxylic oligopeptide bola-amphiphiles and other analogous compounds. The product, suberarnide(GG)2, was purified using its inherent ability to self-assemble in an acidic solution. Novel asymmetrical bola-amphiphiles composed of dipeptide head groups linked via an aliphatic (I)-amino acid, serving as a hydrocarbon spacer, were also synthesized. Two small libraries of bola-amphiphiles were established - the first involved variation in to-amino acid length and the other variation in the C-terminal amino acid. The bolaamphiphiles were self-assembled in either 0.1% trif1uoroacetic acid or 0.1% triethylamine. Electron microscopy revealed the formation of a variety of higher order supramolecular architectures based on ~-sheet self-assembly. FT-IR spectrometry indicated that interlayer and intralayer hydrogen bond networks, together with strong selfassociation, promoted by the hydrophobic effect and, in certain instances, electrostatic interactions, are responsible for the variety of supramolecular architectures. Variations in the higher order structures can be attributed to amino acid composition, specifically length of m-amino acid, nature of the C-terminal amino acid and the optimised solvent conditions used for the self-assembly process. A third library of novel 'hybrid' bola-amphiphilic peptide surfactants, in which a cationic tripeptide motif from antimicrobial peptides was combined in a hybrid molecule containing a oi-amino acid residue, was established. These bola-amphiphiles displayed potent antimicrobial activity against both Gram-positive and Gram-negative bacteria; the analogues were as active or more active than the leader peptides yet, remarkably, displayed little or no appreciable haemolytic activity. These organopeptide bolaamphiphiles thus demonstrated selective toxicity towards bacteria. The hydrophobicity imparted by the co-amino acid has contrasting effects on haemolysis and antimicrobial activity of the peptide analogues. The other unique feature of these peptides and their analogues is the fact they self-assembled into complex supramolecular architectures, composed primarily of ~-sheets. Their self-assembly is primarily governed by hydrophobic interactions together with inter and intralayer hydrogen bonding. Electron microscopy clearly revealed higher order structures for both peptides and analogues. The generation of higher order supramolecular architecture is dependent on optimisation of ~- sheet self-assembly whereas antimicrobial activity is dependent on the balance between net positive charge and optimum hydrophobicity of the peptide hybrids. This study has demonstrated that it is possible to design hybrid peptide surfactants capable of producing higher order supramolecular architecture and improving the antimicrobial activity whilst reducing the haemolytic effect. The study and design of these versatile 'purpose-built' bio-inspired surfactants heralds a novel approach, one that shows tremendous potential.
AFRIKAANSE OPSOMMING: Die afgelope dekade het bola-amfifiliese peptiede baie aandag geniet weens hulle rolle as potensiële modelle van gefunksionaliseerde membrane en as 'n nuwe generasie surfaktante. In die soeke na nuwe surfaktante is 'n peptiedornimetiese benadering gevolg om 'n biblioteek van nuwe "hibried" bola-amfifiliese peptiedsurfaktante van sapesien B en 'n simmetriese oligoglisien bola-amfifil af te lei. Die biblioteek is in verskillende reekse onderverdeel. Elke reeks is doelmatig vervaardig om ondersoek in te stel na twee aspekte, nl. die rangorde van die supramolekulêre strukture en die potensiële antirnikrobiese aktiwiteit. Fmoc-poliamied gebaseerde soliedefase-peptied-sin-tese is aangewend vir die sintese van die bola-amfifile en hulle is met behulp van hoë doeltreffendheid vloeistofchromatografie gesuiwer. Die peptiedhibriede is gekarakteriseer met behulp van elekrosproei massaspektrometrie, kern-magnetiese resonansie, verskillende modusse elektronrnikroskopie, Fourier-transform infrarooispektrometrie en, in sommige gevalle is verdere studies met sirkulêre dichroïsme en bioaktiwiteitstoetsing uitgevoer. Die bola-amfifilsuberamiedtflfij--model is met behulp van peptiedfragment-konden-sasie gesintetiseer gegrond op soliedefase-peptiedsintese. Dit sintese vind in twee rigtings plaas (N~C en C~N) en is veelsydig aangesien dit die sintese van sowel nuwe dikar-boksielbola- amfifile as ander analoë verbindings moontlik maak. Die produk, suber-arnied(GG)2, is gesuiwer met behulp van die verbinding se inherente vermoë tot self-montering in suur oplossings. Nuwe assimetriese bola-amfifile, saamgestel uit dipeptiedkopgroepe, gekoppel via 'n alifatiese ro-aminosuur, wat as koolwaterstofspasieerder dien, is ook gesintetiseer. Twee klein bola-amfifilbiblioteke is saamgestel - die een het variasies in die ro-aminosuur se lengte omvat en die ander een variasies in die C-terrninale aminosuur. Selfmontering van die bola-amfifile het plaasgevind in of 0,1 % trifluorasynsuur Of 0,1 % trietielamien. Elektronrnikroskopie het die bestaan van 'n verskeidenheid hoërorde supramolekulêre strukture, gegrond op p-plaatselfmontering, aangetoon. Uit FT-IR-spektrometrie blyk dit dat inter - en intralaag waterstofbinbdingsnetwerke en sterk selfassosiasie, 19. word bevorder deur die hidrofobiese effek en, in sekere gevalle, elektrostatiese interaksies, is verantwoordelik vir die verskeidenheid supramolekulêre strukture. Variasies in die hoërorde strukture kan toegeskryf word aan aminosuursamestelling, in besonder die lengte van die ro-aminosuur, die aard van die C-terminale aminosuur en die geoptimiseerde oplosmiddelkondisies wat gebruik is vir die selfmonteringsproses. 