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- Jiang, Kun
(author)
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Modulating the structure-function relationship of mucin materials
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Mucus covers the epithelium surfaces playing a key role in the barrier, hydration, lubrication and bioactivity functions for the human body, and mucins are the fundamental components of mucus which provide such functions. However, many details in how these functions are related to the many structural features of mucins are still unknown. In this thesis, strategies were developed to modify mucin structures in defined ways to gain insights and controls over several key functions of this molecule. We altered functional motifs on the mucin molecules, and changed the conformation of mucin networks to modulate the accessibility of such functional motifs. Then functional outcomes, including macrophage responses, diffusion of molecules through mucin networks, and mucin lubricity were studied. This thesis is organized into three parts according to the three functions. We found that the responses of macrophage to mucins were modulated by changing the location of crosslinks between mucins and by the immobilization of mucins onto surfaces. Mucins’ bottle-brush structure allowed their crosslinking either via the protein backbone or via the glycan side chains with similar crosslinking density. With the same crosslinking structure, the placement of mucin thin film on hard substrate and soft substrate led to different macrophage responses, and only mucin coating on soft gel induced similar immune responses as that of mucin gels. A better understanding of how mucin bioactivity, and specifically immune-modulating properties, can be modulated, provide new strategies to develop biomaterials with defined bioactivities. This work could also inspire innovative treatments to modulate mucin bioactivities in vivo to treat mucin-related diseases such as for mucinous cancers. The diffusion of molecules through mucin gels was modulated by changing the gel network and their affinity filtration capacity. By locating the crosslinking sites on the protein backbone or the glycan chains, the gel network remained the same with similar diffusion profiles of dextrans. However, with simple change of mucin concentration, molecules of different size diffused faster in gels with lower mucin concentration. The affinity filtration of mucin gel was modulated by removing sialic acids, which acts as binding sites for molecules or cells via electrostatic interactions or specific binding, and the binding can slow down diffusion of cells or molecules. By altering sialic acid contents, the diffusion of charged dextran was modulated and the penetration of sperms was increased. With the understanding, mucin gels with controlled permeability can be designed for loading drugs or encapsulating tissue. And strategies can be developed in the future for treating mucus barrier related disease in vivo, such as inflammatory bowel disease.The hydration & lubrication of mucin coating was modulated with changed mucin structure and removal of associated impurities. The hydration of mucin coatings remained the same, but their lubricity was lost by removing negatively charged sugars. However, the components associated with mucin, such as DNA, compensated for the missing of negative sugars. Commercial PGM was found with damaged glycosylation and missing peptide domains, and its lubricity was lost completely compared to lab-purified mucins. With this knowledge, mucin structure can be modulated for desired hydration and lubrication performance, and this can inspire to develop strategies for restoring their hydration and lubricity in vivo during diseases, such as dry eye.
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| 2. |
- Koskela, Salla, 1990-
(author)
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Lytic polysaccharide monooxygenases for green production of cellulose nanomaterials
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Cellulose is the main structural polymer in wood, and its potential in the form of nanomaterial building blocks, nanocelluloses, has now been recognized. Nanocelluloses, including cellulose nanofibers (CNFs) and cellulose nanocrystals (CNCs), have become increasingly important in development of modern sustainable materials. Nanocelluloses are typically produced from wood pulp fibers by chemical pre-treatments that deposit charged functional groups onto cellulose microfibril surfaces, thereby promoting disintegration of the fiber cell wall during mechanical fibrillation. Due to environmental risks related to the use of harsh chemical treatments, it is crucial to develop greener, nature-inspired alternatives. As renowned decomposers of wood, fungi secrete cellulose-active enzymes that work in aqueous reaction conditions. Of these, lytic polysaccharide monooxygenases (LPMOs) have piqued a special interest in green production of nanocellulose owing to their ability to introduce charged carboxyl groups onto cellulose surfaces. However, little is known about the properties of LPMO-oxidized nanocelluloses, their mechanical performance in bulk materials, and the mechanism how LPMOs facilitate fibrillation of the wood fiber cells.This PhD thesis aimed to dissect the potential of C1-oxidizing LPMOs in the production of nanocelluloses and to clarify the mechanism of LPMO oxidation that facilitates the disintegration of wood cell wall. LPMOs with and without attached carbohydrate-binding modules (CBMs) were recombinantly produced in Pichia pastoris and studied for the production of CNFs and CNCs, which were further processed into bulk materials. The morphology and properties of the nanocelluloses, and the optical and mechanical properties of the bulk materials were characterized. In addition, delignified wood with a preserved cellular structure was used as a model substrate for LPMO oxidation, and the LPMO-induced changes in the wood cell wall structure were investigated using advanced scattering techniques.The results on CNF production showed that LPMO-oxidized wood pulp fibers can be transformed into discrete and colloidal CNFs by mild mechanical disintegration, analogous