'n Derde biblioteek nuwe "hibried" bola-amfifiliese peptiedsurfaktante, waarin 'n kationiese tripeptiedmotief uit antimikrobiale peptiede gekombineer is met 'n m-aminosuurresidu, is geskep. Sommige van hierdie bola-amfifile het 'n kragtige antimikrobiese aktiwiteit teenoor sowel Gram-positiewe as Gram-negatiewe bakterieë gertoon. Die analoë strukture was aktief, of selfs meer aktief as die voorste peptiede maar het, verbasend genoeg, nie 'n beduidende hemolitiese aktiwiteit vertoon nie. Hierdie organopeptied bola-amfifil het dus 'n selektiewe toksisiteit teenoor bakterieë vertoon. Die hidrofo-bisiteit, as gevolg van die ui-aminosuur, het 'n resiproke effek op hemolise en die antimikrobiese aktiwiteit van die peptiedanaloë. Die ander uitstaande kenmerk van die peptiede en hulle analoë is die vermoë om te selfmonteer en komplekse supramolekulêre strukture, bestaande hoofsaaklik uit ~-plate, te vorm. Hierdie selfmontering word in hoofsaak beheer deur hidrofobiese interaksies asook inter - en intralaagwaterstofbinding. Elektronmikroskopie het duidelik hoërorde strukture getoon by sowel dié peptiede as hulle analoë. Die ontwikkeling van hoërorde supramolekulêre struktuurvorms is afhanklik van die optimalisering van die ~-plaatselfmontering. Daarteenoor is die antimikro-biese aktiwiteit afhanklik van die balans tussen die netto positiewe lading en die opti-male hidrofobisiteit van die peptiedhibriede. Hierdie studie het getoon dat dit moontlik is om hibriedsurfaktante te ontwerp wat hoërorde supramolekulêre strukture te produseer en om die antimikrobiese aktiwiteit te verbeter terwyl die hemolitiese effek verminder word. Die studie en ontwerp van hier-die veeldoelige, "doelmatig-gesintetiseerde" biogeïnspireerde surfakante stel 'n unieke benadering daar, wat oor groot potensiaal beskik.

L'attention de la communauté scientifique, ainsi que le développement, pour les bioprocédés dédiés à la culture et à l'expansion de cellules souches mésenchymateuses (MSCs) pour la thérapie cellulaire et la médecine régénérative a considérablement grandi pendant ces dernières décennies. Une plus ample compréhension du lien entre la structure, la fonction et les propriétés des suspensions de cellules mésenchymateuses est devenue de première importance. Dans cette thèse, nous présentons tout d'abord les résultats d'une étude expérimentale portant sur l'écoulement de suspensions concentrées de cellules vivantes d'origine mésenchymateuse pour une grande gamme de concentration cellulaire. Nous caractérisons l'évolution de la viscosité relative en fonction de la contrainte de cisaillement appliquée pour des fractions volumiques cellulaires allant de 20 à 60%. Ces matériaux ont des empreintes rhéologiques compliquées mais très reproductibles, incluant des comportements de fluide à seuil, rhéofluidifiants ainsi que des fractures liées à la contrainte de cisaillement. Les propriétés rhéologiques de la suspension sont ensuite étudiées avec l'addition d'acide hyaluronique (HA), une biomolécule avec des séquences d'adhésion pour des récepteurs à la surface des cellules étudiées. Nous montrons que l'addition d'acide hyaluronique modifie substantiellement le comportement de la suspension et nous permet de contrôler les propriétés d'écoulement de la suspension à toutes les fractions volumiques. Cytométrie de flux et imagerie confocal à l'appui, nous montrons que l'effet observé est dû à un important changement dans la formation d'agrégats cellulaires dans la suspension, et donc dans l'envergure du réseau correspondant. La troisième partie de cette thèse porte sur l'ajout de polyéthylène glycol, une molécule qui n'est pas naturellement présente dans l'organisme mais fréquemment utilisée dans la formulation d'hydrogel. En utilisant trois types de PEG, l'influence de la charge des molécules est étudiée. Les résultats montrent que la charge est un paramètre important dans le contrôle des propriétés d'écoulement de suspensions cellulaires, car déterminant dans la formation et la compacité des agrégats. En considérant les agrégats comme des objets fractals, nous montrons qu'en prenant en compte les modifications de fractions volumiques avec le cisaillement, nous pouvons obtenir une courbe maitresse pour l'ensemble des conditions testées, et en extraire la force d'adhésion moyenne entre les cellules, au travers une population de plusieurs millions de cellules. Cette étude livre de nouveaux aspects sur la complexité des propriétés en écoulement de suspensions de cellules méchymateuses, adhérentes et concentrées, sur leur sensibilité à l'ajout de molécules, qu'elles soient naturellement présentes dans les tissues ou non, ainsi qu'une nouvelle méthode pour mesurer la force d'adhésion entre les cellules.

Fluorescence-based techniques are widely used in bioscience, offering a high sensitivity and versatility. In this work, fluorescence electronic energy migration/ transfer is applied to measure intramolecular distances in two types of systems and under various conditions. The main part of the thesis utilizes the process of donor-acceptor energy transfer to probe distances within the ribosomal protein S16. Proteins are essential to all organisms. Therefore, it is of great interest to study protein structure and function in order to understand and prevent protein malfunction. Moreover, it is also important to try to study the proteins in an environment which resembles its natural habitat. Here two protein homologs were investigated; S16Thermo and S16Meso, isolated from a hyperthemophilic bacterium and a mesophilic bacterium, respectively. It was concluded that the chemically induced unfolded state ensemble of S16Thermo is more compact than the corresponding ensemble of S16Meso. This unfolded state compaction may be one reason for the increased thermal stability of S16Thermo as compared to S16Meso. The unfolded state of S16 was also studied under highly crowded conditions, mimicking the environment found in cells. It appears that a high degree of crowding, induced by 200 mg/mL dextran 20, forces the unfolded state ensemble of S16Thermo to become even more compact. Further, intramolecular distances in the folded state of five S16 mutants were investigated upon increasing amounts of dextran 20. We found that the probed distances in S16Thermo are unaffected by increasing degree of crowding. However, S16Meso shows decreasing intramolecular distances for all three studied variants, up to 100 mg/mL dextran. At higher concentrations, the change in distance becomes anisotropic. This suggests that marginally stable proteins like s16Meso may respond to macromolecular crowding by fine-tuning its structure. More stable proteins like S16Thermo however, show no structural change upon increasing degree of crowding. We also investigated the possibility of local specific interactions between the protein and crowding agent, by means of fluorescence quenching experiments. Upon increasing amounts of a tyrosine labelled dextran, a diverse pattern of fluorescence quantum yield and lifetime suggests that specific, local protein-crowder interactions may occur. In a second studied system, electronic energy migration between two donor-groups, separated by a rigid steroid, was studied by two-photon excitation depolarization experiments. Data were analysed by using recent advances, based on the extended Förster theory, which yield a reasonable value of the distance between the two interacting donor-groups. To the best of our knowledge, this is the first quantitative analysis of energy migration data, obtained from two-photon excited fluorescence.

Macromolecular biomaterials often require covalent crosslinking to achieve adequate stability and mechanical strength for their given application. However, the use of auxiliary chemicals may be associated with long-term toxicity in the body. Oppositely-charged polyelectrolytes (PEs) have the advantage that they can self-crosslink electrostatically and those derived from marine organisms are an inexpensive alternative to glycosaminoglycans present in the extracellular matrix of human tissues. A range of different combinations of PEs and preparation conditions have been reported in the literature. However, although there has been some work on complex formation between chitosan (CS) and carrageenan (CRG), much of the work undertaken has ignored the effect of pH on the consequent physicochemical properties of self-crosslinked polyelectrolyte complex (PEC) gels, films and scaffolds. Chitosan is a positively-charged polysaccharide with NH3+ side groups derived from shrimp shells and, carrageenan is a negatively-charged polysaccharide with OSO3- side groups derived from red seaweed. These abundant polysaccharides possess advantageous properties such as biodegradability and low toxicity. However, at present, there is no clear consensus on the cell binding properties of CS and CRG or CS-CRG PEC materials. The aim of this study was to explore the properties of crosslinker-free PEC gels, solvent-cast PEC films and freeze-dried PEC scaffolds based on CS and CRG precursors for medical applications. The objective was to characterise the effect of pH of the production conditions on the physicochemical and biological properties of CS-CRG PECs. Experimental work focused on the interaction between PEs, the composition of PECs, the rheological properties of PEC gels and the mechanical properties of PEC films and scaffolds. In addition, cell and protein attachment to the PEC films was assessed to determine their interactions in a biological environment. For biomedical applications, these materials should ideally be stable when produced such that they can be processed to form either a film or a scaffold and have mechanical properties comparable to those of collagenous soft tissues. FTIR was used to confirm PEC formation. Zeta potential measurements indicated that the PECs produced at pH 2-6 had a high strength of electrostatic interaction with the highest occurring at pH 4-5. This resulted in stronger intra-crosslinking in the PEC gels which led to the formation of higher yield, solid content, viscosity and fibre content in PEC gels. The weaker interaction at pH 7-12 resulted in higher levels of CS incorporated into the complex and the formation of inter-crosslinking through entanglements between PEC units. This resulted in the production of strong and stiff PEC films and scaffolds appropriate for soft tissue implants. The PECs prepared at pH 7.4 and 9 also exhibited low swelling and mass loss, which was thought to be due to the high CS content and entanglements. From the range of samples tested, the PECs produced at pH 7.4 appeared to show the optimum combination of yield, stability and homogeneity for soft tissue implants. Biological studies were performed on CS, CRG and PECs prepared at pH 3, 5, 7.4 and 9. All of the PE and PEC films were found to be non-cytotoxic. When the response of three different cell types and a high binding affinity protein (tropoelastin) was evaluated; it was found that the CS-CRG PEC films displayed anti-adhesive properties. Based on these experimental observations and previous studies, a mechanistic model of the anti-adhesive behaviour of PEC surfaces was proposed. It was therefore concluded that the CS-CRG PECs produced might be suitable for non-biofouling applications.