to chemical pre-treatments such as 2,2,6,6-tetramethylpyperidine-1-oxy radical (TEMPO)-mediated oxidation. Importantly, these CNFs were well individualized with an average width of 4 nm, resembling that of cellulose microfibrils in wood. Such CNFs were obtained from softwood holocellulose- and kraft pulp fibers with a hemicellulose content of 16–19%, but not from dissolving pulp with a lower hemicellulose content of 4%. Nanopapers prepared from the LPMO-oxidized CNFs were transparent and they demonstrated tensile strengths of ca. 260 MPa and Young’s moduli of ca. 17 GPa. The water suspensions of LPMO-oxidized CNFs also exhibited acid-triggered gelation behavior due to the enzymatically introduced carboxyl groups.LPMO oxidation was also found applicable in the preparation of CNCs from microcrystalline cellulose. The LPMO-oxidized CNCs had a needle-like morphology and they formed stable colloidal suspensions in water that demonstrated flow-induced birefringence. Solution cast films showed that the CNCs bearing C1 carboxyl groups possessed the pivotal ability to undergo self-assembly into an anisotropic phase. As some LPMOs are appended to a non-catalytic CBM, the effect of this module on nanocellulose production was also determined. CBM was found to increase the release of carboxyl groups from cellulose microfibril surfaces in the form of soluble cello-oligosaccharides. By contrast, a non-modular LPMO introduced more carboxyl groups to the cellulose surfaces, up to 0.53 mmol g-1 on CNFs, and 0.70 mmol g-1 on CNCs. Indeed, a non-modular LPMO was found advantageous in production of both CNFs and CNCs.Despite the important role of LPMOs for natural and biotechnological degradation of wood biomass, the LPMO-induced changes in the wood cell wall structure have remained unknown. In this work, these changes were characterized for the first time. It was shown that a C1-oxiding LPMO can modulate cellulose microfibrils and disrupt the wood cell wall ultrastructure by modifying cellulose surface chemistry. After the LPMO oxidation, the average distance between cellulose microfibril centers increased from 4.1 nm to 10.7 nm, signifying the separation of microfibrils in a microfibril bundle. This result revealed a previously unidentified role for C1-oxidizing LPMOs in degradation of cellulose at the nanoscale. Remarkably, LPMO-treated wood veneers could be further compressed into anisotropic, transparent films with an ultrahigh tensile strength of 824 MPa.In summary, this PhD thesis clarified the potential of C1-oxidizing LPMOs in green production of nanocelluloses and showed that LPMO oxidation is a suitable method to obtain high-performing isotropic and anisotropic bulk materials from wood. On the basis of the obtained findings, a new model was also proposed which elucidates the mechanism of cellulose degradation at the nanoscale. This study broadened the understanding of LPMOs including their biological- and biotechnological significance and provided new insights into the use of LPMOs for the preparation of cellulose-based nanomaterials.
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| 3. |
- Yuan, Yusheng, 1992-
(author)
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Development of Functionalized Protein Materials
- 2022
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Doctoral thesis (other academic/artistic)abstract
- Many proteins are available as side-streams from food production, and in some cases even from industrial waste-streams. This means that proteins are available in large scale and at a relatively low price. As protein are highly complex molecules it is interesting to try to use protein as starting materials in for applications in materials science. Most proteins have the ability to self-assemble into nanofibrils. These fibrils have a regular repeating substructure that consists of β-strands running perpendicular to the fibril axis, resulting in cross-β sheets that run parallel along the fibril axis. The extended β-sheets structure results in the formation of hydrophobic grooves that can act as potential binding sites organic molecules. This means that the functionality of the material may be modified by addition of e.g. light emitting molecules or drug molecules. By such functionalization the protein material may accordingly be suitable for applications such as light-conversion materials (e.g. for use as coatings of light emitting diodes (LEDs)) or for drug-delivery. For such applications, the protein fibrils must be processes into macroscopic structures such as films or gels. Against this background, we employ the food proteins hen egg white lysozyme and β-lactoglobulin as model proteins for fibrillation and functionalization. Through a mechanochemical process the hydrophobic dyes can conveniently be combined with proteins, that can be converted into functionalized protein nanofibrils by liquid-phase self-assembly. By employing protein fibrils functionalized with three dyes, we have been able to form films that enables conversion of UV light to white light (and can thus be employed as a coating on UV-LEDs) with protein fibrils functionalized with multiple dyes. By mixing biodegradable polymers with functionalized protein fibrils, luminescent bioplastic films can be prepared that are processable when wet; a cut film will also self-heal if water is applied. We have also turned functionalized protein fibrils into gel states, including hydrogels or aerogels. In the case of protein fibrils functionalized with Hydantoins (a type of drug molecule) hydrogels were prepared, and the release of the drug was investigated. In addition, aerogels can be prepared from hydrogels by freeze drying, and in this manner lightweight functionalized aerogels are achieved. By functionalization with an electrically conductive polymer, an elastic conductive aerogel is formed that employed as a piezoelectric pressure sensor. In summary a wide range of materials have been prepared suitable for various applications demonstrating the flexibility of the developed functionalization methodology and that the structural richness of protein self-assembly can be employed to prepare a wide variety of types of materials of varying functionality.
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