Self-assembly of polysaccharide derivatives at liquid/solid interfaces was studied by surface plasmon resonance spectroscopy (SPR) and quartz crystal microbalance with dissipation monitoring (QCM-D). Carboxymethyl cellulose (CMC) adsorption onto cellulose surfaces from aqueous solutions was enhanced by electrolytes, especially by divalent cations. A combination of SPR and QCM-D results showed that CMC formed highly hydrated layers on cellulose surfaces (90 to 95% water by mass). Voigt-based viscoelastic modeling of the QCM-D data was consistent with the existence of highly hydrated CMC layers with relatively low shear viscosities of ~ 10-3 Nâ ¢sâ ¢m-2 and elastic shear moduli of ~ 105 Nâ ¢m-2. Adsorption of pullulan 3-methoxycinnamates (P3MC) and pullulan 4-chlorocinnamates (P4CC) with different degrees of cinnamate substitution (DSCinn) onto cellulose, cellulose acetate propionate (CAP), poly(L-lactic acid) (PLLA), and methyl-terminated self-assembled monolayer (SAM-CH3) surfaces was also studied by SPR and QCM-D. Hydrophobic cinnamate groups promoted the adsorption of pullulan onto all surfaces and the adsorption onto hydrophobic surfaces was significantly greater than onto hydrophilic surfaces. SPR and QCM-D results showed that P3MC and P4CC also formed highly hydrated layers (70 to 90% water by mass) with low shear viscosities and elastic shear moduli. Finally, cellulose adsorption and activity on pullulan cinnamate (PC) and cellulose blend films were studied via QCM-D and in situ atomic force microscopy (AFM). The hydrophobicity of PC surfaces was controlled by adjusting the degree of cinnamate substitution per anhydroglucose unit (DSCinn). It was found that cellulase showed weak adsorption onto low DSCinn PC surfaces, whereas cellulase adsorbed strongly onto high DSCinn PC surfaces, a clear indication of the role surface hydrophobicity played on enzyme adsorption. Moreover, cellulase catalyzed hydrolysis of cellulose/PC and cellulose/polystyrene (PS) blend surfaces was studied. The QCM-D results showed that the cellulase hydrolysis rate on cellulose in cellulose/PC blend surfaces decreased with increasing DSCinn. AFM images revealed smooth surfaces for cellulose/PC (DSCinn = 0.3) blend surfaces and laterally phase separated morphologies for cellulose/PC (DSCinn â ¥ 0.7) blend surfaces. The combination of QCM-D and AFM measurements indicated that cellulase catalyzed hydrolysis was strongly affected by surface morphology. The cellulase hydrolysis activity on cellulose in cellulose/PS blend surfaces was similar with cellulose/PC blend surfaces (DSCinn â ¥ 0.7). These studies showed self-assembly of macromolecules could be a promising strategy to modify material surfaces and provided further fundamental understanding of adsorption phenomena and bioactivity of macromolecules at liquid/solid interfaces.
Ph. D.

In Biochemistry, the term “multivalency” defines a concept of simultaneous, multiple recognition/binding events between two (macro)molecular counterparts. Enabling enhanced binding strength even in the case of intrinsically moderate-potent ligands, this concept has found rather wide application in biomolecular engineering and drug design. In the present work, focused on the development of multivalent modulators of biological and biotechnological processes, oligomerization of functional biomolecules was achieved by their genetic fusion or enzyme-mediated ligation to oligomerization domains, as well as by non-covalent coiled-coil interactions of modified proteins with multivalent bioparticles. Summarized in three peer-reviewed publications given in the cumulative part, the results of this doctoral research contribute to the toolbox of synthetic and biotechnological methods for the generation of multivalent architectures with tailor-made properties. Conceptually, the study can be separated into two independent research branches, the first one exploring effects of avidity by scaffold-based oligomerization of therapeutically relevant target-binding molecules, and the second one dealing with immobilization of orthogonal biocatalytic cascades on bioparticles. In our previous investigation it was shown that engineered cystine-knot miniproteins based on the scaffold of trypsin inhibitor McoTI-II from the squash plant Momordica cochinchinensis are able to bind a therapeutically relevant target, namely cytotoxic T lymphocyte antigen 4 (CTLA-4), however, with low affinity. The potency of these binders can be improved either applying rather sophisticated and time-consuming affinity maturation, or by inducing avidity effects upon multimerization. The latter approach was considered in the present work due to the fact that dimeric CTLA 4 protein is presented on the cell surface in high copy numbers. Within the frame of this work, binding molecules of peptidic nature, among them particular oligopeptides and cystine-knot miniproteins, were attached to oligovalent scaffold proteins by genetic fusion yielding stable oligomers upon recombinant expression. As expected, oligomerization of low-affinity CTLA-4 binder cystine knot MC-CT-010 (Kd = 3.7 µM) on the Fc part of human IgG and the C-terminal oligomerization domain of human C4b binding protein (C4BP) lead to a significant improvement of its functional binding affinity. Indeed, a more than 400-fold improved Kd of 8 nM was determined for the heptavalent fusion construct comprising the C4BP scaffold and MC-CT-010 binder. In order to extend the repertoire of oligomerization methods and to ensure tailoring of multivalent architectures in a modular way, enzyme-catalyzed conjugation of functional molecules with the desired oligovalent scaffolds was applied. To this end, the Fc and C4BP scaffolds were N- or/and C-terminally functionalized with peptidic recognition tags enabling subsequent sortase A-catalyzed ligation with the ligands of interest bearing a respective counterpart. This Lego®-like strategy allowed for the fast enzyme-promoted conjugation of functional monomers at the desired positions within the scaffold. Being applied to death receptor 5 (DR5) targeting peptides (DR5TPs) and the modified Fc and C4BP scaffolds, this approach yielded dimeric, tetrameric and heptameric constructs possessing improved binding capacity towards DR5. These results are of special value as DR5 is overexpressed on cancer cells and, being crosslinked, induces an apoptotic signaling cascade. Interestingly, the strongest binding to DR5 in vitro was observed when the DR5TP ligand was attached to the carboxytermini of C4BP in a linear fashion. Furthermore, this engineered heptad revealed a remarkable biological activity, being able to specifically induce apoptosis in living COLO205 cancer cells (EC50 = 3 nM). We ascertained that ligand number per scaffold molecule as well as their position and spatial orientation is crucial for the biological activity of DR5-targeting oligomers. In general, the established platform allowed for the fast oligomerization of functional probes with further investigation of the steric factors, as well as issues of ligand density, which can influence binding and bioactivity. In addition to covalently bound oligomeric constructs, a non-covalent coiled-coil interaction was used to fabricate enzyme-loaded bioparticles able to promote orthogonal biocatalytic cascades. The carrier particles were derived from a recombinant polyhydroxyalkanoate synthase (PhaC) fusion protein, displaying a multitude of negatively charged Ecoil helices on their surface. Immobilization of enantioselective NADH-dependent alcohol dehydrogenase from Rhodococcus erythropolis and a formate dehydrogenase from Candida boidinii was achieved through the interaction of their engineered Kcoil domains with the respective Ecoil counterpart on the surface of PhaC particles. The resulting multimeric, multifunctional system enabled a catalytic cascade for the stereoselective production of chiral alcohols from ketones – an important step in the manufacturing of pharmaceuticals and fine chemicals. In the frame of our study, the presence of immobilized proteins on PhaC particles was revealed by atomic force microscopy imaging, and the resulting system appeared fully functional. Thus, complete conversion of p-chloroacetophenone to (S)-4-chloro-α-methylbenzyl alcohol by ADH with parallel cofactor regeneration by FDH was confirmed by GC-MS analysis of the reaction products. Moreover, the enantioselectivity of ADH was not affected by the immobilization onto the particles, as was confirmed by GC-MS analysis applying the chiral stationary phase (ee > 99 %). Interestingly, application of an aqueous biphasic system allowed us to use highly concentrated solutions of hydrophobic substrates in organic solvents. In addition, this multienzyme construct could be easily tailored depending on the functional task as the active components could be presented on bioparticles in different ratios, and their orientation can be controlled by the position of the coiled-coil components.

Carboxylated macromolecules such as alginate and glycoproteins are abundant components of modern shallow marine sediments where they are secreted by bacteria and marine infauna. Both organic compounds are proton and metal reactive; hence, they have the potential to facilitate metal sorption and biomineralization reactions. In this study, lab experiments were coupled to field-based sampling to assess the role that these compounds play in microbial mats, with particular emphasis on the hypersaline lagoons of Los Roques, Venezuela. Here I applied a surface complexation approach to model proton and Cd adsorption behaviour of both uronic acid-rich alginate and mucin. Measured total site concentrations, available for metal adsorption, demonstrate that these compounds have the potential to induce metal partitioning in early diagenetic microenvironments. Field results from Venezuela are consistent with Mg- and trace metal- enrichment that follows a likely correlation with the degradation states of microbial biomass trapped during accretion of modern microbialites.

Inside the biological milieu, nanoparticles come in myriad shape and size those upon interaction with different biomolecules form nano-biomolecular complexes. The interface formed as a result of nanoparticle and biomolecular interactions determines fate of both the nanoparticle and biomolecules inside the biological milieu. Accordingly, investigating the interaction pattern at different interfaces will help in optimizing the use of nanoparticle for relatively wider biomedical applications. Hence, the thesis intends to study the effects of different photocatalytic nanoparticle interfaces on biological membranes, like prokaryotic and eukaryotic membranes, and biomacromolecules, like nucleic acid and protein. To this end, photocatalytic nanoparticles, such as zinc oxide (ZnONP), iron oxide (IONP) and silver (AgNP) nanoparticles, were synthesized using chemical synthesis or green synthesis methods. Initially, the effects of interfacial potential and interfacial functional groups were studied against Gram-positive and Gram-negative bacteria. The studies demonstrated that the interfacial potential and surface functionality significantly affect interaction pattern at the interface, which defines anti-bacterial/cytocompatible property of nanoparticles. In addition, second part of the thesis explored the effect of nanoparticle surface defects on cytotoxic and antimicrobial propensities of nanoparticle. The study revealed that energy band gap reduction significantly enhances the oxidative stress in cells, leading viable cells into non-viable cells. The second part, unlike the first part of the thesis where the focus was cell membrane functionality, focused on the interface effects on nucleic acid. Third objective of the thesis observed photocatalytic nanoparticle interaction with antimicrobial peptide (AMP), like nisin, and its effect on the peptide conformational and functional dynamics. The interaction leading into nisin assembly onto AgNP interface enhanced the efficacy of peptide by many folds, without significant change in peptide conformation. Whereas in fourth objective, interaction with globular protein, like lysozyme, showed that the assembly onto ZnONP interface led into conformational rearrangement that hinders the amyloidogenic propensity of lysozyme in studied conditions. Nevertheless, with increase in ZnONP fraction in the conjugate mixtures, the protein attains relatively more regular conformation than partially unfolded conformation at pH 9. Insignificant conformational changes in lysozyme assembled onto ZnONP interface was observed at pH 7.4. Thus, the findings, altogether, suggested that the physico-chemical properties of photocatalytic nanoparticle interface significantly affect the fate of biomembrane and biomacromolecules inside the biological milieu.

abstract: Time-resolved serial femtosecond crystallography is an emerging method that allows for structural discovery to be performed on biomacromolecules during their dynamic trajectory through a reaction pathway after activation. This is performed by triggering a reaction on an ensemble of molecules in nano- or microcrystals and then using femtosecond X-ray laser pulses produced by an X-ray free electron laser to collect near-instantaneous data on the crystal. A full data set can be collected by merging a sufficient number of these patterns together and multiple data sets can be collected at different points along the reaction pathway by manipulating the delay time between reaction initiation and the probing X-rays. In this way, these ‘snapshot’ structures can be viewed in series to make a molecular movie, allowing for atomic visualization of a molecule in action and, thereby, a structural basis for the mechanism and function of a given biomacromolecule. This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained.
Dissertation/Thesis
Doctoral Dissertation Chemistry 2018

Research Doctorate - Doctor of Philosophy (PhD)
There is an urgent need for substantial investigation of non-platinum drugs with higher activity and improved selectivity to address the problem associated with the use of platinum-based compounds as therapeutic agents. In light of this, diphenyltin(IV), dimethyltin(IV) and tin(IV) compounds were synthesised from the Schiff bases of three series of dithiocarbazate (S-2-methylbenzyldithiocarbazate (S1), S-4-methylbenzyl dithiocarbazate (S2), S-benzyldithiocarbazate (S3)) and two series of thiosemicarbazides (4-methyl-3-thiosemicarbazide and 4-phenyl-3-thiosemicarbazide) with aldehydes, 2-hydroxy-3-methoxybenzaldehyde (oVa) or 2,3-dihydroxybenzaldehyde (catechol). The tin(IV) compounds formed were found to have a general formula of [R2Sn(ONS)] and [Sn(ONS)₂] (where R = Me and Ph). The compounds were fully characterised by physicochemical and spectroscopic methods. The spectroscopic results supported the coordination geometry in which the Schiff bases behaved as tridentate ONS donor ligands coordinating via azomethine nitrogen, thiolo sulphur and phenoxide oxygen atoms. A total of 11 crystal structures of the expected compounds were solved in this work. In order to verify the experimental data, the compounds were optimised using the density functional theory (DFT) method with the B3LYP hybrid exchange correlation functional with LanL2DZ pseudopotential on tin and 6-311G(d,p) Pople basis set for all other atoms. Diphenyltin(IV) compounds showed the most promising cytotoxicity with IC50 values ranging between 0.016 – 4.40 μM against a panel of twelve cancer cell lines (RT-112, EJ-28 (bladder), HT29 (colon), U87, SJ-G2, SMA (glioblastoma), MCF-7 (breast), A2780 (ovarian), H460 (lung), A431 (skin), Du145 (prostate), BE2-C (neuroblastoma) and MIA (pancreatic)). The three diphenyltin(IV) compounds of the oVa series were able to induce the production of Reactive Oxygen Species (ROS) and acted as a cell apoptosis inducer. Good binding interactions for all the diphenyltin(IV) compound series were observed and supported by molecular docking analysis, where hydrogen, electrostatic and hydrophobic binding interactions were observed. This highlights the important of two phenyl groups coordinated directly to the tin ion to enhance the cytotoxicity by strong π-π stacking interactions to biomacromolecules. Diphenyltin(IV) compounds could bring hope in the field of drug development against various diseases including cancers.

Cyclodextrins (CDs) are macrocycles composed of several glucose units bound through α-1,4 glycosidic linkages. They can be chemically modified to display functional groups on their primary or secondary rim. CDs display these groups in defined geometries ideally suited to bind biomacromolecules. Moreover, CDs have a hydrophobic cavity that allows them to form stable host-guest complexes with lipophilic molecules. This combination of functionality and guest binding ability makes CDs important scaffolds for the design of functional supramolecular systems. This thesis explored the interaction of heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-β-cyclodextrin (1) with many hydrophobic guest molecules. The binding constants of CD host-guest interactions were measured using ITC and fluorometry-based approaches. These studies revealed 1 to form the highest affinity 1:1 cyclodextrin-guest complexes reported to date. This thesis then explored the use of CD inclusion complexes as biomacromolecular sensors. CD 1 and its derivatives were used to develop self-assembling sensors. First, a library of polycationic CDs with differing charge distribution was synthesized. The sensing motif was synthesized by covalently linking a quinolinium fluorophore to lithocholic acid (LCA). The CD-based binding motifs and the LCA-based sensing motif self-assemble through host-guest interactions (i.e. 1 binding to LCA displays a Ka = 5.52 × 107 M-1). These inclusion complexes were then used as an array of self-assembling sensors capable of differentiating between pure and contaminated samples of heparin (anticoagulant). To capitalize on the promise of CD 1 a new technique was explored to functionalize a single amine of 1. The technique relies on an S to N acyl transfer from a guest molecule to a CD host resulting in the mono-acylation of the host. The importance of the linker between the guest and the reactive acylating agent was fully explored. Furthermore, two CD probes are synthesized and are shown to display differential fluorescent responses with a small series of proteins.

Xu, Xiao-ping.
"March 1999."
Thesis (Ph.D.)--Chinese University of Hong Kong, 1999.
Includes bibliographical references (p. 152-166).
Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web.
Mode of access: World Wide Web.
Abstracts in English and Chinese.

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This review summarises the recent developments in the synthesis and applications of polymers derived from malic acid. There has been an increased interest in the design of sustainable and biodegradable polymers as a result of the drive to use renewable feedstocks as an alternative to petrochemicals in addition to their significant potential in biomedical applications. Synthetic strategies to access polymers from malic acid based on both condensation and ring-opening polymerization, across a broad range of conditions, are reviewed along with their advantages and limits. The role that such materials are studied for in biomedical applications is discussed, and their environmental impact based on the biodegradability of the malic polymer backbone is outlined.
The Royal Society, EPSRC, BBSRC

Controlling conformation of macromolecules, both in solution and solid state, has remained an exciting challenge till date as it confronts the entropy driven random coil conformation. Folded forms of biomacromolecules, like proteins and nucleic acids, have served as role-models to the scientists in terms of designing synthetic foldamers. The folded functional forms of proteins and nucleic acids have been shown to rely heavily on various factors, like directional hydrogen bonding, intrinsic conformational preferences of the backbone, solvation (e.g. hydrophobic effects), coulombic interactions, charge-transfer interactions, metal-ion complexation, etc. Chapter-1 discusses various designs of synthetic polymers explored by research groups world-over to emulate the exquisite conformational control exercised by biomacromolecular systems. Our laboratory has been extensively involved since 2004 in designing charge-transfer complexation induced folding of flexible donor-acceptor (DA) polymeric systems, such as those shown in Scheme 1. It was observed that such polymers adopt a folded conformation in polar solvents, like methanol, in the presence of an excess of an appropriate alkali metal ion. To explore folding in the solid state, Jonas and co-workers recently showed that a polyethylene-like polyester with long alkylene segments containing periodically located pendant propyl group forms a semicrystalline morphology with alternating crystalline and amorphous regions primarily because of the periodic folding of the backbone due to the steric exclusion of the propyl branches from the crystalline domains. In order to explore immiscibility-driven folding of polyethylene-like polyesters, Roy et al. designed a periodically grafted amphiphilic copolymer (PGAC) containing long alkylene segments (mimicking polyethylene) and pendant oligoethyleneglycol chains at periodic intervals (Scheme 2). Scheme 2: Proposed folding of a periodically grafted amphiphilic copolymer It was demonstrated that immiscibility between the hydrocarbon backbone and pendant PEG segments drives the polymer to adopt a folded zigzag conformation as shown in Scheme 2. The above synthetic strategy, however, does not permit easy structural variation of the side chain segments because the side-chain segment is covalently linked to the malonate monomer. In Chapter-2, a more general strategy to prepare periodically grafted copolymers has been described. In an effort to do so, we designed a series of clickable polyesters carrying propargyl/allyl functionality at regular intervals along the polymer backbone, as shown in Scheme 3. Scheme 3: Periodically clickable polyesters for the preparation of periodically grafted copolymers The polyesters were prepared by reacting either 2-propargyl-1,3-propanediol, 2,2-dipropargyl-1,3-propanediol or 2-allyl-2-propargyl-1,3-propanediol with an alkylene diacid chloride, namely 1,20-eicosanedioic acid chloride, under solution polycondensation conditions. Since these polyesters carry either, one propargyl, two propargyls or one propargyl and one allyl group on every repeat unit, it provides us an opportunity to synthesise exact graft copolymers with one side chain, two side chains or even two dissimilar side chains per repeat unit. In Chapter-3, the periodically clickable polyesters were reacted with MPEG-350 (PEG 350 monomethyl ether) azides using Cu(I) catalyzed azide-yne click reaction to generate periodically grafted amphiphilic copolymers (PGAC) carrying crystallizable hydrophobic backbone and pendant hydrophilic MPEG-350 side-chains (Scheme 4). Since the PGACs carry either one or two pendant MPEG-350 chains on every repeat unit, it allowed us to examine the effect of steric crowding on the crystallization propensity of the central alkylene segment. Scheme 4: Functionalization of periodically clickable polyesters with MPEG 350 azide by azide-yne click reaction From DSC studies, it was observed that increase in steric crowding at junctions resulting from increased side-chain volume hinders effective packing of the hydrocarbon backbone. As a result, both transition temperatures and the enthalpies associated with these transitions decreases. SAXS and AFM studies revealed the formation of lamellar morphology with alternate domains of PEG and hydrocarbon. Based on these observations, we proposed that self-segregation between hydrophobic backbone and hydrophilic side-chains induce the backbone to adopt a folded zigzag conformation (Scheme 5). Scheme 5: Schematic depiction of self-segregation induced folding of PGAC and their assembly on mica surface (AFM image) In order to study the effect of solvent polarity on conformational evolution of the periodically grafted amphiphilic copolymers, we randomly incorporated pyrene in the backbone of the polymer by reacting a small fraction (~ 5 mole %) of the propargyl groups with pyrene azide. Fluorescence study of the pyrene labelled polymer showed that increase in solvent polarity increases the intensity of the excimer band dramatically; this suggests the possible collapse of the polymer chain to the folded zigzag form. In an extension of this work, the PGAC was further used as template to synthesise layered silicates that appears to replicate the lamellar periodicity seen in the polymer. In order to study the effect of reversing the amphiphilicity on self-segregation, in Chapter-4, we synthesised a series of clickable polyesters carrying PEG segments of varying lengths, namely PEG 300, PEG 600 and PEG 1000, along the polymer backbone. The polymers were prepared by trans-esterification of 2-propargyl dihexylmalonate with different PEG-diols. These polyesters were then clicked with docosyl (C22) azide using Cu(I) catalyzed azide-yne click reaction to generate the desired periodically grafted amphiphilic polymers carrying crystallizable hydrophobic pendant chains at periodic intervals; the periodicity in this case was governed by the length of the PEG diols (Scheme 6). Scheme 6: PGACs carrying hydrophilic PEG backbone and crystallizable hydrophobic pendant docosyl chains Varying the average periodicity of grafting provided an opportunity to examine its consequences on the self-segregation behavior. Given the strong tendency of the pendant docosyl segments to crystallize, DSC studies proved useful to analyse the self-segregation; DOCOPEG 300 clearly exhibited the most effective self-segregation, whereas both DOCOPEG 600 and DOCOPEG 1000 showed weaker segregation. Based on the observations from DSC studies, we proposed that the PEG backbone adopts a hairpin like conformation (Scheme 7). Scheme 7: Proposed self-segregation through hairpin like conformation of backbone PEG segments In order to confirm the bulk morphology, we carried out small angle X-ray scattering (SAXS) and atomic force microscopic (AFM) studies. The SAXS profiles confirmed the observations from DSC studies, and only DOCOPEG 300 exhibited well-defined lamellar ordering. Thus, it is clear that the length of the backbone PEG segment (volume-fraction) strongly influences the morphology of the PGACs. Based on the inter-lamellar spacing from SAXS and the height measurements from AFM studies (Scheme 8), we proposed that these polymers form lamellar morphology through inter-digitation of the pendant docosyl side-chains. The observations from Chapters 3 and 4 suggested that the crystallization of the backbone has a dramatic effect on the conformation of the polymer backbone. In order to explore the possibility of independent crystallization of both backbone and pendant side-chains, the periodically clickable polyesters, described in Chapter-2, were quantitatively reacted with a fluoroalkyl azide, namely CF3(CF2)7CH2CH2N3 using Cu(I) catalyzed azide-yne click reaction; Chapter-5 describes these polyesters carrying long chain alkylene segments along the backbone and either one or two perfluoroalkyl segments located at periodic intervals along the polymer chain (Scheme 9). DSC thermograms of two of the samples showed two distinct endotherms associated with the melting of the individual domains, while the WAXS patterns confirm the existence of two separate peaks corresponding to the inter-chain distances within the crystalline lattices of the hydrocarbon (HC) and fluorocarbon (FC) domains; this confirmed the occurrence of independent crystallization of both the backbone and side chains. Scheme 10: Left-variation of SAXS profile of all three polymers as a function of temperature, Right- molecular modelling of representative FC-HC-FC triblock structures. Interestingly, a smectic-type liquid crystalline phase was observed at temperatures between the two melting transitions. SAXS data, on the other hand, revealed the formation of an extended lamellar morphology with alternating domains of HC and FC (Scheme 10). The inter-lamellar spacing calculated from SAXS matches reasonably well with those estimated from TEM images. Based on these observations, we proposed that the FC modified polymers adopt a folded zigzag conformation whereby the backbone alkylene (HC) segment becomes colocated at the center and is flanked by the perfluoroalkyl (FC) groups on either side, as depicted in Scheme 11. Melting of alternate HC domains first leads to the formation of a smectic-type liquid crystalline mesophase, wherein the crystalline FC domains retain the smectic ordering; this was confirmed by polarizing light microscopic observations. Scheme 11: Schematic presentation of self-segregation induced folding of polymer chains; and hence crystallization assisted assembly of these singly folded chains to form lamellar structure One interesting challenge would be to create unsymmetrical folded structures, wherein the top and bottom segments of the zigzag folded form would be occupied by two different segments, such as PEG and FC, whereas the backbone alkylene segment would form the central domain; this would lead to the possible formation of consecutive domains of PEG, HC and FC through immiscibility driven self-segregation process. In Chapter-6, several approaches to access such systems have been described; one such design that could have resulted in the successful synthesis of a periodically clickable polymer carrying orthogonally clickable propargyl and allyl groups along the backbone in an alternating fashion is depicted in (Scheme 12). The parent polyester was successfully synthesized and the propargyl group was first clicked with the FC-azide to yield the FC-clicked polyester; however, several attempts to click MPEG-SH onto the allyl groups using thiol-ene click reaction failed. Scheme 12: Scheme for the synthesis of alternating orthogonally clickable polymer In order to accomplish our final objective, we chose to first prepare the FC-clicked diacid chloride and polymerize it with an azide-alkyne clickable macro-diol, as depicted in Scheme 13; this approach was successful and yielded the desired clickable polyester bearing the FC segments at every alternate location. This polymer was then clicked with PEG-750 azide to yield the final targeted polymer that carries mutually immiscible FC and PEG-750 segments at alternating positions along the polymer backbone. The occurrence of self-segregation of FC, PEG-750 and the alkylene backbone (HC) was first examined by DSC studies, which appeared to suggest the presence of three peaks, although these were not very well-resolved. Scheme 13: Schematic for the synthesis of the polymer carrying FC and PEG 750 alternatingly along the backbone A schematic depiction of the anticipated organization of such unsymmetric folded macromolecules is shown in Scheme 15; it is evident that because of mutual immiscibility, the layers will be organized such that the FC domains of adjacent layers will be together and similarly the PEG domains of adjacent layers will also be together. Such an organization would lead to an estimated spacing that would correspond to a bilayer of the folded structures. Interestingly, SAXS study (Scheme 14) reveals the formation of lamellar morphology with a d-spacing of 14.6 nm. Scheme 14: Figure 6.10: SAXS profile of the polymer PE-FC-PEG 750 In order to gain an estimate of the expected inter-lamellar spacing, the end-to-end distance of a model repeat-unit was computed to be ~ 9.4 nm. It is, therefore, evident that the inter-lamellar spacing of 14.6 nm seen in the SAXS is significantly larger and must represent a bilayer type organization (Scheme 15). In this regard it is important to say that the organization of these alternatingly functionalized folded chains should give a variety of d-spacings. Because of highest electron density contrast of FC among PEG, HC and FC, we proposed that the d-spacing calculated from the SAXS profile corresponds to ‘d4’ in Scheme 15. This first demonstration of the formation of zigzag folded unsymmetric entities bearing dissimilar segments on either side of the folded chain holds exciting potential for a variety of different applications and beckons further investigations. Scheme 15: Schematic for the proposed self-assembly of the singly folded polymer chains